Receptor Tyrosine Kinase c-Kit Signalling in

Hematopoietic Progenitor Cells

Charlotte Edling

Department of Medical Biosciences, Pathology, Umeå University, Sweden

Umeå 2006

Umeå University Medical Dissertations

New Series no. 1058 ISSN: 0346-6612 ISBN: 91-7264-180-0

Edited by the Dean of the Faculty of Medicine

Copyright © 2006 by Charlotte Edling

Printed in Sweden by Solfjädern Offset AB, Umeå 2006

RTK C-KIT SIGNALLING IN HEMATOPOIETIC PROGENITOR CELLS _______________________________________________________________________

ABSTRACT

The receptor tyrosine kinase (RTK) c-Kit is expressed in hematopoietic stem and progenitor cells,

mast cells and in several non-hematopoietic tissues. In the hematopoietic system c-Kit and its

ligand Steel Factor (SF, aka Stem Cell Factor) are critical for proliferation, survival and

differentiation. Mutations in either receptor or ligand lead to lethal anaemia, hematopoietic stem

cell defects, mast cell deficiency and a series of non-hematological defects.

The aims of the studies included in this thesis are to describe the signalling pathways

downstream c-Kit in hematopoietic stem/progenitor cells and to further analyse the role of c-Kit

signalling in fundamental biological functions.

To study c-Kit signalling in the hematopoietic system we have employed hematopoietic

stem cell-like cell lines which share many properties with primary hematopoietic stem cells in

vitro and in vivo, including surface markers, multipotentiality, capacity for self-renewal and long

term repopulation. In paper I we demonstrate that upon SF activation the RTK c-Kit is

autophosphorylated and downstream signalling mediators are transiently activated. Surprisingly

we find that the c-Kit mediated activation of the MAPK pathway is dependent on the activation

of phosphoinositide 3-kinase (PI3K) in hematopoietic progenitor cells and that differentiation of

these progenitors to mast cells results in a signalling switch where Raf activation changes from

PI3K dependent to PI3K independent. We here establish that PI3K activity is required for

viability and proliferation of hematopoietic progenitor cells. In paper II we studied the

conventional protein kinase C (cPKC) involvement in c-Kit signalling. We observe that the

cPKCs can phosphorylate c-Kit on serine 746 and that this phosphorylation negatively regulates

the activation of the receptor. We demonstrate that inhibition of this negative phosphorylation

results in dramatically increased protein kinase B (PKB) activation and as a consequence

inhibition of cPKCs rescues cells from starvation induced apoptosis. Moreover we exhibit that the

cPKCs are necessary for full activation of extracellular signal-regulated kinase (Erk) and that

impaired PKC activity leads to hampered proliferation. In paper III we demonstrate that in

addition to the cPKCs also the novel PKCδ is required for Erk activation and proliferation.

Furthermore we present results indicating that PKCδ negatively regulates differentiation of bone

marrow.

In conclusion, with the studies in this thesis we display details in the signalling pathways

induced upon RTK c-Kit activation and we demonstrate that c-Kit has significant effects on

hematopoietic cell-physiology.

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CHARLOTTE EDLING 2006 _______________________________________________________________________

TABLE OF CONTENTS

ABSTRACT ..................................................................................................................... 1 PAPERS IN THIS THESIS............................................................................................... 4 ABBREVIATIONS ........................................................................................................... 5 INTRODUCTION.............................................................................................................. 7

Phosphorylation mediated signalling ....................................................................................7 Protein phosphorylation .................................................................................................................... 7 Receptor Tyrosine Kinases................................................................................................................ 8 The Src homology 2 domain.............................................................................................................. 8 Phosphatases...................................................................................................................................... 9 Endocytosis......................................................................................................................................... 9 Regulation of signal transduction..................................................................................................... 9 Protein kinase inhibitors .................................................................................................................. 10

Receptor tyrosine kinase c-Kit .............................................................................................10 Identification of receptor and ligand............................................................................................... 10 Expression and Mutations ............................................................................................................... 11 Imatinib mesylate.............................................................................................................................. 11 SF, the c-Kit ligand ........................................................................................................................... 12 C-Kit structure ................................................................................................................................... 12 Isoforms and splice variants ........................................................................................................... 13 Activation of c-Kit ............................................................................................................................. 13 Protein binding sites on c-Kit .......................................................................................................... 14

APS and Cbl .................................................................................................................................. 14 Src .................................................................................................................................................. 15 Shp1/2 ............................................................................................................................................ 15 Grb2/7 and Shc ............................................................................................................................. 15 P85, PI3K regulatory subunit....................................................................................................... 15 PLCγ ............................................................................................................................................... 16

Signalling mediators downstream of c-Kit ..........................................................................16 Ras and the Raf/Mek/Erk cascade................................................................................................... 16

Ras activation ............................................................................................................................... 16 Raf activation and phosphorylation sites .................................................................................. 17 Mek and Erk activation................................................................................................................. 18 Regulation of the Raf/Mek/Erk cascade ..................................................................................... 19

The PI3K pathway ............................................................................................................................. 19 The lipid kinase PI3K.................................................................................................................... 19 PKB activation .............................................................................................................................. 20 PKB substrates ............................................................................................................................. 20 Regulation of the PI3K pathway.................................................................................................. 21

The Protein Kinase C family ............................................................................................................ 22 The hematopoietic system ....................................................................................................23

The hematopoietic stem cell............................................................................................................ 23 Defining the Hematopoietic stem cell ........................................................................................ 23 Regulation of HSC self-renewal .................................................................................................. 24

The hematopoietic hierarchy ........................................................................................................... 25 The mast cell lineage ................................................................................................................... 25 The roles of c-Kit and SF in the hematopoietic system ........................................................... 27

Model systems for hematopoietic cells.......................................................................................... 27 The Hematopoietic progenitor cell lines .................................................................................... 28

AIMS .............................................................................................................................. 29

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RTK C-KIT SIGNALLING IN HEMATOPOIETIC PROGENITOR CELLS _______________________________________________________________________

RESULTS AND DISCUSSION ...................................................................................... 30

Paper I .....................................................................................................................................30 Erk activation is PI3K dependent in progenitor cells but not in differentiated cells ................. 30

Crosstalks reported in the literature .......................................................................................... 32 Erk is required for proliferation of HPCs........................................................................................ 33 Activation of the PI3K pathway is crucial for survival .................................................................. 34

Paper II ....................................................................................................................................35 PKC activates Raf, but not Ras or PKB in the HPC lines ............................................................. 35 PKC is required for Erk activation and proliferation ..................................................................... 36 The cPKC mediates serine phosphorylation of c-Kit .................................................................... 36 Conventional PKCs inhibit PKB activation and promote apoptosis ........................................... 37

Paper III ...................................................................................................................................38 Inhibition of PKCδ does not affect c-Kit ......................................................................................... 38 Rottlerin prevents starvation induced apoptosis .......................................................................... 38 Inhibition of PKCδ causes G1 cell cycle arrest.............................................................................. 39 PKCδ influences differentiation of bone marrow........................................................................... 40

CONCLUSIONS............................................................................................................. 41 ACKNOWLEDGEMENTS.............................................................................................. 42 REFERENCES............................................................................................................... 43 ORIGINAL PAPERS………………………………………………………………………….I-III

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PAPERS IN THIS THESIS _______________________________________________________________________

PAPERS IN THIS THESIS This thesis is based on the following papers referred to in the text by their

roman numerals (I-III).

Paper I

“Activation of the MAP-kinase pathway by c-Kit is PI-3-kinase dependent in hematopoietic progenitor/stem cell lines”

Wandzioch E*, Edling CE*, Palmer RH, Carlsson L and Hallberg B.

Blood. 2004 Jul 1;104(1):51-7

*contributed equally Paper II

“Hematopoietic progenitor cells and mast cells utilize PKC to activate Erk and suppress PKB/Akt activity in response to c-Kit stimulation.”

Edling CE, Pedersen M, Carlsson L, Rönnstrand L, Palmer RH and Hallberg B.

Submitted to British Journal of Haematology Paper III

"PKCδ is required for proliferation and can negatively regulate differentiation in c-Kit expressing hematopoietic cells"

Edling CE, Rönnstrand L and Hallberg B. manuscript

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ABBREVIATIONS _______________________________________________________________________

ABBREVIATIONS

aPKC atypical PKCs APS adaptor containing PH and SH2 domains ATP adenosine triphosphate CMP common myeloid progenitors cPKC conventional PKCs CSF-1 colony-stimulating factor-1 DAG diacylglycerol Erk extracellular signal regulated kinase ES embryonic stem FACS fluorescence-activated cell sorting FoxO forkhead box, class O GAP GTPase activating protein GDP guanine diphosphate GEF guanine nucleotide exchange factor GIST gastrointestinal stromal tumour GM-CSF granulocyte-macrophage colony stimulating factor GNNK glycine-asparagine-asparagine-lysine Grb2 growth factor receptor-bound protein 2 GSK3 glycogen synthase kinase-3 GTP guanine triphosphate GTPase guanosine triphosphatase HEK human embryonic kidney HPC hematopoietic progenitor cell HSC hematopoietic stem cell HZ4-FeSV Hardy-Zuckerman 4 feline sarcoma virus IGF insulin-like growth factor Ig-like immunoglobulin-like IL-3 interleukin-3 IP3 inositol-1,4,5-triphosphate JM juxtamembrane KSR kinase suppressor of Ras Lhx2 LIM (Lin11, Isl1 and Mec3) homeobox 2 lin- lineage negative LSK lin- Sca+ c-Kit+ LT long-term MAPK mitogen-activated protein kinase MCP mast cell progenitor Mek MAPK/Erk kinase MGF mast cell growth factor MP1 Mek partner-1 MPP multipotential progenitor mTor mammalian target of Rapamycin nPKC novel PKC PDGF platelet-derived growth factor

5

ABBREVIATIONS _______________________________________________________________________

PDK phosphoinositide-dependent protein kinase PH pleckstrin homology PHLPP PH domain leucine-rich repeat protein phosphatase PI3K phosphoinositide-3 kinase PIP2 phosphatidylinositol 4,5 diphosphate PIP3 phosphatidylinositol 3,4,5 triphosphate PKA protein kinase A PKB protein kinase B PKC protein kinase C PLCγ phospholipase C γ PMA phorbol 12-myristate 13-acetate PP2A protein phosphatase-2A PTB phosphotyrosine binding PTEN phosphatase and tensin homologue deleted on chromosome ten Raf rapidly growing fibrosarcomas Ras rat sarcoma RBD Ras-binding domain RKIP Raf kinase inhibitor protein RTK receptor tyrosine kinase S serine Sca stem cell antigen SF steel factor SFK Src family kinases SH2 Src homology 2 Shc SH2-containing transforming protein C1 SHIP SH2-containing phosphatase Shp1/2 SH2 domain-containing phosphatase 1 and 2 Sl steel Sos son-of-sevenless ST short-term T threonine W white spotting Y tyrosine

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INTRODUCTION _______________________________________________________________________

INTRODUCTION

Phosphorylation mediated signalling Protein phosphorylation Eukaryotic cell functions are to a large extent regulated by protein kinases. Protein

phosphorylation is involved in intracellular and intercellular communication which controls

pivotal processes such as metabolism, cell cycle progression, differentiation, apoptosis,

cytoskeletal architecture and gene expression in the cell.

Phosphate in proteins was described for the first time one hundred years ago by Levene and

Alsberg [1], however the enzymatic phosphorylation reaction was not described until fifty years

later by Kennedy and Burnett [2]. In the 1970s studies by Krebs, Cohen and others had

established that serine/threonine phosphorylation was a rapid post-translational key mechanism in

signal transduction [3] and in 1979 Hunter and colleagues identified tyrosine phosphorylation.

They furthermore demonstrated that this new form of phosphorylation could be linked to

malignant transformation [4].

Today, the protein kinases constitute a large protein family. In fact, almost 2% of the eukaryotic

genes contain protein kinase domains [5]. The protein kinase family members are well conserved

through the molecular evolution based on sequence and structure. The importance of the protein

kinases are further underlined by the number of diseases that are caused by mutations in protein

kinase genes [6].

Protein kinases act by catalysing the transfer of an ATP (adenosine triphosphate) gamma-

phosphate to hydroxyl groups of the amino acids serines, threonines or tyrosines on target

proteins. The phosphorylation of a target protein in most cases results in a modification of the

conformation that changes the enzyme activity or facilitates physical interaction with other

molecules. Phosphorylation of proteins is a reversible mechanism enabling proteins to switch

from an inactive state to an active state in the course of a millisecond [7,8].

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INTRODUCTION _______________________________________________________________________

Receptor Tyrosine Kinases Following the discovery that proteins could be tyrosine phosphorylated Hunter and others

displayed that the viral protein Src transformed cells through tyrosine phosphorylation.

Continuation on this line of research has established that the receptors for several growth factors

are in fact tyrosine kinases and that abnormal activation or expression can induce cellular

transformation [9]. It is now predicted that, based on the sequences of the catalytic domains, of

the total 518 protein kinases 90 are tyrosine kinases. These are further divided in 58 receptor

tyrosine kinases (RTK) and 32 non-receptor tyrosine kinases [6].

The RTKs are transmembrane protein tyrosine kinases with an extracellular part comprising the

ligand binding domain and an intracellular part containing the kinase domain. The extracellular

and intracellular domains are connected via a single helix through the cell membrane. The

intracellular domains are much conserved among the RTKs, contrary to the extracellular regions

which display high divergence and are able to interact with different ligands. All RTKs, except

the Insulin receptor, are monomers in the cell membrane. However ligand binding induces

dimerisation of the receptors which is followed by autophosphorylation of their cytoplasmic

domains. The dimerisation induced activation differs among the RTKs, some are activated by a

single ligand binding to two receptors simultaneously, and then additional receptors can bind to

the complex for further stabilisation. Other receptor-ligand complexes are constructed by two

ligands and two receptors to enable full activation [10].

The Src homology 2 domain The autophosphorylation of the receptor results in intracellular proteins containing Src homology

2 (SH2) domains recognizing and transiently interacting with specific phosphotyrosine motifs on

the receptor. The SH2 domain was first identified by Pawson and colleagues in 1986 as a non-

catalytic region that could modify both kinase activity and substrate recognition [11]. A few years

later Kuriyan and Eck solved the structure for the Src SH2 domain and described in detail how it

could recognise and bind phosphorylated tyrosines [12,13]. Individual SH2 domains can

distinguish between the specific autophosphorylation sites because of their ability to recognise

residues C-terminally of the phosphotyrosine and depending on preference they will bind [14]. In

addition to the SH2 domain it has been revealed that proteins can contain a phosphotyrosine

binding (PTB) domain in connection to the SH2 domain that also enables binding to

phosphorylated tyrosines, although, with less specificity. Upon activation and intrinsic

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INTRODUCTION _______________________________________________________________________

phosphorylation of the RTK, cytoplasmic mediators containing SH2 domains are recruited to the

phosphotyrosines on the receptor and these interactions activate the effector proteins which

subsequently mediate the signal via downstream pathways. These signals mediate the effects of,

for example; growth factors, hormones, or antigens which are vital for the cell [9].

Phosphatases Activation by phosphorylation is closely regulated by different mechanisms. The protein

phosphatases play a key role in regulating the action of kinases since they catalyze the removal of

phosphate groups from phosphorylated substrates. The first protein tyrosine phosphatase, PTP1B,

was isolated and described by Tonks et al. in 1988 [15,16]. Since then the family of phosphatases

has been shown to constitute a wide range of enzymes with different specificity. The

phosphatases are divided into two major classes: protein serine/threonine phosphatases and

tyrosine phosphatases. In contrast to the kinases there is no sequence similarity in the catalytic

domain between the two classes. The protein tyrosine phosphatases are defined by the signature

motif, (H/V)C(X)5R(S/T), in the catalytic domain. Mechanistically they act by transferring the

phosphate group to the cysteine residue before hydrolysation with water. In addition to enzyme

regulation, phosphates can also be removed in a nonenzymatic hydrolytic reaction [17,18].

Endocytosis Phosphorylation and dephosphorylation mechanisms are crucial to regulate activity and inactivity

of proteins. Beside this instant mechanism of regulation, proteins, and especially receptors,

become down regulated after activation via internalisation by endocytosis. Upon activation of a

RTK a clathrin coated vesicle is formed at the signalling site and the receptor is internalised in

the vesicle and transferred via the early and late endosomes for degradation in the lysosome. The

endocytosis process is ongoing constantly in the cell. Activated receptors get rapidly degraded

and inactive receptors get recycled at a low rate [19].

Regulation of signal transduction A major question in the field of cell regulation is how signals can be transduced with the required

specificity. Despite the fact that many receptors are expressed in several tissue types and that

different receptors can utilise the same downstream pathways, the effect of the receptor activation

can still induce a specific biological response. The answer probably lays in that the system of cell

types, receptors, ligands and pathways comprise an extremely complex network that enables

enough variation to fulfil specificity for all purposes. Factors involved in determining the signal

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INTRODUCTION _______________________________________________________________________

transduction specificity include the balance between kinase and phosphatase activity, cellular

localisation of proteins, binding of scaffold proteins, multisite versus single site phosphorylation,

duration and strength of signals, number of available receptors and combinations of receptors and

signalling molecules [9,10,20].

Protein kinase inhibitors Since all kinases require ATP for there enzymatic activity they can all be inhibited by blocking

the domain where the ATP binds. The catalytic domain, including the ATP binding pocket, is

highly conserved among the protein kinases. Despite this fact, it has proven possible to develop

specific inhibitors because, especially in the inactive state, the kinases present distinct features

that can be targeted by small binding molecules. The available protein kinase inhibitors are well

used tools for biochemical analyses of protein functions and some are now successfully used in

cancer therapy [21].

Receptor tyrosine kinase c-Kit Identification of receptor and ligand In 1986 Besmer et al. identified a new viral oncogene in the Hardy-Zuckerman 4 feline sarcoma

virus (HZ4-FeSV) that had been isolated from a feline fibrosarcoma. The amino acid sequence

revealed homology with the tyrosine kinase gene family and it was shown to be closely related to

v-fms. This oncogene was designated v-Kit [22]. A year later, the human cellular homologue, c-

Kit, was identified and characterized by Yarden et al. [23]. They reported that the c-Kit gene

product is structurally related to the receptor for macrophage colony-stimulating factor-1 (CSF-1

or c-fms) and the receptor for platelet-derived growth factor (PDGF) that both had been recently

identified [24,25]. They further demonstrated that the c-Kit gene encoded a transmembrane

glycoprotein of 145kDa which was capable of autophosphorylation on tyrosine residues and

suggested, based on the structural likeness to the PDGF and CSF-1 receptors, that c-Kit (also

referred to as stem cell factor receptor or CD117) was a growth factor receptor for an unidentified

ligand [23]. In 1988 c-Kit was exhibited to be encoded by the white spotting (W) locus on

chromosome 5 in mice (chromosome 4 in human [26]) [27,28]. A couple of years later a novel

mast cell growth factor (MGF) was identified and demonstrated to be a ligand for the c-Kit

receptor [29] and at the same time the MGF coding sequence was cloned and mapped to the Steel

(Sl) locus in the distal region of mouse chromosome 10 [30]. The c-Kit ligand has been assigned

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INTRODUCTION _______________________________________________________________________

several different names: kit ligand, MGF, stem cell factor and Steel Factor (SF). In this text it will

be referred to as Steel Factor (SF).

Expression and Mutations The mapping of c-Kit and SF explained the similar phenotypes caused by mutations in the W and

Sl locuses that had been described several years earlier [31]. Normal c-Kit expression is observed

in several tissues including: mast cells, hematopoietic stem and progenitor cells, interstitial cells

of Cajal in the gastrointestinal tract, melanocytes, germ cells and neurons [32]. The expression in

the hematopoietic system will be described more in a later section. Various types of loss-of-

function and gain-of function mutations have been reported at the W locus. The loss-of-function

mutations in mice are clearly connected to the c-Kit expressing tissues. Abnormalities due to

impaired c-kit function in mice are depletion of mast cells [33] and interstitial cells of Cajal [34],

severe anaemia due to damaged erythropoiesis [31], piebaldism [31] and reduced hearing ability

[35] due to lack of melanocytes and sterility in both males and females [31].

There is a range of different types of gain-of-function mutations of c-Kit including point

mutations, deletions and duplications. Frequently occurring point mutated residues are valine 560

in the juxtamembrane (JM) domain and asparagine 816 in the kinase domain [36]. The activating

mutants of c-Kit confer with constitutional tyrosine phosphorylation and downstream activation

independent of ligand binding. These mutations expose a strong oncogenic potential of c-Kit.

Tumours caused by constitutively activated c-Kit are different types of mast cell neoplasms,

gastrointestinal stromal tumours (GISTs), germ cell tumours and some leukemias. In addition

aberrant expression of c-Kit has been observed in some tumours such as small cell lung

carcinomas [32].

Imatinib mesylate As discussed above there are several tyrosine kinase inhibitors available. Interruption of

signalling pathways are said to be the new generation of tumour therapy. One such inhibitor that

is used with remarkable success is imatinib mesylate, formerly known as STI571 (commercially

Gleevec/Glivec). Initially this drug was developed at Ciba-Geigy (now Novartis) as a specific

inhibitor of the tyrosine kinases Bcr-Abelson (Abl) and the PDGF receptor [37]. Extended

research has revealed that imatinib mesylate is also a potent inhibitor of c-Kit, but not of the

closely related c-fms, flt and tek tyrosine kinases [38]. Imatinib mesylate is approved and

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INTRODUCTION _______________________________________________________________________

indicated for treatment of chronic myeloid leukaemia (CML) that is caused by expression of Bcr-

Abl and of c-Kit expressing GISTs [US and European drug administrations].

SF, the c-Kit ligand SF exists in a soluble and a membrane-anchored isoform, due to alternative RNA splicing and

proteolytic processing [39]. The soluble form consists of 165 amino acids and it functions as a

noncovalent homodimer. SF dimers bind soluble or membrane bound c-Kit with high specificity

and affinity [40].

Figure 1. Schematic structure of c-Kit, amino acids apply to human c-Kit

C-Kit structure signal sequence

C-terminal tail

transmembrane segment

juxtamembrane domain

activation loop

distal kinase domain

kinase insert domain

Ig-like motifs

proximal kinase domain

1

5

4

3

2

1-22

685-761

521-543

Y568

Y936

Y721

Y570544-581

Y703

Y823

Y730762-937

582-684

938-976

810-839

signal sequence

C-terminal tail

transmembrane segment

juxtamembrane domain

activation loop

distal kinase domain

kinase insert domain

Ig-like motifs

proximal kinase domain

1

5

4

3

2

1-22

685-761

521-543

Y568Y568

Y936Y936

Y721Y721

Y570Y570544-581

Y703Y703

Y823Y823

Y730Y730762-937

582-684

938-976

810-839

The c-Kit receptor is categorised as a type III

receptor tyrosine kinase. The members in

this subfamily features unique

characteristics; an extracellular part

containing five immunoglobulin-like (Ig-

like) motifs and a tyrosine kinase domain

that is divided in a proximal and a distal part

by an insert sequence of variable length. In

addition to these characteristics the c-Kit

structure is composed of domains that are

common among most RTKs. See figure 1.

A signal sequence is located in the N-

terminal end. This sequence is followed by

the five Ig-like motifs. The second and third

Ig-like motifs together constitute the ligand

binding pocket [41]. The fourth Ig-like motif

contains the dimerisation site. Deletion of

the fourth Ig-like motif completely abolishes

dimerisation and the following signal

transduction. A defective dimerisation site

results in accelerated ligand dissociation,

indicating that the ligand affinity is

dependent on dimerisation of the receptors

[42].

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INTRODUCTION _______________________________________________________________________

The intracellular part of c-Kit is composed of the autoinhibitory juxta-membrane domain and the

two kinase domains with the insert between them. Deletion of the juxtamembrane domain causes

increased induction time for activation of c-Kit and addition of an exogenous juxtamembrane

domain peptide inhibits the trans phosphorylation [43]. The kinase domains, with the activation

loop located in the distal kinase domain, are responsible for catalysing the transfer of a phosphate

group from ATP to the substrate [44].

Isoforms and splice variants In addition to the above described transmembrane form of c-Kit there is a soluble truncated form

of c-Kit. The truncated c-kit is constituted of a single distal kinase domain and it is expressed in

male germ cells [45]. Since this isoform is severely truncated it lacks functional kinase activity.

Even so, it can still mediate signals and has been shown to be of importance for spermatogenesis

[46].

In both human and mouse alternative mRNA splicing has resulted in c-Kit isoforms that differ in

a four amino acid sequence, Glycine-Asparagine-Asparagine-Lysine (GNNK), located

extracellulary near the transmembrane segment [47]. The ratio between the two isoforms varies in

different tissues, but the GNNK- isoform is usually more abundant [48]. A difference in

transformation ability and downstream signalling depending on isoform has been suggested. The

GNNK- form appears to induce anchorage-independent growth and tumourigenicity to a higher

extent than the GNNK+ from [49]. Moreover, the SF stimulated c-Kit GNNK- form is more

highly tyrosine phosphorylated, and Erk and Shc are more strongly activated due to increased and

more rapid Src activation [50]. In addition to these splice forms, human c-Kit splice variants that

differ in the presence or absence of a single serine residue in the kinase insert domain have been

described [48], although whether this has any impact on signalling is not known.

Activation of c-Kit Activation of c-Kit is initiated when a SF dimer simultaneously binds two monomeric c-Kit and

thereby promote dimer formation [41]. Dimerisation enables autophosphorylation in trans on

tyrosine residues in the intracellular domain. Commonly the first tyrosine residues to be

phosphorylated are those located in the activation loop. This results in stabilisation of the receptor

in its most active enzyme form [51]. However, in the RTK type III subfamily the tyrosine

residues in the juxtamembrane are phosphorylated first. Studies of the crystal structure of c-kit

have revealed why tyrosines 568 and 570 in the juxtamembrane domain are phosphorylated

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INTRODUCTION _______________________________________________________________________

before the tyrosine 823 in the activation loop. These initial phosphorylations of the

juxtamembrane domain alter the static configuration that sterically blocks the activation loop

from converting to its extended active conformation. Subsequently the tyrosine 823 is

phosphorylated to stabilise the activation loop in the active enzyme form [52,53]. When c-Kit is

fully activated the phosphorylated tyrosine residues in the juxtamembrane domain, kinase

domains and the kinase insert domain facilitate docking sites for substrates encompassing SH2.

The conformation change of the activation loop allows access to the bound ATP and the gamma

phosphate group from ATP is transferred to hydroxyl groups on the substrate [10].

Protein binding sites on c-Kit Upon activation the RTK c-Kit is phosphorylated at specific intracellular tyrosine (Y) residues to

which downstream signalling mediators bind and subsequently get activated. See table 1. In

addition to the effectors included in Table 1, there are many different proteins that are recruited to

the activated receptor and take part in constituting signalling complexes, but do not directly

associate with the receptor (for reference see Roskoski, 2005 [54]).

Phospho-site Recruited proteins Mechanism Y568/570 APS, Src family kinases, Shp1/2, Cbl, Shc autophosphorylation Y703 Grb2 autophosphorylation Y721 p85 subunit of PI3K autophosphorylation Y730 PLCγ autophosphorylation Y823 activation loop autophosphorylation Y900 p85/Crk phosphorylated by Src Y936 Grb2/7, APS, Cbl autophosphorylation

Table 1. Phospho-sites in human c-Kit, protein name and phosphorylation mechanism

APS and Cbl

APS (adaptor containing PH and SH2 domains) is an adaptor protein which associates with c-Kit

on Y568 and Y936. APS activation is involved in induction of internalisation and degradation of

c-Kit by interacting with Cbl [55]. Cbl acts as an E3 ubiquitin-protein ligase, transferring

ubiquitin from ubiquitin-conjugating enzymes to substrates promoting their degradation by the

proteasome [56].

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INTRODUCTION _______________________________________________________________________ Src

The Src family kinases (SFK) are suggested to be involved in many different biological

functions, including survival, proliferation, differentiation and motility [57]. They associate with

Y568 and Y570, either directly or via the adaptor proteins APS. Activation of c-Kit induces

rapidly increased SFK kinase activity [58,59]. They have further been shown to be responsible

for phosphorylating Shc (SH2-containing transforming protein C1) and thereby enabling

recruitment of the Grb2/Sos complex (growth factor receptor-bound protein 2/Son-of-Sevenless)

[60]. Recruitment and activation of this complex subsequently results in activation of Ras [61].

Moreover, recent data demonstrates that Cbl can bind directly to Y568 and Y936, but it requires

phosphorylation by SFK members for activation leading to down-regulation of c-Kit [62].

Shp1/2

Shp1 and 2 (SH2 domain-containing phosphatase 1 and 2) are cytosolic phosphotyrosyl

phosphatases which negatively regulate growth factor signalling. Shp1 is mainly expressed in

hematopoietic and epithelial cells, while Shp2 is ubiquitously expressed [63]. In vivo studies

indicate that Shp1 binds to c-Kit at Y570 while Shp2 binds at Y568. They act by

dephosphorylating the receptor directly, or inhibit c-kit signalling indirectly by

dephosphorylating other receptor-associated protein-tyrosine kinases [64].

Grb2/7 and Shc

Grb2 and 7 are adaptor proteins. Grb2 binds c-Kit both at Y703 and Y936, and Grb7 only at

Y936 [65]. Grb2, in complex with Sos and Shc, is the link that mediates the growth factor signal

from the receptor to Ras (rat sarcoma) [61]. Grb2 is also shown to be involved in the recruitment

of Cbl to c-Kit [66].

P85, PI3K regulatory subunit

Phosphoinositide-3 Kinase (PI3K) is a heterodimeric lipid kinase constituting two subunits: p85

and p110 [67]. PI3K signalling mechanisms and effects will be discussed in a later section. SF

stimulation of cells leads to rapid activation of PI3K, and PI3K has been demonstrated to interact

with the kinase insert domain of c-Kit [68]. More exactly, the regulatory subunit (p85) of PI3K

binds to Y721 of c-Kit [69] causing increased kinase activity of the catalytic subunit. The p85-

receptor interaction is suggested to be negatively regulated by the SFK member Lyn since PI3K

is constitutively associated with c-Kit in Lyn-deficient bone marrow mast cells [70].

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INTRODUCTION _______________________________________________________________________

Furthermore, the p85 subunit is also known to associate with the adapter proteins Cbl and Crk

[71]. This complex, possibly without Cbl, is thought to bind, via p85, the Y900 residue after it

has been phosphorylated by Src or a downstream mediator. Mutation of this site results in weaker

induction of DNA synthesis. [72].

PLCγ

Phospholipase C γ (PLCγ) belongs to an enzyme family that catalyses the hydrolysis of

phosphatidylinositol-4,5-biphosphate (PIP2) to generate the second messengers diacylglycerol

(DAG) and inositol-1,4,5-triphosphate (IP3). DAG activates protein kinase C (PKC) family

members and IP3 triggers the release of Ca2+ by binding specific receptors on the endoplasmatic

reticulum [73]. Rottapel et al. showed that both PLCγ and PI3K were activated upon SF

stimulation of murine mast cells [74] and overexpression studies have suggested Y730 to be the

site for PLCγ association with c-Kit [75,76].

Signalling mediators downstream of c-Kit Ras and the Raf/Mek/Erk cascade Ras activation

Ras belongs to the Ras superfamily that contains over 150 human members. Ras is a small

guanosine triphosphatase (GTPase), like all members in the Ras superfamily it binds guanine

nucleotides with high affinity and it possesses low intrinsic GTPase activity. Ras is active when

bound to GTP (guanine triphosphate) and it is inactive when bound to GDP (guanine

diphosphate). Exchange from GDP to GTP causes conformational changes that result in the GTP-

bound form exhibiting a higher affinity for effector targets. The switch from inactive to active

and back is tightly regulated by GEFs (guanine nucleotide exchange factors) and GAPs (GTPase

activating proteins). GEFs promote the switch from GDP to GTP, hence result in activation.

GAPs increase the intrinsic GTPase activity resulting in inactivation. [77].

Upon activation of c-Kit the adapter proteins discussed above are recruited to the phosphorylated

receptor. Grb2 is constitutively associated with Sos, which is a GEF for Ras. The colocalisation

and complex-building of Grb2, Sos, Shc and possibly other adaptor proteins increase Ras activity

[78].

16

INTRODUCTION _______________________________________________________________________

The Ras superfamily members can induce mitogen-activated protein kinase (MAPK) cascades.

The MAPKs constitute a network of cascades transmitting stimuli-induced signals. The MAPK

pathways contain a kinase cascade comprising a MAP kinase kinase kinase, a MAP kinase

kinase, and a MAP kinase. In this thesis we have mainly focused on the classical MAPK cascade

that is commonly activated downstream of RTKs. This pathway constitutes Raf (rapidly growing

fibrosarcomas), Mek (MAPK/Erk kinase), and Erk (extracellular signal regulated kinase) [79].

Raf activation and phosphorylation sites

Activation of Raf is complex and not fully understood. However it involves membrane

recruitment, dimerisation or oligomerisation, protein-binding, conformational changes and

phosphorylation [80]. The activation process is initiated by the binding of GTP-bound Ras to the

Ras-binding domain (RBD) of Raf [81]. This interaction recruits Raf to the plasma membrane

where it gets activated [80,82]. There are three isoforms of Raf; a-Raf, b-Raf and c-Raf. C-Raf

was cloned first [83] and has been studied most intensively.

C-Raf is tightly regulated by phosphorylation. Protein kinase A (PKA) is believed to negatively

regulate c-Raf by phosphorylation on three different serine (S) residues; S43, S233 and S259.

PKA is a cyclic-AMP-dependent kinase and these sites have been demonstrated to be

phosphorylated upon increase of cyclic-AMP levels in the cell [84]. S259 is also suggested to be

phosphorylated by protein kinase B (PKB, also known as Akt) resulting in suppression of c-Raf

activity [85]. Phosphorylation of S43 sterically hinders binding of Ras to Raf which inhibits

activation [86]. Phosphorylated S233 and S259 both create binding sites for the scaffold protein

14-3-3. Binding at these sites blocks the activation process of c-Raf [84,87].

In addition to the three negative sites there are five sites, located within or near the kinase

domain, that stimulate c-Raf activity. Phosphorylation of S338 is required for activation of c-Raf,

however, there is a discrepancy in the literature regarding which kinase is targeting this site.

Some studies suggest that it is the serine/threonine(S/T)-specific protein kinase PAK [80].

However, the PAKs are demonstrated to phosphorylate c-Raf located in the cytosol, in a Ras

independent manner, and are therefore probably not responsible for the growth-factor stimulated

activation at the membrane. Furthermore growth-factor stimulated S338 phosphorylation is not

inhibited by dominant negative PAK [88]. The SFKs have been shown to phosphorylate Y341, a

site which is required for activation [82,89,90]. Phosphorylation of T491 and S494 are essential

since they are located in the activation loop of c-Raf and mutations of these sites have been

17

INTRODUCTION _______________________________________________________________________

observed to inhibit activation [91]. Finally, phosphorylation of S621 mediates binding of 14-3-3

to the C-terminal end which is also necessary for activation [87,92].

Mek and Erk activation

The Raf proteins are serine/threonine kinases with just one established substrate; Mek1/2 (two

isoforms). Active Raf transmits the signal from Ras to Mek1/2 by phosphorylating Mek1/2 at two

sites, S217 and S221, in the activation loop [93]. Mek1/2 is a dual specificity kinase, targeting

both serine/threonine and tyrosine residues [94-96]. In addition, Mek1/2 is very specific, it targets

only one substrate, the serine/threonine kinase Erk1/2, which is activated by phosphorylation of

both T202 and Y204 in the threonine-glutamine-tyrosine motif in its activation loop [97]. Erk1/2

has an extensive range of substrates including nuclear transcription factors, cytoskeletal proteins,

kinases and phosphatases. Activation of the Raf/Mek/Erk cascade therefore regulates many cell

functions. Its main effects are the promotion of proliferation and differentiation [98].

Shc

KSR

RafP P P

14-3-3

MekPP

ErkP P

RasGTP

MP1

Nuclear and cytosolic substrates

PPPP

PPP

Grb2 Sos

SF SF

P

14-3-3

Shc

KSR

RafP P P

14-3-3

MekPP

ErkP P

RasGTP

MP1

Nuclear and cytosolic substrates

PPPP

PPP

Grb2 Sos

SF SFSF SF

P

14-3-3

Figure 2. Activation of Ras and the Raf/Mek/Erk cascade.

18

INTRODUCTION _______________________________________________________________________

Regulation of the Raf/Mek/Erk cascade

The Raf/Mek/Erk cascade is regulated and stabilised by various proteins. The kinase suppressor

of Ras (KSR) is a scaffold protein that is constitutively associated with Mek, but can also bind

Raf and Erk upon extracellular stimulation [99]. KSR acts by organising the kinases together in a

module. Following activation, KSR gets dephosphorylated by the protein phosphatase-2A (PP2A)

and the KSR-Raf/Mek/Erk-module is localised in proximity to Ras at the plasma membrane

enabling mediation of the signal [100]. The interaction between Mek and Erk is further

coordinated by the scaffold protein MEK partner-1 (MP1), resulting in enhanced activation. MP1

forms heterodimers with a small protein called p14, which targets the MP1-Mek-Erk complex to

endosomes. Erk is initially activated at the membrane, however the translocation to the

endosomes is believed to sustain the activation [101].

There are some proteins that have been described to negatively regulate the Raf/Mek/Erk

cascade. The 14-3-3 proteins are expressed in abundance and can bind several different proteins.

They mainly act by sequestering proteins, for example Raf and KSR, in the cytosol when the cell

is not under the influence of extracellular stimuli [100]. The interaction between Raf and Mek is

negatively regulated by the Raf kinase inhibitor protein (RKIP). RKIP inhibits Mek

phosphorylation by binding to both Raf and Mek and thereby preventing their physical

interaction [102].

Research over the last few years suggests that MAPK cascades are more complex than has

previously been thought. For example, the Raf isoforms are now suggested to have quite different

functions. It has been demonstrated that b-Raf actually has much higher kinase activity than c-

Raf and a-Raf, and that b-Raf might activate c-Raf. Furthermore, c-Raf is suggested to have other

targets than Mek1/2 and that it might itself act as a scaffold protein for the MAPK cascade

[80,100]. In addition, recent research connect b-Raf, but not c-Raf, with several types of

malignancies which is suggested to be explained by the higher kinase activity of b-Raf [103,104].

The PI3K pathway The lipid kinase PI3K

There are several classes of phosphoinositide-3 kinases. Here the class IA PI3Ks will be

discussed, since these are the PI3Ks that are linked to RTK activation. The PI3Ks are

heterodimers consisting of a catalytic and a regulatory subunit. In mammals there are three genes

19

INTRODUCTION _______________________________________________________________________

encoding the class IA catalytic subunit isoforms, p110α, p110β and p110δ, and three genes

encoding the regulatory subunit isoforms p85α, p85β and p55γ [105].

PI3K is a lipid kinase that catalyses the phosphorylation of phosphatidylinositol 4,5 diphosphate

(PIP2) to phosphatidylinositol 3,4,5 triphosphate (PIP3). These inositol phospholipids are located

in the cell membrane and acts as second messengers by facilitating binding sites for proteins

containing Pleckstrin homology (PH) domains [105]. Some of the PH domain containing proteins

bind phospholipids with high affinity. The residues for binding phospholipids effectively are

located in the N-terminal part and the motif includes basic amino acids which directly interact

with the inositol phosphate groups of the phosphatidylinositol [106].

PKB activation

Protein kinase B and phosphoinositide-dependent protein kinase (PDK) are serine/threonine

kinases containing PH domains. Upon receptor autophosphorylation and subsequent PI3K

catalysed generation of PIP3, PKB and PDK are attracted to the membrane where the interaction

with PIP3 occurs via their PH domains [107,108]. PKB gets activated by phosphorylation at two

sites; T308 in the activation loop and S473 in the carboxy-terminal regulatory region [109]. PDK,

which is localised in close proximity to PKB due to the interaction with PIP3, phosphorylates

PKB on T308 [110]. The kinase that is responsible for phosphorylating PKB on S473 is still not

fully clarified. However, a recent study suggests that the complex mTor (mammalian target of

Rapamycin)-Rictor directly phosphorylates S473 of PKB, further it is demonstrated that

inhibition or downregulation of mTor inhibits PKB activation [111,112].

PKB substrates

In resting cells PKB is localised in the cytosol, and upon activation it becomes localised to the

cell membrane. When activated, PKB detaches from the phospholipid and is translocated to the

cytosol and nucleus [113,114]. Active PKB phosphorylates several different downstream targets,

mainly involving metabolism and cell survival [105]. The first protein to be defined as a PKB

substrate was Glycogen synthase kinase-3 (GSK3) [115]. Phosphorylation of GSK3 inactivates

the protein and thereby glycogen synthesis is promoted [116]. PKB substrates affecting cell

survival include BAD (Bcl2/BclX-antagonist, causing cell death), and the FoxO (Forkhead box,

class O) transcription factors. Both these targets are inactivated by PKB phosphorylation and

hence are inhibited from inducing apoptosis [117].

20

INTRODUCTION _______________________________________________________________________

P

PKBP

P

Survival and metabolism

SF SF

PPPP

PPPP

PP

PP

P

PDK

mTorRictor

?

FoxOBAD

P

GSK3

PP

P

PTENSHIP

PI3K

P

14-3-3

P

P

PKBP

P

Survival and metabolism

SF SFSF SF

PPPP

PPPP

PP

PP

P

PDK

mTorRictor

mTorRictor

?

FoxOBAD

P

GSK3

PP

P

PTENSHIP

PI3K

P

14-3-3

P

Figure 3. Activation of the PI3K pathway via the c-Kit receptor.

Regulation of the PI3K pathway

The activation of the PI3K pathway is suppressed, or terminated, by two different phosphatases,

PTEN (phosphatase and tensin homologue deleted on chromosome ten) and SHIP (SH2-

containing phosphatase). PTEN counteracts the activity of PI3K by hydrolysing PI(3,4,5)P3 back

to PI(4,5)P2. PTEN is ubiquitously expressed and a major regulator of the PI3K pathway,

furthermore PTEN is known as a tumour suppressor implicated in a wide variety of human

cancers [118]. SHIP acts by hydrolysing PI(3,4,5)P3 to PI(3,4)P2 and thereby suppressing the

PI3K activity. SHIP expression is primarily restricted to hematopoietic cells and stem cells,

which makes it particularly important for c-Kit signalling [119]. Furthermore, the PI3K pathway

is suppressed by PHLPP (PH domain leucine-rich repeat protein phosphatase), a phosphatase that

targets the S473 phosphorylation on PKB. This dephosphorylation results in triggered apoptosis

and inhibited tumour growth [120].

21

INTRODUCTION _______________________________________________________________________

The Protein Kinase C family The PKCs are serine/threonine kinases that are involved in a multitude of signalling pathways.

The PKC family comprises at least ten related isoforms which are classified into three subgroups

depending on structural and biochemical properties; the conventional PKCs (cPKC), the novel

PKCs (nPKC), and the atypical PKCs (aPKC) [121]. The table below describes how the isoforms

are subgrouped, and summarises how they are regulated (Table 2).

Subgroup: Classical PKCs Novel PKCs Atypical PKCs Isoforms: α, βΙ, βΙΙ, γ δ, ε, η, θ ζ, ι Regulated

by: Phosphatidylserines DAG/Phorbol esters

Ca2+ Phosphorylation

Phosphatidylserines DAG/Phorbol esters

Phosphorylation

PhosphatidylserinesPhosphorylation

Table 2. The PKC isoforms and their regulation All PKC isoforms require the negatively-charged phospholipid phosphatidylserine for activation.

Phosphatidylserine resides within the plasma membrane and, in the presence of DAG, PKC binds

phosphatidylserine with higher affinity than other anionic lipids [122]. DAG and IP3 are second

messengers which are generated by RTK activated PLCγ catalysed cleavage of PI(4,5)P2, as

mentioned in a previous section [73]. The regulatory domain of cPKCs and nPKCs constitute

binding-sites for DAG and other phorbol esters. Following interaction, formation of a

hydrophobic surface occurs that enables part of the protein to penetrate the lipid bilayer of the

membrane where the phosphatidylserines are located [122]. The aPKCs lack essential residues in

the binding-site for DAG and are therefore ‘atypical’ [123]. IP3 causes release of Ca2+ which

leads to increased Ca2+ levels in the cytosol [73]. The cPKCs contain five conserved aspartic acid

residues which constitute a Ca2+ binding site [124]. Binding to Ca2+ increases the cPKC affinity

for phosphatidylserines, and thereby further promotes activation [125]. In order to become fully

activated PKC is phosphorylated at three sites [126]. PDK catalyses the phosphorylation of the

threonine in the activation loop in most PKC isoforms. This initial phosphorylation is not

activating, however, it promotes autophosphorylation at two other sites which stabilises the

protein in a protease and phosphatase resistant conformation. The fully phosphorylated protein is

catalytically competent but inactive due to an inhibitory conformation. Upon binding to

phosphatidylserines the conformation is altered, which enables PKC to interact and phosphorylate

substrates [127]. The wide range of substrates include receptors, enzymes, cytoskeletal proteins,

nuclear proteins and transcription factors [128].

22

INTRODUCTION _______________________________________________________________________

The hematopoietic system The hematopoietic system functions to continuously provide mature blood cells from the

embryonic stages and on throughout the life-time. Prior to birth the development of the

hematopoietic system is initiated in the yolk sac, and then progresses in the fetal liver. Shortly

after birth the bone marrow (BM) becomes the principal site for blood development. The

hematopoietic activity in the BM is extensive since mature blood cells are relatively short-lived

and need replacement continuously. In the BM billions of new mature blood cells are generated

every day [129].

The hematopoietic stem cell The blood comprises a variety of cells with diverse functions, however, they all originate from

the small pool of hematopoietic stem cells (HSC) that reside in the BM. Stem cells are rare in

most tissue types and the HSCs are no exception, in the BM less than 0.05% are estimated to be

HSCs [130].

Defining the Hematopoietic stem cell

HSCs are, by definition, cells that possess the capability to self-renew and to generate mature

progeny of all blood cell lineages by differentiation [131]. The HSCs are divided in long-term

(LT) and short-term (ST) HSCs depending on self-renewal capacity. The LT-HSCs are capable of

unlimited self-renewal, while the ST-HSCs regeneration time seems to be more constrained,

based on experiments analysing the ability of reconstituting the hematopoietic lineages in lethally

irradiated mice [132,133]. The HSC phenotype is characterised by physical and functional

properties. The method of fluorescence-activated cell sorting (FACS) has revealed a set of

surface markers that discriminate the HSCs from their differentiating progeny. The isolation is

based on a combination of negative and positive selection. HSCs express low or undetectable

levels of surface markers associated with committed mature cells which are described as lineage

negative (lin-). The positive selection is based on different markers, most consistently stem cell

antigen (Sca) and c-Kit, which by themselves are not HSC specific, but in combination coincide

with the HSC phenotype. Selection for lin- Sca+ c-Kit+ (LSK) cells results in a population highly

enriched for HSCs with long-term activity [134]. Functionally, the established method to define

HSCs is to demonstrate sustained capacity for regeneration of the hematopoietic system

following transplantation into immuno-compromised recipients. Transplantation experiments

23

INTRODUCTION _______________________________________________________________________

indicating the presence of stem cell properties were successfully performed already some 50

years ago by Ford et al [135] and, Till and McCulloch [136].

Regulation of HSC self-renewal

Hematopoiesis is tightly regulated by intrinsic and extrinsic events that control the lineage

commitment and maturation of the hematopoietic cells. Fundamental for this control is the

regulation of the HSC that is the initiating factor for the blood development. The question in

focus is how the balance between self-renewal and differentiation of the HSCs is maintained and

regulated.

The process of differentiation of HSCs is suggested to be based on asymmetric cell division,

where a single division results in the formation of one daughter cell that maintains the stem cell

capacity and one daughter cell that is committed to differentiation. This hypothesis is supported

by several in vitro and in vivo studies showing that HSCs and multipotential progenitors can

divide asymmetrically [137]. Recent data demonstrate that the most primitive hematopoietic cells

to a high extent divide asymmetrically, while the more committed progenitors have a tendency to

foremost expand their own cell fate by forming two equal daughter cells with the same

developmental capacity [138].

During development, the amount of stem cells increases, suggesting that the system, at the time,

favours symmetric division to expand the HSC pool. In adults, however, the asymmetric division

results in sufficient amount of self-renewing cells to sustain the hematopoiesis throughout life

and to produce enough mature blood cells. During physiological stress, like bleeding, this balance

is hypothesised to be shifted towards more symmetric division favouring differentiation and

production of mature blood [139].

The stem cell niche has shown to be an important extrinsic factor that regulates how the HSC

divides. The concept of the niche is based on primitive cells, restricted to a specific location,

being regulated by physical interaction with the surrounding cells and matrix components. The

sustained contact between the stem cells and the supporting cells can determine the fate of the

daughter cells [139].

Identification of specific extrinsic factors that promote self-renewal of HSCs is a hot topic. Such

factors would enable long-term ex vivo expansion of HSCs, which indeed has extensive clinical

24

INTRODUCTION _______________________________________________________________________

potential. One of the more promising suggestions to play such a role is Wnt. Members of the Wnt

family can be produced by both hematopoietic cells and their stromal cell environment, i.e. the

niche [140]. Furthermore, Wnt proteins can promote self-renewal of HSCs in vitro, and increase

the ability to reconstitute the hematopoietic system in lethally irradiated mice [141].

The hematopoietic system in general is largely regulated by different cytokines and transcription.

These include, for example, interleukins, erythropoietin and SF, and are normally expressed by

stromal cells within the hematopoietic tissues and they exert their function through receptors on

the hematopoietic cells [142].

The hematopoietic hierarchy The hematopoietic system is organised as a hierarchy where the long-term HSCs give rise to the

short-term HSCs, which in turn give rise to the multipotent progenitors, which subsequently

generate the mature cells of the different lineages via more committed precursor cells (see figure

4). As the HSCs progress from long-term to short-term to multipotent progenitor they lose their

self-renewal capability but become more responsive to growth factors [133].

The mast cell lineage

Mast cells retain a high expression of c-Kit during differentiation from HSCs. As mentioned in a

previous section, c-Kit expression is required for mast cell development. Mast cells play a central

role in immediate hypersensitivity and chronic allergic reactions. Following recognition of an

antigen, the reaction occurs within minutes and multiple signalling pathways are activated via the

IgE receptor (FcεR1). This response leads to degranulation and production of various cytokines

[143]. Unlike other mature blood cells, mast cells normally circulate through the vascular system

as progenitors and complete their development within the connective tissues. For a long time it

was actually believed that the mast cells were a component of the connective tissue. However, it

is now clear that the mast cells develop from the HSCs [143]. How this differentiation process is

lineated has until recently been unclear. It has been proposed that the mast cells are myeloid cells

and hence are derived from the common myeloid progenitors (CMP). Recent evidence though

indicates that the mast cell progenitor (MCP) arises from the multipotential progenitor (MPP) as a

separate lineage [144]. See figure 4.

25

INTRODUCTION _______________________________________________________________________

LT-HSC ST-HSC

B cell

Megakaryocyte

NK cell

Macrophage

Granulocyte

Trombocytes

T cell

Erytrocytes

Mast cell

MPPCMP

MCP

CLP

Pro-B

Pro-T

GMP

MEP

Monocyte

Figure 4. Organisation of the mammalian hematopoietic hierarchy. LT-HSC, long-term

hematopoietic stem cell, ST-HSC, short-term hematopoietic stem cell, MMP,

multipotential progenitor, CLP, common lymphoid progenitor, CMP, common myeloid

progenitor, MCP, mast cell-committed progenitor, GMP, granulocyte/macrophage

progenitor, MEP, megakaryocyte/erythrocyte progenitor. Adopted from Reya 2001 [130]

and Chen 2005 [144].

26

INTRODUCTION _______________________________________________________________________ The roles of c-Kit and SF in the hematopoietic system

In the hematopoietic system c-Kit is expressed on HSCs and progenitors at all developmental

stages [145], on mast cells [146,147], and to some extent on granulocytes and lymphoid cells

[148]. SF, though, is expressed by mast cells [149], fibroblasts [147], stromal cells [150], and on

several cell types in the airways [148]. Signalling via c-Kit influences hematopoiesis in numerous

different ways, extensively reviewed by Broudy 1997 [151]. Inhibition of SF/c-Kit interaction

with a monoclonal antibody abrogates the hematopoietic development, and diminishes the

progenitor pool in the BM, indicating that SF signalling is required for self-renewal of

progenitors at different stages [145,152] and SF is also shown to promote progenitor cell survival

[153]. In HSCs SF promotes survival [154] and sustained self-renewal [155]. Furthermore, SF in

combination with interleukin-11 is shown to favour the long-term repopulating activity of BM

cells [156] and SF also synergises with other cytokines to support colony growth of both

granulocyte-macrophage and erythroid units [151]. In mast cells SF is demonstrated to regulate

survival, proliferation, migration and mediator release [148].

Model systems for hematopoietic cells To investigate the effects of c-kit signalling in hematopoietic cells in a physiologically relevant

environment it would be preferable to use non-transformed primitive cells of human origin. Since

that is problematic for practical and technical reasons different model systems are used.

Studying c-Kit in mast cells is fairly uncomplicated since cultures can be derived from both

embryonic stem (ES) cells and BM and then cultured and expanded for a few weeks without

transforming the cells [157,158]. However, to find suitable systems to study HSC or progenitors

is more complex considering that factors like surface marker expression, cytokine response and

ability to self-renew and differentiate should be fulfilled. In a recent paper Olsen et al. have

reviewed several specific hematopoietic progenitor lines [159]. Additionally there are a few

HSC-like cell lines that have been used to study c-Kit signalling. Bernstein et al. have generated

HCS-like cell lines able to self-renew and commit to both lymphoid and myeloid lineage by

immortalizing ES cells with constitutive Notch signalling [160]. In our studies we have used cell

lines known as hematopoietic progenitor cells (HPC) developed in the laboratory of Leif Carlsson

at Umeå University, and described below.

27

INTRODUCTION _______________________________________________________________________ The Hematopoietic progenitor cell lines

The hematopoietic progenitor cell (HPC) lines are immortalised SF-dependent hematopoietic

stem cell-like cell lines. They are generated by transduction of the Lhx2 (LIM (Lin11, Isl1 and

Mec3) homeobox 2) gene into murine embryonic stem (ES) cells [161] or HSCs derived from

adult BM [162]. The Lhx2 gene encodes a transcription factor that is expressed in the fetal liver

during active hematopoiesis [163] and Lhx2 homozygous mutant mice die before birth due to

severe anaemia [164]. Lhx2 is also believed to play a role in the control of asymmetric cell

division and differentiation of specific cell types, reviewed and discussed in the thesis by

Wandzioch 2004 [165]. The experimental procedure for generating the cell lines is reviewed in

detail by Carlsson et al. [166] and discussed further in the thesis by Pinto do Ó 2002 [167]. The

HPC lines feature several of the characteristics common for early hematopoietic progenitor/stem

cells, including response to cytokines, transcription factor expression and cell surface markers

[161,162,168]. Like wild type HSCs they also display the capacity to differentiate into several

different mature blood cells (e.g. erythrocytes, megakaryocytes, macrophages, granulocytes and

mast cells) in response to cytokine stimulation in vitro [161] and the BM derived HPCs can

reconstitute the hematopoietic system in lethally irradiated mice [162]. Furthermore, they self-

renew by an unknown cell nonautonomous mechanism [168]. These features, in combination

with the possibility for long-term in vitro culture, make the HPC lines a useful model system for

studying c-Kit signalling.

28

AIMS _______________________________________________________________________

AIMS

The aim of this thesis was to analyse the signalling downstream the receptor tyrosine kinase c-Kit

in immature and differentiated hematopoietic cells and to investigate the effects of the activated

signalling pathways on proliferation, survival and differentiation.

The specific aims were:

• To identify what proteins are initially activated upon SF stimulation of c-Kit.

• To study if, and how, the known signalling pathways interact.

• To compare c-Kit signalling in hematopoietic progenitor cells with differentiated

hematopoietic cells.

• To further investigate the effects of PKC in hematopoietic cells.

• To gain insights in the mechanisms behind c-Kit-dependent proliferation and survival of

hematopoietic cells.

29

RESULTS AND DISCUSSION _______________________________________________________________________

RESULTS AND DISCUSSION Paper I “Activation of the MAP kinase pathway by c-Kit is PI3 kinase dependent in hematopoietic progenitor/stem cell lines”

• SF induces transient activation of c-Kit, receptor binding proteins (Grb2, Shc), Ras and

MAPK pathway (Raf, Mek, Erk), and PI3K pathway (PKB).

• SF stimulated Erk activation is PI3K dependent in HPCs, but not in differentiated

hematopoietic cells.

• The PI3K pathway interconnects with the MAPK cascade at the level of Raf.

• Erk and PI3K activity are required for proliferation of HPCs.

• Inhibition of PI3K induces apoptosis.

Erk activation is PI3K dependent in progenitor cells but not in differentiated cells To investigate c-Kit signalling in hematopoietic cells we have used the hematopoietic stem cell-

like cell lines, denoted HPCs. The characteristics of these cells are described in the introduction

section. In Paper I we firstly depict the proteins that are activated upon SF stimulation in this

HSC-like model system with endogenously expressed proteins. To study the signalling

downstream c-Kit in further detail we have used different inhibitors to block the activity of

specific proteins in the known signalling pathways. Surprisingly, we found that activation of Erk

was dependent on PI3K activity since usage of the PI3K specific inhibitors LY294002 [169] and

Wortmannin [170] effectively blocked Erk phosphorylation as well as PKB phosphorylation.

Further analysis showed that the MAPK cascade was targeted at the level of Raf, since Ras

activation was not influenced by PI3K inhibition. Thus, SF stimulated activation of Raf/Mek/Erk

in the HPCs requires PI3K signalling.

In order to find clues to explain the purpose of this crosstalk between the pathways we compared

the c-Kit signalling in these hematopoietic progenitor cell lines with more mature hematopoietic

cells. The mature cells we used were generated by differentiation of the HPCs (ES or BM

generated HPCs) to myeloid committed cells or mast cells. We also used mast cells differentiated

directly from wild type BM. Comparison of the different cell types revealed that the crosstalk was

30

RESULTS AND DISCUSSION _______________________________________________________________________

differentiation stage-specific, since activation of Raf/Mek/Erk was not PI3K dependent in the

differentiated cells. Thus, a molecular switch takes place in the signalling when the cells

differentiate from hematopoietic progenitors to mature cells.

SF SF

PPPP

PPPP P

PP P

PP

PKB P

P

PDKP

PI3K

LY294002wortmannin

Ras GTP

RafP P P

MekPP

ErkP P

ShcGrb2 Sos

mTorRictor

PFoxO BAD

P

SurvivalProliferation

PD98059SF SF

PPPP

PPPP

SF SFSF SF

PPPP

PPPP P

PP P

PP P

PP P

PP

PKB P

P

PDKP

PI3KPI3K

LY294002wortmannin

Ras GTPRas GTP

RafP P P

MekPP

ErkP P

ShcGrb2 Sos

mTorRictor

mTorRictor

PFoxO BAD

PBAD

P

SurvivalProliferation

PD98059

Figure5. Overview of the PI3K and the MAPK signalling pathways and the proposed

crosstalk.

In our attempts to explain the mechanistics behind the molecular switch that occurs during

differentiation, we have investigated changes in mRNA and protein levels of some involved

mediators. Our unpublished data indicate that upon differentiation the level of phosphorylated

PKB decreases and at the same time the level of phosphorylated Erk seems to increase. The PKB

decrease might be a result of the increase in SHIP that we have observed in mast cells compared

to HPCs. Together these data suggest that the levels of some important mediators change during

differentiation and it is possible that this might change the balance between the pathways and also

cause the molecular switch.

31

RESULTS AND DISCUSSION _______________________________________________________________________

As discussed in the introduction section, SF, together with the stem cell niche, is required for

HSC self-renewal and maintenance of the HSC pool [145,152,154,155]. The HPCs though, are

transduced with Lhx2, which maintains the cells in an immature state. It is plausible that Lhx2

induces expression of an unknown stem cell specific factor that in combination with SF, induces

self-renewal. Furthermore, such factor might also be responsible for maintaining Erk activation

PI3K dependent in the immature cells.

A possible function of the differentiation-stage specific crosstalk might be to enable enhanced

control of the balance between proliferation and differentiation. In undifferentiated cells the

crosstalk might function to ensure adequate expansion and prevent pre-mature differentiation of

HSC/HPCs. However, the exact mechanism and function of the crosstalk presented in this study

is yet to be explained.

Crosstalks reported in the literature

Reviewing the literature on the subject of crosstalks between the PI3K and MAPK pathways

reveals that interconnections are actually observed in several different cell types, however with

contrasting mechanisms and outcomes.

Already in 1994 it was reported that PI3K could be a direct target for Ras activation in in vitro

experiments and in COS cells [171,172]. In c-Kit signalling this does not seem to be the case and

PI3K has clearly been demonstrated to become activated upon direct binding to the receptor [69].

However, Zhang et al. have recently made observations in erythroid progenitor cells indicating a

role for K-Ras in PKB activation [173]. Downstream of Ras several mechanistically different

interconnections have been reported. Essentially the field can be divided into two parts; negative

and positive regulation of the MAPK pathway by PI3K.

The groups of Moelling and Glass presented evidence in 1999 that PKB can directly

phosphorylate Raf on serine 259 in human embryonic kidney (HEK) 293 cells stimulated with

insulin-like growth factor (IGF) and that this has an inhibitory effect on Raf activation [85].

Furthermore, they demonstrated that this inhibitory phosphorylation was differentiation stage-

specific, since PKB inhibition of Raf was observed in myotubes, but not in myoblast precursors

[174]. In continuation this crosstalk has also been observed in PDGF stimulated vascular smooth

muscle cells where abrogation of PI3K/PKB lead to a phenotypic modulation [175]. Furthermore,

32

RESULTS AND DISCUSSION _______________________________________________________________________

Moelling et al. have presented results implicating that this type of crosstalk is regulated by ligand

type, concentration and time course and that the function might be to regulate the balance

between proliferation and differentiation [176]. In enterocytic cells it has been demonstrated that

inhibition of PI3K enhances Erk activation when the cells are confluent but not subconfluent.

This correlates with differentiation since upon confluence the cells switch from proliferating to

differentiating [177].

Contrary to the above findings that PKB has an inhibitory effect on Raf there are several reports,

in addition to our study, demonstrating that PI3K activity is required for activation of the MAPK

pathway. Partly confirming our results Chiu et al. have tested a range of hematopoietic cell lines

and found that in some cells MAPK activation was blocked by the PI3K inhibitors LY294002

and wortmannin, but the result differed depending on inhibitor. In this series of experiments the

cells were stimulated with granulocyte-macrophage colony stimulating factor (GM-CSF),

interleukin-3 (IL-3) or serum which might explain the varying results [178]. In erythroid

progenitor cells Ras and the downstream MAPKs were blocked by PI3K inhibitors upon

erythropoietin stimulation, however in contrast to our results, this was not the case upon SF

stimulation in the same cells [179]. In a myeloid progenitor cell line it has been demonstrated that

Mek and A-Raf, but not C-Raf require PI3K for activation upon IL-3 stimulation. This distinction

between the Raf isoforms suggests an additional regulation of the signalling [180]. In T cells a

variant of the crosstalk has been reported, here PI3K overexpression enhances Erk activation and

wortmannin inhibits Erk activation. However, these effects were not observed in signalling

components upstream of Erk [181].

In non-hematopoietic cells there are a several reports that concur with the above findings even

though cell types, stimulation and exact mechanisms diverge [182-191]. Contrary to our findings

in HPCs there are observations indicating that the crosstalk may be receptor concentration

dependent, so that low receptor concentration results in PKB dependent Erk activation, but not if

the receptor is expressed in abundance [185,189].

Erk is required for proliferation of HPCs As authentic HSCs, the HPC lines are cytokine dependent, withdrawal of SF leads to inhibited

proliferation and ultimately cell death ([161] and unpublished data). To investigate the biological

consequences of interfering with the MAPK pathway we used a specific Mek inhibitor and

monitored proliferation and survival. In Paper I we demonstrate that blocking the activity of Mek

33

RESULTS AND DISCUSSION _______________________________________________________________________

results in a dramatic decrease of the rate of growth indicating that activation of the MAPK

pathway is required for proliferation. Further we demonstrate that Mek inhibition only slightly

increases the death rate. Thus, even though the MAPK pathway is required for proliferation it is

not necessary for survival of the HPCs. These data confirm that a main function of the MAPK

pathway is to regulate cell growth [98]. However, in HSCs SF is not enough to induce self-

renewal [154]. Since the HPC lines are transduced with Lhx2 which keep the cells immature, it is

possible that MAPK signalling together with the effects of the transformation promotes self-

renewal in the HPCs.

Activation of the PI3K pathway is crucial for survival The PI3K pathway in hematopoietic cells is, as discussed in the introduction section, an anti-

apoptotic pathway, crucial for the viability of hematopoietic cells [192-194]. In addition to being

involved in survival, PI3K activation has been demonstrated to be a regulator of different mast

cell functions [195,196], macrophage growth [197] and stem cell renewal [198,199]. In Paper I

we utilised the Trypan blue exclusion assay to analyse the survival upon PI3K inhibition with

LY294002. Our results demonstrate a dramatic and rapid increase of cell death under such

conditions. Actually, the rate of survival after PI3K inhibition was as low as upon full withdrawal

of SF (data not shown). The data presented here supports a critical role for PI3K in HPC

physiology. The severe effects following inhibition of PI3K in the HPCs might be a consequence

of the crosstalk since in practice both MAPK and PI3K pathways are inhibited with the single

inhibitor. However, inhibition of PI3K in mast cells induces apoptosis to the same extent as

starvation which suggests that the PI3K pathway alone is necessary for survival of these cells. It

is noticeable though that the effect of starvation and PI3K inhibition is much delayed in mast

cells compared to HPCs (unpublished data).

34

RESULTS AND DISCUSSION _______________________________________________________________________

Paper II “Hematopoietic progenitor cells and mast cells utilize conventional PKC to suppress PKB/Akt activity in response to c-Kit stimulation”

• PKCs are activated upon SF and PMA stimulation of HPCs.

• Raf and downstream mediators, but not Ras and PKB, are activated by PMA.

• PMA induced Raf activation is PI3K independent in the HPCs.

• The cPKCs are required for full Erk activation.

• The cPKCs mediate phosphorylation of S746 on c-Kit.

• Inhibition of the cPKCs leads to increased tyrosine phosphorylation of c-Kit.

• Inhibition of the cPKCs leads to increased PKB phosphorylation and activation in both

HPCs and mast cells.

• The cPKCs are required for HPC proliferation.

• Inhibition of the cPKCs increases the survival rate upon starvation induced apoptosis.

PKC activates Raf, but not Ras or PKB in the HPC lines To investigate the role of PKC in HPCs we started by analysing what proteins are activated by

PMA (phorbol 12-myristate 13-acetate). PMA is a DAG analogue and therefore able to activate

the conventional and novel PKCs [200]. In Paper II we show that PMA treatment of starved

HPCs activates the Raf/Mek/Erk cascade independently of c-Kit and Ras. Furthermore, the PI3K

pathway is not activated by PMA. This is in agreement with studies from other cell systems

showing that PKC phosphorylates Raf directly and thereby activates Raf independently of Ras

[201-204]. As an extension to Paper I we further studied the PI3K dependence of Raf activation.

Here we show that PMA induced Raf activation is PI3K independent in HPCs. This is in contrast

to the SF induced activation, at the level of phosphorylation status of S338 on Raf, which is

known to be important for activation [80]. In addition, comparison of PMA and SF induced

activation of Raf can be used to evaluate the specificity of the crosstalk presented in Paper I.

Since only RTK induced Raf activation is PI3K dependent it is clear that the crosstalk is c-Kit

signalling dependent and not due to unspecific inhibitor effects.

35

RESULTS AND DISCUSSION _______________________________________________________________________

PKC is required for Erk activation and proliferation As discussed above, it is evident that PKCs are activated both by PMA and SF in the HPCs.

However, to study the effects of PKC on c-Kit signalling we have utilised some of the inhibitors

available to block PKC activity. In Paper II we have focused on the conventional PKCs (cPKC)

and in Paper III the effects of the novel PKCδ are investigated. In Paper II we demonstrate that

not only can PKC activate Raf/Mek/Erk, but also that PKC is required for Erk activation.

Ro318220 is a pharmacological inhibitor, which specifically targets the cPKCs [205,206]], and

we demonstrate that pre-treatment with Ro318220 prior to SF stimulation blocks Erk

phosphorylation. We have also used long-term treatment of PMA to inhibit the PKCs and study

the effects on c-Kit signalling. While PMA is known as an activator of PKCs, long-term

treatment depletes the PKC pool and can therefore be used to inhibit the pathway [207]. In

concurrence with Ro318220 treatment we have observed that long-term treatment with PMA

blocks Erk activation in the HPCs. Contrary to this result, Blume-Jensen et al. did not observe an

decrease in Erk phosphorylation upon PKC inhibition in SF stimulated cells [208]. This

discrepancy might be explained by the fact that they use porcine aortic endothelial cells

transfected with c-Kit instead of a model system that examines the endogenous c-Kit receptor.

In Paper I we establish an important role of the Raf/Mek/Erk cascade in the regulation of

proliferation of HPCs. Since we in this study observed that PKC is required for Erk

phosphorylation we decided to monitor proliferation of the HPCs under the influence of

Ro318220. PKC inhibition of HPCs in culture results in total block of growth, but does not seem

to induce significant cell death. This result is similar to what we observed with the Mek inhibitor

in Paper I, which is not surprising since a common effect of these inhibitors is inhibition of Erk

activation.

The cPKC mediates serine phosphorylation of c-Kit Blume-Jensen et al suggested that PKC can phosphorylate c-Kit on serine residues [209]. Further,

they identified S741 and S746 as the major sites targeted by PKC [210]. However, these data are

based on in vitro studies and experiments on the porcine aortic endothelial cells transfected with

and therefore overexpressing c-Kit. In order to investigate whether PKC could phosphorylate c-

Kit in the HPCs as well, we used a novel phospho-specific antibody targeting S746 on c-Kit. In

Paper II we demonstrate that this site is phosphorylated upon SF stimulation and that PKC

inhibition abrogates this phosphorylation. Furthermore, we demonstrate that inhibition of PKC

36

RESULTS AND DISCUSSION _______________________________________________________________________

mediated serine phosphorylation leads to increased overall tyrosine phosphorylation of the c-Kit

receptor, which is in agreement with the studies of Blume-Jensen et al. [209]. Together these data

indicate that PKC and c-Kit constitute a negative feedback loop where c-Kit activates PKC which

subsequently phosphorylates c-Kit on serine residues which negatively regulate c-Kit activation.

A similar type of feedback loop has recently been reported in studies on a chimeric form of the

RTK RET, where they show that PKCα associates with Y1062 on RET resulting in decreased

tyrosine phosphorylation and decreased mitogenicity [211]. Thus, this feedback loop appears to

be a novel mechanism for regulating RTK activation.

Conventional PKCs inhibit PKB activation and promote apoptosis In addition to the results discussed above we have observed that PKC inhibition dramatically

increases PKB activation. Initially we analysed the phosphorylation status of PKB following

inhibition with Ro318220 or depletion of PKCs with PMA long-term treatment and observed that

phosphorylation of PKB is increased, and prolonged, upon SF stimulation of HPCs. To confirm

the effect of PKC on PKB we performed in vitro PKB activity assays, which demonstrate that

PKC severely hampers PKB activation since PKC inhibition with Ro318220 increases PKB

activity 5-6 times in both HPCs and mast cells. This finding might be a consequence of increased

c-Kit activation that is caused by abrogation of the proposed PKC/c-Kit negative feedback loop.

We have observed increased phosphorylation of Y719 in c-Kit and the hypothesis is further

supported by experiments in the porcine aortic endothelial cells indicating increased association

efficiency between c-Kit and PI3K upon PKC inhibition [208]. To further analyse the possible

biological effects of PKC inhibition, and specifically the dramatically increased PKB activity, we

decided to monitor the viability of HPCs. FACS analyses of propidium labelled HPCs show that

Ro318220 treatment rescues cells from starvation induced apoptosis. We believe that this is an

effect of the increased PKB activity in combination with decreased Erk activity, since PKB

signalling is known to block activation of proapoptotic mediators and Erk promotes proliferation

(see introduction and discussion above).

It is noteworthy that the limited numbers of studies on the subject of PKC effects on PKB are

quite contradictory. In a recent report Grandage et al. demonstrated that one PKC inhibitor

(Gö6976) actually inhibits IL-3 and GM-CSF induced signalling, proliferation and survival.

Further they put forward the hypothesis that this inhibitor has no effect on c-Kit signalling [212].

However, in the non-hematopoietic cells A549 and HEK293, it is reported that three different

PKC inhibitors (Ro318220, bisindolylmaleimide VIII, and LY379196), increase PKB activity

37

RESULTS AND DISCUSSION _______________________________________________________________________

and that PMA attenuates the same [213]. Another recent report supports PKC as a negative

regulator of PI3K signalling, but, contrary to our results, suggests that PKC targets PI3K directly

and not via a receptor [214]. It is evident that PKC can influence PKB activity; however it is also

clear that the mode of operation is dependent upon cell system, and possibly, the inhibitor used.

Paper III “PKCδ is required for proliferation and can negatively regulate differentiation in c-

Kit expressing hematopoietic cells”

• Inhibition of PKCδ rescues HPCs from starvation induced apoptosis.

• Inhibition of PKCδ causes G1 arrest in HPCs within 4 hours.

• Long-term treatment with Rottlerin results in cell death of the HPCs.

• Low dose Rottlerin treatment of BM cells accelerates the differentiation process.

• PKCδ is required for SF stimulated Erk activation.

• Inhibition of PKCδ does not affect c-Kit phosphorylation.

Inhibition of PKCδ does not affect c-Kit

In paper II we studied the effects of the conventional PKCs on c-Kit signalling in HPCs. In paper

III we used Rottlerin, an inhibitor that specifically targets PKCδ [215] to investigate how a

member of the novel PKCs affect hematopoietic cells.

Contrary to the conventional PKCs, the novel PKCδ is not required for phosphorylation of S746

on c-Kit, and inhibition with Rottlerin does not increase the overall tyrosine phosphorylation

(data not shown). Furthermore, we show that PKCδ inhibition does not seem to increase PKB

activity, which is concurrent with the lack of effect on the receptor. These differences underline

that effects of the PKC family of serine/threonine kinases are highly diverse.

Rottlerin prevents starvation induced apoptosis

Even though PKCδ is not indicated in c-Kit regulation, we demonstrate that inhibition of PKCδ

with Rottlerin in hematopoietic cells has surprising effects. At first, Rottlerin treatment is

observed to greatly prevent starvation induced apoptosis as measured by FACS analysis of

propidium iodide incorporated cells. However, even though the FACS analysis indicates very low

38

RESULTS AND DISCUSSION _______________________________________________________________________

numbers of apoptotic cells, morphological analysis of the cells tells otherwise. Under the

microscope it is clear that cells are affected by the treatment. HPCs that are starved normally

remain quite small and uniform, with a visibly disintegrated cell membrane, however Rottlerin

treated cells vary more in size and contain large vacuoles. This might indicate that the treatment

induces a mitotic catastrophe (described in [216]) with delayed cell death that is not apparent

with FACS analysis. Hence, we demonstrate that treatment with Rottlerin initially prevents

apoptosis, but that long-term treatment kills the cells. Our results suggest that even though

Rottlerin is a well used inhibitor, it might provide results that are deceiving if not properly used.

In other hematopoietic cells (HL60, Jurkat and RAW264.7 cell lines) Liao et al. have reported

that Rottlerin indeed induces apoptosis in a time and dose dependent manner [217]. In their report

they show that the induced apoptosis is mediated by dissipation of membrane potential,

redistribution of cytochrome C and induction of the caspase cascade [217].

Inhibition of PKCδ causes G1 cell cycle arrest

The rescue from starvation induced apoptosis seen on the histograms is accompanied by a clear

G1 arrest, which might explain why the cells seem viable since it is possible that cell division is a

prerequisite for induction of apoptosis under these circumstances. Since no increase in PKB

phosphorylation was observed upon Rottlerin treatment the apoptotic inhibition observed cannot

be explained by increased PKB activity, as with inhibition of the cPKCs (Paper II). Thus, in

Paper III we show that inhibition of PKCδ induces a PKB independent G1 arrest that blocks

proliferation and prevents induction of apoptosis for up to about ten hours. The observation that

Rottlerin induces G1 arrest is supported by a recent report that shows that PKCδ stimulates G1

phase cell cycle progression in thyroid cells, but subsequently induces S phase arrest and caspase-

dependent apoptosis [218]. Their presentation of a PKCδ induced cell cycle-dependent cell death

supports our hypothesis that proliferation is a prerequisite for apoptosis. The study from Liao et

al. also supports a proliferation repressing effect of Rottlerin [217].

When monitoring the proliferation rate of HPCs under the influence of Rottlerin the cell numbers

were at first stable, but started to decrease after about ten hours, indicating that PKCδ is required

for proliferation and that long-term treatment at the used dose is toxic for the HPCs. In trying to

explain the observed G1 arrest and blocked proliferation we analysed the effects of Rottlerin on

the MAPK pathway downstream of c-Kit. As expected PKCδ, like the cPKCs, was required for

Erk activation.

39

RESULTS AND DISCUSSION _______________________________________________________________________

PKCδ influences differentiation of bone marrow

Our data and others suggest that proliferation and survival are closely linked and it is presumable

that differentiation is a third event that is somehow linked to the first two. To test if PKCδ might

be involved in the differentiation of hematopoietic cells we cultured freshly purified BM

according to protocols for mast cell differentiation (see Paper I), but with the addition of a low

dose of Rottlerin. Surprisingly, we observed that the cells seemed to differentiate faster than in

the absence of Rottlerin. Observations like this have not been reported previously, and the

mechanism behind the accelerated differentiation of BM cells is unknown. It is possible that it is

associated with the inhibited proliferation. Seemingly, the low dose that was used in this

experiment was non-toxic to the cells, but promoted differentiation over proliferation perhaps due

to the inactivation of Erk. This finding further adds to the complexity of PKCδ function.

40

CONCLUSIONS _______________________________________________________________________

CONCLUSIONS

• The canonical signalling pathways, MAPK pathway and PI3K pathway, interconnect in a

differentiation stage-dependent manner in hematopoietic progenitor cells.

• Activation of the PI3K pathway is crucial for the viability of hematopoietic progenitor

cells and mast cells.

• Conventional and novel PKC family members are important regulators of c-Kit signalling

and of hematopoietic cell physiology.

In conclusion, the data presented in this thesis demonstrate that the signalling downstream c-Kit,

and possibly receptor tyrosine kinases in general, is more complex than earlier studies have

suggested. Our data contribute to the idea that receptor tyrosine kinase signalling can be regulated

in a cell stage specific manner in addition to ligand stimulation, concentration and expression

patterns. Furthermore, we show that the receptor itself can be targeted by kinases which add to

the number of regulatory mechanisms. Consequently, these data furthermore underline the

significance of employing appropriate model systems to study signalling downstream receptor

tyrosine kinases. In the hematopoietic cells, where c-Kit plays an important role in progenitors

but not in mature cells, this has great relevance for the understanding of the balance between self-

renewal and differentiation.

41

ACKNOWLEDGEMENTS _______________________________________________________________________

ACKNOWLEDGEMENTS

Finally, the thesis is written and all that remains is to acknowledge the people that made the whole PhD journey possible or just more enjoyable. Great thanks to my supervisor, Bengt, who convinced me to start in the group way long ago. Thanks for teaching me a whole lot about hardcore signalling and for always being enthusiastic about the research and for always trying to see good results no matter the blackness of the film. Moreover, you are the only one I know that find western blots hot, even if they’re not radio-labelled. And thanks for giving me super skies to vasaloppet, that really helped! Ruth, my co-supervisor, many thanks for coming up with smart ideas for the project during the years and special thanks for reading and correcting the thesis and the papers I finally managed to write. Leif Carlsson group, and specially Ewa, who taught me how to manage the cells, provided me with all sorts of factors and for collaborating on our fab blood paper! Lars Rönnstrand and Malin Pedersen for collaboration and for providing antibodies. The Hallberg group; Maria, Anna, Emma, Lubna, Hailing; Thanks a lot, you are a great bunch to have in the lab and as friends! Please disturb me any time now… And thanks to all other students, that passed during the years and made the lab an interesting place to work to say the least. Especially Pawel who actually helped with the project. The RHP group who made Friday morning meetings more populated and more fun. I have to admit that I’m now almost convinced; fruitflies are cool! Caroline, you’re soon finished too! And yes, I think I now know a little about telomerase and bugs as well, thanks Sofie and Margareta! The Med Bio Dep – thanks for creating a very nice place to do research. Folket i korridoren som gör att det är trevligt att gå och fika även på icke-torsdagar☺. Även molbiol folket som man får hänga med fast man blev förflyttad! Sekreterarna/intendenten/diskpersonalen som verkligen tagit hand om den Hallbergska gruppen. Mången administrativt problem och högar med disk har ni fixat bort åt oss. Tack. Stort tack till en samling fantastiska vänner som gjort mitt liv de senaste åren så mycket roligare och som man kan lita på vid alla tänkbara tillfällen. Helga och Marie, jag är tacksam att ni ser till att vi håller kontakten trots att jag blev fast lite längre i norr än vad som var tanken. Linda, tack för att du roat och supportat mig genom flertal mail och besök. Sofia T, nu är vi äntligen färdiga! Nu ska det bli svänga av! Maja, tack för att du varit en sån bra jobb-orelaterad vän. Så till gänget, jag är otroligt tacksam att jag har er till vänner. Ni gör verkligen helgen till fest och vardagen uthärdlig (när det behövs). Sofia, alltid på min sida (förutom i spel), Sara-Rara, du är super! Anna, tur att du inte blev läkare på en gång, för då hade jag missat en fin kompis. Christina, tack detsamma för delade intressen mm☺ Petra, jobba inte ihjäl dig, jag skulle bli så ledsen då . Maria E, det är roligt att ha dig som vän, Gussing också och Gunnarn fast du alltid envisas. Jeanette, du är alltid snäll med mig, David, ack så retsam (men det får man ta om man vill leka med de stora fiskarna). Kicki och Erik, ni är hyvens ni! Magnus, Martina, Johan, Linda, (London lockar lite nu trots allt) Niklas, Maria, Håkan, Henke, Katrin, CharlotteP, KristinaL, jag är glad att många ännu är kvar, saknar dock er som redan flyttat. Min familj, som alltid stöttar mig i alla avseenden och åtminstone låtsas förstå vad jag gör. Alla ni nämda, och en del onämnda, som hjälpt mig fixa de här åren. Tack.

42

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