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Identification and characterization of novel substrates/interacting partners for the protein tyrosine phosphatase PRL-2 Nau Nau Wong Goodman Cancer Center and the Department of Biochemistry, McGill University. Montreal July 2011 A thesis submitted to McGill University in partial fulfillment of the requirements of the Master degree © Nau Nau Wong, 2011

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Identification and characterization of novel substrates/interacting partners for the protein tyrosine phosphatase PRL-2

Nau Nau Wong Goodman Cancer Center and the Department of

Biochemistry, McGill University. Montreal

July 2011 A thesis submitted to McGill University in partial fulfillment

of the requirements of the Master degree

© Nau Nau Wong, 2011

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Table of Contents Pages

Abstract 1 Résumé 2-3 Acknowledgements 4

Chapter I: Introduction and background 5-25 Phosphorylation and phosphatase 4 PRLs family 7 PRLs structure 8 PRLs phosphatase activity 12 Involvement of PRLs in disease malignance 13 Regulation of PRLs 16 Identified PRLs interacting partners and substrates 18 Cellular pathway regulates by the PRLs family 21 PRL inhibitors 24 Project goals 25

Chapter II: Methods and materials 26-33 Cell culture and transfection 26 Stable transfection 26 Affinity Purification of Mass Spectrometry (AP-MS) 26 Silver-staining 29 Plasmids construction 29 Immunoprecipitation and pull down 30 PCR mouse genotyping 32 Western blot of thymus tissues 32 Murine embryonic fibroblasts (MEF) 33

Chapter III: Results 34-57 Identification of PRL-2 interacting partners/substrates 34 CNNM family members interact with PRL-2 41 PRL-1,2,3 all interact with CNNM3 42 Endogenous interaction of PRL-2 and CNNM3 in several cell 43 lines CNNM3 ACD domain is required for interaction 44 All PRLs interact with CNNM3 via the CBS domains 47 Global phosphorylation did no promoted CNNM3 interaction 52 with PRL-2CSDA trapping mutant PRLs mutations disrupting their interaction with CNNM3 53

PRL-2 knockout mouse model 54 Immortalize MEF cell line 56 Reduced body weight of knockout PRL-2 mouse 57

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Chapter IV: Discussion 58-71 Identification of PRL-2 interacting partners using AP-MS 58 CNNM3 interaction with PRL-2 and PRL family 61 PRL-2 binds to CNNM3 at its CBS domains 62 PRL-2 catalytic domain mutations disrupts CNNM3 interaction 68 Model for CNNM3 PRL-2 interaction 69 Physiologic role of PRL-2 71

Conclusion 73

References 74-86

List of Figures

Figure 1: Classification of phosphatase family 6 Figure 2: PRL family 7 Figure 3: Conserved structural domain of PRLs 9 Figure 4: Crystal structure of PRL-1,3 11 Figure 5: Schematic of the AP-MS experiment 28 Figure 6: HEK293 stable cell lines 35 Figure 7: Western blot analysis evaluating the IP efficiency 35 Figure 8: Silver-staining of eluted protein from flag IP 36 Figure 9: CNNM3 interaction with PRL-2 43 Figure 10: PRL-1,2,3 interaction with CNNM3 43 Figure 11: Endogenous interaction of PRL2 and CNNM3 44 Figure 12: Schematic of ACDP structure domains 45 Figure 13: ACD and both CBS domains are important for PRL-2 46

binding Figure 14: PRLs all interact with CNNM3 at its CBS domains 47 Figure 15: Point mutations within the CNNM3 CBS domains 50 Figure 16: D396A and G433D mutation in CBS domains 50

abolished interaction with PRL-2 Figure 17: G433D mutation in full length CNNM3 abolished 52

interaction with PRL-2 Figure 18: increase in global phosphorylation level did not 53 prompt PRL-2 CNNM3 interaction Figure 19: PRL2 C110S and D96A mutation decrease PRL-2 54

interaction with CNNM3 Figure 20: Schematic of gene trap mouse and its PCR genotype 55 Figure 21: Western blot of PRL-2 KO mouse thymus tissues 56 Figure 22: Western blot of PRL-2 KO mouse MEF cell line 57 Figure 23: Body weight of mice measured at 4 weeks time 57

point Figure 24: ACDP family 61 Figure 25: Alignment of CBS domain of CNNM3 with other CBS 67 containing proteins Figure 26: PRL-2 mutation used in this study 69

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Tables Table 1: Previously published PRLs substrates and interacting 20 partners Table 2: Primer used in the study 31 Table 3: Non-specific interacting proteins identified in AP-MS 38 Table 4: Potential interacting partners of PRL-2 40 Table 5: Potential interacting partners of PRL-2 CS/DA mutant 40 Table 6: PRLs binding partners identified by large-scale mass 41 spectrometry mapping of human protein-protein interactions Table 7: LC MS/MS peptides unique to each CNNM family 42 Table 8: CBS mutations identified in different CBS domains 49

containing proteins Table 9: CBS domain containing proteins and effect of mutation 64

within their CBS domain

List of Abbreviation

AP-MS Affinity Purification Mass Spectrometry AML Acute myeloid leukemia co-IP Co-immunoprecipitation CRC colorectal carcinoma CNNMs Ancient Conserved Domain Protein Family CRC Colorectal carcinoma DSPs Dual specificity phosphatases EGR-1 Early growth response EMT Epithelial-mesenchymal transition ERK1/2 Extracellular signal-regulated protein kinase ½ Flag beads Flag-M2-Agarose (Sigma)

FTase Farnesyltransferase

GST Glutathione-s-transferase IP Immunoprecipitation KO Knockout Mouse model MKPs Mitogen-activated protein kinase phosphatase MMPs Matrix metalloproteinases NLS nuclear localization signal NCL Neuronal Ceroid Lipofuscinoses PD Pull down PRLs Phosphatase of Regenerating Family PRL1 Phosphatase of regenerating liver 1 PRL2 Phosphatase of regenerating liver 2 PRL3 Phosphatase of regenerating liver 3 PTKs Kinases PPs Protein Phosphatases PTEN Phosphatase and tensin homologue deleted on chromosome 10 PTPs protein tyrosine phosphatases TCL Total cell lysate TGFβ Transforming growth factor beta

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Abstract

The reversible process of protein phosphosphorylation by kinases and

phosphatases regulates essentially every aspect of cellular processes.

PRLs (Phospshatase of Regenerating Liver) are dual specificity

phosphatases, belonging to the protein tyrosine phosphatase family. PRL

family members (PRL-1, PRL-2 and PRL-3) possess several oncogenic

properties and play an important role in tumoriogenesis and metastasis. In

previous studies using multiple cellular and in vivo models, we confirmed

the role of PRL-2 in cell migration and in the transformation process of

breast cancer. Thus, we seek to identify the cellular pathway, mechanism

of actions, and the physiological function of PRL-2. One of the most

common approaches to elucidating the function of a protein is by

identifying its substrates and/or interacting partners. In this study, we have

identified several interesting PRL-2 interacting proteins using Affinity-

Purification Mass Spectrometry (AP-MS) and we characterized the

interaction with one of these candidates: cyclin M3 (CNNM3). We also

generated a PRL-2 KO mouse models: these PRL2-KO mouse showed

significant weight loss, suggesting PRL-2 might play an essential

physiological role. We believe that the identification of PRL-2 interacting

partners will shed light on the physiological functions of this PTP, and may

lead to the development of new targets for breast cancer therapy.

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Résumé

L’état de phosphorylation des protéines dans la cellule est

essentiellement régulé par les kinases et les phosphatases de façon

réversible. Les PRLs (Phosphatases of Regenerating Liver) sont des

phosphatases à double spécificité appartenant à la famille des protéines

tyrosine phosphatase. La surexpression des membres de la famille PRL

(PRL-1, PRL-2 et PRL-3) est observée dans une grande variété de

cancers. Ceux-ci possèdent de nombreuses propriétés oncogéniques et

jouent un rôle important au niveau de la génération des tumeurs ainsi que

leur dissémination métastasique. PRL-2 est la moins caractérisée de la

famille PRLs. Nos études effectuées à partir de différentes lignées

cellulaires ainsi que modèles in vivo ont démontrées le rôle de PRL-2

dans la migration cellulaire et le développement de tumeurs du sein. Mes

recherches portent sur l’identification des voies de signalisation cellulaire

modulées par PRL2 ainsi que son mécanisme d’action et son rôle

physiologique. L’approche la plus commune pour comprendre la fonction

d’une protéine est d’identifier ses substrats ou partenaire d’interaction.

Dans cette étude, nous avons identifié plusieurs protéines interagissant

avec PRL-2 in vivo par spectrométrie de masse (MS) et nous avons

caractérisé son interaction avec l’un des candidats de MS : CNNM3. Nous

avons aussi généré un modèle de souris knock-out (KO) de PRL-2. La

souris KO manifeste une perte importante de poids suggérant un rôle

physiologique important de PRL-2. Nous sommes persuadés que

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l’identification des substrats physiologiques de PRL2 permettra de mieux

comprendre cette PTP et ainsi assurer le développement de nouvelles

cibles afin de fournir un meilleur traitement contre le cancer du sein.

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Acknowledgement:

I want to offer my sincerest thanks to my great supervisor Dr. Michel

Tremblay for accepting me in his lab and offering me one of the my

greatest learning experiences. My two years of stay in Montreal were full

of joy, not only because Montreal is a great city to live in, but also because

all of the amazing people that I met. I want to thank Dr. Hardy, for he is

always there to lend a helping brain and hand. I have learned so much

from him and he is one of the greatest people I have had the pleasure of

working with. I also want to thank Ailsa for all of her supports; she

supported me through many difficult times; helping me escape from my

evil landlord, feeding me and offering me a words of kindness when my

research hit a rough patch. I want to thank my sister and mother (knowing

that someday in boredom they may might decide to read my thesis). I

thank my mother for letting me to decide what I wanted to do in my life,

although she is clueless as to what science is. I want to thank my sister for

all of her motivational speeches (= verbal abuse), without them I would not

have worked half as hard as I did. I want to thank all of the people in my

karate class, their selfless sacrifice to help me release my stress from

work (I am sorry for all of the bruises!). I want to thank my future children

(in case they feel left out), the thought that someday I might have to make

enough money to feed them motivates me to work hard. Last but not least,

I also want to thank my friend Vanda McNiven, who is always there for

me, to correct my grammar and listen to all my complaints. I want to thank

everyone in the lab, you guys are amazing!

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Chapter I: Introduction and Background

Phosphorylation and phosphatases

Protein phosphorylation regulates nearly all aspects of cellular physiology,

including metabolism, survival/apoptosis, signal transduction, and cell-cell

interactions (26). This major post-translational modification is a reversible

and precisely orchestrated event involving two opposing groups of

proteins: the kinases and phosphatases (69). Deregulation in this

elaborate signal transduction network results in many diseases, including

diabetes, obesity, osteoporosis, neurodegenerative diseases and various

types of carcinomas (82). Early studies viewed kinases as the “on”

switches while phosphatases were viewed as “off” switches because of

their roles in tumorigenesis: kinases such as Ras and Src were

characterized as oncogenes, while phosphatases such as PTEN

(phosphatase and tensin homologue deleted on chromosome 10) were

characterized as tumor suppressors (13)(87).

Recent discoveries recognize an exchangeable role of kinases and

phosphatases: their functions in positive or negative regulation are

dependent on the effect of phosphorylation on the substrate and its

downstream signalling transduction pathways (93). Some phosphatases

can even target and activate kinases by dephosphorylation, suggesting

that phosphatase activities are just as important as their counterparts in

the regulation of signaling pathways (108). Protein phosphatases (PPs)

are grouped into two major families based on their structure: the Ser/Thr

phosphatases and protein tyrosine phosphatases (PTPs) (108). The

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human genome contains 107 PTP genes. The amino acid sequence of the

PTP family members are divergent, but they all share a conserved

catalytic motif [(I/V) HCxAGxxR] (105). PTPs can be further grouped into

Cys-based PTPs (Class I, II and III) and Asp-based phosphatases

depending on the key amino acid involved in the catalysis (65). Type I

cysteine based PTPs make up the largest class and include tyrosine

specific classical PTPs or non-classical dual specificity phosphatases

(DSPs) (65). DSPs are defined by their unique ability to dephosphoryate

at phosphotyrosine and/or phosphoserine and/or phosphothreonine within

a substrate (65).This family of PTP includes 61 members, which are

further categorized into sub-families of phosphatases of regenerating liver

(PRLs), and others such as PTEN and mitogen-activated protein kinase

phosphatase (MKPs). PTEN and MKPs regulate major cascade pathways

and are accountable for various diseases such as cancer (65) (Fig.1).

Figure 1: Classification of protein phosphatase families. Phosphatases of Regenerating Liver (PRLs) belong to the subfamily of dual specific phosphatase (DSPs) within Class I PTPs.

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PRLs family

The PRLs family includes three members: PRL-1 (PTP4A1/PTPCAAX1),

PRL-2 (or PTP4A2/PTPCAAX2/OV-1) and PRL-3 (PTP4A3) (7). PRL-1

was the first in the family to be identified by its modulated expression in

both rat regenerating liver and insulin treated H35 cells (58). The PRL-1

sequence was cloned and its phosphatase activity established four years

after its recognition as an immediate early gene involved in regenerating

liver (22). Subsequently, mPRL-1 and mPRL-3 were found by searching

expressed sequence tag (EST) databases with the PRL-1 sequence

(105). At the same time, PRL-1 and PRL-2 were also identified separately

in a human breast carcinoma cDNA library as a farnesyltransferase

(FTase) substrate protein (16).

PRLs are among the

smallest phosphatases in the

PTP family, it is only ~20kDa

in size. PRLs are located on

three different chromosomes

in humans: 1, 6 and 8

(103)(Fig. 2). Although PRLs

share low sequence similarity with other DSPs, they are highly conserved

within the family and across species (45). Human PRL-2 shares closer

amino acid identity with PRL-1 (87%) than with PRL-3 (76%) (7) (Fig. 4).

Across species, human PRLs display over 40% sequence identity with

lower eukaryotes and above 70% among mammals (45). For instance,

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human and mouse PRL-1 and PRL-2 protein sequences are completely

identical, while the PRL-3 sequence is 96% identical (82)(105). The high

degree of conservation of PRLs suggests that they carry out critical

cellular functions.

The distribution of PRLs in various human tissues has been studied

extensively (50)(23)(105). PRL-1 and 2 have nearly ubiquitous expression

in all human tissues, while PRL-3 displays a more restricted expression

pattern (23). All three PRLs are expressed in skeletal muscle at high

levels (47). PRL-1 has its highest expression in the brain, PRL-2 has high

levels of expression in all tissues, and PRL-3 is primarily expressed in

heart and skeletal muscle, and has only low expression in all other tissues

(23). In conclusion, although the global expression profiles of PRL-1 and -

2 expression attest to their function in a basic process that is common to

many tissues and cell types, their distinct expression levels and patterns

suggests a different regulatory mechanism for PRLs (7)(103).

PRLs structure

PRLs are one of the smallest in size in the PTP family; therefore they do

not contain additional domains or sequence motifs other than conserved

PTP catalytic domains and a prenylation motif. PTP catalytic domains

include a WPD loop and signature motif (VHCXAGXXR). The prenylation

motif CAAX is conserved in all PRLs and they are similar to those found

in small GTPases of the Ras family (106)(Fig. 3).

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Figure 3: Domain structure of the PRLs family.

Prenylation at the CAAX motif is critical for the ability of PRLs to anchor at

the membrane and orient towards the cytoplasm (105). PRLs are the only

human PTPs that undergo prenylation, suggesting that they might have a

unique function compared to other PTPs (26). Another conserved region is

a highly polybasic region that occurs immediately before the CAAX

prenylation motif, which is also crucial for PRLs membrane localization.

Two mechanisms have been proposed to explain its function. First, these

basic residues act as a basic patch that interacts favourably with the acidic

membrane, contributing to membrane localization with the prenylation

motif (84). Second, they are also thought to be a potential nuclear

localization signal (NLS). Interestingly, when PRL-1 is prenylated, the NLS

is masked, which results in membrane localization. In the absence of

prenylation these residues act as nuclear localization signals targeting

PRLs to the nucleus (84).

Crystal structures and NMR assignments have been published for PRL-1

and PRL-3. However, only the secondary structure NMR assignment is

available for PRL-2 (7). In Figure 4, we summarized and applied the

previous published structural data of PRL-1 and PRL-3 to PRL-2 (39)(45)

(65). The secondary structures and overall folding of PRLs are highly

similar to one another: they consist of five β-sheets and six α-helices (7)

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(45). PRLs have the shallowest catalytic cleft of all known phosphatases,

suggesting that they have a broad range of substrate specificity (45). The

surface area near the active site is important in the recognition of PTP

substrates (84). Despite the expected structural similarity between PRL-1

and PRL-3, several variations are found in the active site region: the P

loop and WPD loop do not align between the two structures (39). PRL-3

has more flexible WPD loops due to an extra proline and glycine residue

following the loop (39). In addition, in PRL-3, the side chains of Cys104

and Arg110 is orientated away from the catalytic pockets, creating an

open conformation, while in PRL-1 they are constricted and oriented

towards the pocket (82).

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PRLs Phosphatase activity

The conserved PTP signature motif in PRLs is the first clue linking PRLs

to phosphatases (22). Other than this motif, PRLs do not share any

significant similarity to any PTP described previously. However, PRLs do

demonstrate phosphatase activities (108). First, PRL-1 was able to

dephosphorylate p-nitrophenyl phosphate substrate and this phosphatase

activity can be inhibited by PTP inhibitor orthovanadate. Second,

consistent with other PTPases, a critical cysteine mutation within the

conserved catalytic domain completely abolished its activity (22).

Although PRLs have low sequence identity with other DSPs (<30%),

they are still classified as a member of DSPs because of their

secondary structure element and overall fold (45). PRLs have the

closest sequence similarity to PTEN and Cdc14, but they have the

closest structure similarity to VHR, PTEN, MKP and KAP (7)(82).

Functionally, it is still not clear if PRLs dephosphorylate at tyrosine

and serine/threonine. Although the tyrosine phosphatase inhibitor

(orthovanadate) inhibits PRL-1, Ser/thr phosphatase inhibitors such

as okadaic acid, sodium fluoride, and calyculin A have no inhibitory

effect (50). In addition, PRLs show strong preference for

phosphotyrosine-containing peptides, whereas they have no activity

with phosphothreonine-containing peptides (22)(102). PRLs

phosphatase activities are essential for enhanced cell growth and cell

transformation, therefore the identification of PRLs substrates is

critical for clarifying its mechanism (46).

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Although classical PTPs and DSPs dephosphorylate very different

residues, they share the same general catalytic mechanism (45). PRLs

dephosphorylation involves two essential amino acids within two

conserved areas: asparatic acid in the WPD loop and cysteine within the

CX5R catalytic pocket (39)(Figure 3). Dephosphorylation starts with the

insertion of the substrate phospho-amino acid into the catalytic pocket.

Upon substrate docking, the WPD loop adopts a closed conformation and

covers the active site like a “flap” (39). The conserved cysteine

nucleophile attacks the substrate thioester, forming a covalent thio-

phosphate intermediate (13). Aspartic acid in the WPD loop then acts as a

general acid, donating a proton to the leaving phosphate group. The same

aspartic acid then acts as a base to assist the nucleophilic attack by a

water molecule, releasing the substrate and inorganic phosphate (7)(13).

Involvement of PRLs in disease malignance

Particular interest in the PRLs family is generated mostly by its association

with several aspects and types of human carcinomas. Elevated

expression of PRLs is involved in tumor growth, angiogenesis, metastasis

and poor prognosis (31) (70)(99)(104). PRL-3 is the most studied member

of the PRLs due to its key role in mediating the progression and

metastasis of colorectal carcinoma (CRC). PRL-3 expression is virtually

undetectable in normal colon epithelial, but its expression is escalated

from advanced primary tumors to CRC liver metastasis (40). PRL-3 is also

involved in melanoma metastasis (96), breast carcinoma (74), gastric

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carcinomas (70), liver carcinoma (96), and ovarian cancer (92). PRL-3

expression has been established as an excellent prognostic factor in

predicting the development of liver and lung metastasis (7). Similarly,

PRL-1 overexpression is also found in several cancers, including

melanoma (93), pancreatic cancer (81), lung cancer cell lines (1) and

esophageal squamous cell carcinoma (93). PRL-2 overexpression is

detected in pancreatic cancer (81), prostate cancer (94), acute myeloid

leukemia (AML) (99), and lung cancer (97). PRL-2 is involved in prostate

tumor progression, its expression is up-regulated in both prostate tumor

cells and advanced prostate cancer (94). PRL-2 is also associated with

hematopoietic malignancies in several studies. Overexpression of PRL-2

contributes to poor prognosis of acute myeloid leukemia (AML). Its

chromosomal anomaly is frequently linked to leukemia/lymphoma and it is

upregulated in Hodgkin’s lymphoma cell line (4)(99). We also showed that

PRL-2 expression is elevated in laser capture microdissected primary

breast tumour epithelial cells when compared to matched normal tissues

in our previous study (33). Clearly, all clinical data point to the involvement

of PRLs in cancer progression. PRLs contributes to several hallmarks of

cancers, including increased cell proliferation, migration/invasion,

angiogenesis and the inhibition of apoptosis (40)(95). Aberrations of PRLs

expression was studied in numerous cell lines using soft agar, matrigel

invasion, transwell migration and wound healing assays. Unyielding

results show that overexpression of PRLs altered cell morphology,

increased colony formation, promoted cell growth, migration and wound

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healing, and enhanced cell adhesion and spread. Conversely, the

knockdown and catalytic dead PRLs mutant reduced the motility and

invasive property (4)(16)(46)(48)(50)(57)(59)(69)(81)(84)(92)(96)(97)

(104). We have also shown PRL-2 overexpression increases colony

formation and cell migration in TM15 and DB7 cells (33). PRL-3

participates in angiogenesis. PRL-3 recruits endothelial cell forming blood

vessels to support bigger tumor cells and it also down-regulates the

secretion of IL-4, a known negative vasculogenesis (31). To date, no

evidence has suggested the involvement of PRL-1 or -2 in angiogenesis.

The physiological role of PRLs in vivo has also been studied in mouse

models. Tail vein injections of several overexpressing PRLs cell lines

promote tumor formation and metastasis in nude mice (16)(96)(104). We

have also demonstrated that injection of PRL-2 overexpressing cells in the

mouse mammary fat pad promotes tumor formation (33). Beside its

oncogenic role, it is not a surprise to find that PRLs are also involved in

other biological processes. PRL-1 is involved in embryonic development,

its expression is elevated during the development of mouse neuronal,

gastrointestinal and skeletal tissue (95). In addition, oxidative stress in

cone photoreceptors increases PRL-1 expression, suggesting an

involvement of PRL-1 in the phototransduction cascade (102). PRL-3

mediates angiotensin II (AngII), a factor that triggers a series of signaling

events leading to cardiac hypertrophy (50). Curiously, PRL-2 expression is

upregulated during hibernation in bats brain tissue, suggesting that PRL-2

maybe involved in maintaining normal cycle of nerve cells in the

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hibernating bat (101). PRL-2 was also identified in Neuronal Ceroid

Lipofuscinoses (NCLs) mouse models as a protein critical for the neuronal

growth of cone-cytoskeletal dynamics (89).

Regulation of PRLs

PRLs activity is regulated at several levels: transcription,

phosphorylation, localization, oligomerization and oxidation.

PRLs genomic sequences are highly conserved except at the 5’ non-

coding region, suggesting PRL members are differentially controlled at the

transcriptional level (105). Several transcription factors were identified to

regulate PRL expression. PRL-1 and 3 are direct transcriptional targets of

p53 since they both contain a p53 binding site within their promoters (6).

In addition, an increase of p53 expression increased PRL-1 and 3 mRNA

levels in a dosage dependent manner (56). PRL-1 also contains a binding

motif for a growth activated transcription factor known as early growth

response (Egr-1)(82). In NIH3T3 cells, overexpression of Egr-1 increases

PRL-1 gene transcription (68). PRL-3 is a direct target of the TGFβ

signaling pathway; the binding of Smad3/4 to the PRL-3 promoter site

downregulates its transcription (40). Since the TGFβ signal is frequently

lost in colorectal carcinoma, PRL-3 upregulation in CRC is proposed to be

regulated by the TGFβ pathway (40). As of yet PRL-2 transcription

regulation has not been described.

PRLs subcellular localization is controlled by conserved PRLs CAAX

domains and polybasic regions. The farnesylation of the CAAX domain is

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essential for their subcellular localization to the membrane and the

endosomal compartment. Prenylation deleted mutants (SAAX) and

farnesyl transferase inhibitor (FTI) treatment redistributes PRLs to the

nucleus (13). Also, PRL-1 localizes in a cell cycle dependent manner; in

non-mitotic cells, it localizes to the membrane and endoplasmic reticulum

while in mitotic cells it relocalizes to the centrosome and spindle apparatus

(93). Interestingly, PRLs promote invasion and motility and cell cycle

control in a farnesylation dependent manner. FTI treatment and PRL-1

SAAX mutants induced defects in mitosis and cytokinesis. In addition, they

also thoroughly inhibit the invasion and motility in SW480 cells (26)(93).

Deletion of the polybasic region of PRL-1 not only redistributed the

enzyme from the membrane to the cytoplasm it also abolished cell growth

and migration (84). Together, these results suggest that subcellular

localization of PRLs is important for their functions in regulating cell

growth, invasion and migration. Oxidation regulates the phosphatase

activity of PRL-1 and PRL-3 by of the formation of an intramolecular

disulfide bridge between cys49 and cys104 (45)(102). This disulfide bond

imposes a conformational constraint on the phosphatase active site,

inhibiting catalysis and substrate binding (65). Disulfide bridge formation

was suggested as a mechanism to prevent the permanent inhibition of

PRLs by irreversible oxidation (45). Interestingly, oxidative stress induces

PRL-1 disulfide bridge formation and inactivated phosphatase activity is

observed in mouse retinas stimulated with continuous illumination (45).

Given the conservation of the two cysteines in PRL-1 and 3, we expect

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that PRL-2 activity could also be modulated by oxidative stress.

Trimerization of PRL-1 is novel among the PTPs (84). Trimerization is

postulated to be essential for PRL function as PRL-1 structure has always

been crystallized in its trimeric form (84). Mutation within the trimeric

interface disrupted PRL-1 trimerization, abolishing the proliferation and

migration phenotype in a phosphatase activity independent manner (39)

Previous studies suggested that the trimerization and membrane

localization cooperate with each other to provide stronger adhering forces

to the membrane (39). In addition, trimerization could also increases the

affinity and substrate specificity of PRL-1 to its substrates (84). The

trimeric interface structure is conserved in all PRLs therefore

oligomerization might be a preserved and common regulatory mechanism

(84).

Identified PRLs interacting partners and substrates

PRLs interacting partners and substrates are involved in diverse aspects

of cellular function (Table 1). PRL-3 interacts with transcription factor ATF-

5 and elongation factor EF2 to regulate protein synthesis (62)(69). PRL-3

also mediates EMT processes with several cell adhesion proteins

(integrins and CDH22) and components of cytoskeleton remodelling

(tubulin, stathmin and keratin). PRL-1 and PRL-3 both interact with α-

tubulin, the major constituent of microtubules (93). PRL-3 also interacts

with stathmin, a regulator of the microtubule dynamic (109). Keratin-8 is a

component of the intermediate filament, and reorganization of keratin

mediates cell migration (57). Phosphorylation regulates the activation of

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keratin, nucleolin, integrin and Ezrin and PRLs overexpression has been

found to decreases all their phosphorylation levels (27)(57)(67)(78)(97).

PRL-2 has one known interacting partners, geranylgeranyl transferase

(βGTTII). PRL-2 competes with the α-subunit of GGTII for βGGTII binding,

inhibiting the GGTIIα/β subunit association. The GGTIIα/β complex is

important for the prenylation of Rab, therefore PRL-2 is thought to regulate

the vesicle trafficking through Rab mediated protein recycles (79). Erzin is

the first substrate identified for both PRL-2 and PRL-3 (27)(97). PRL-2 and

3 dephosphorylate specific Tyr and Thr sites on Erzin. Erzin is involved in

tumor invasiveness; a previous study showed that PRL-3 mediates its

angiogenesis property through dephosphorylation of Erzin (27).

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Cellular pathway regulated by the PRLs family

Given the role of PRLs in tumorigenesis, it is not surprising to find that

several cellular pathways mediated by the PRL family are related to

cell proliferation, apoptosis and metastasis (31)(104).

PRLs play a critical role in mediating the progression of cells through

mitosis. Overexpression of PRL-1 and -2 promotes a cell population

transition from the G1 to S phase in a p53-dependent pathway (95).

PRL-1 reduces p53 levels by increasing its proteosomal degradation

either directly by the activation of MDM2, or indirectly by the up regulation

of PIRH2 (56). Decreased amounts of p53 diminishes the activity of

downstream p21Cip/Waf1 (p21), a cyclin kinase inhibitors of CDK2. In

time, the enhanced CDK activity allows the cell to bypass the G1

restriction point and entry into S phase (95). PRL-3 knockdown also

inhibits cell proliferation by arresting the cell cycle at the G1 phase. Cell

cycle arrest is regulated by a p53-dependent and a PI3K/AKT dependent

pathway (6). A decrease in PRL-3 level up-regulates p19ART, a MDM2

sequestration protein. A reduction in the amount of MDM2 sequestered

therefore increase p53 stability. In addition, a decrease in PRL-3 activates

the PI3K/AKT pathway and increases phosphorylation of FOXO, a

transcription factor. FOXO then up regulates cell cycle regulatory genes,

such as p21/p27 (6). PRL-2 expression does not alter total p53 levels,

suggesting that PRL-2 may exert its cell cycle control in a p53

independent manner (97).

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Metastasis is a multi-step process in which cancer cells spread from the

primary tumor to distant locations. Epithelial-mesenchymal transition

(EMT) is an central step in metastasis - it is a process in which cancer

cells gain an increase in cell motility and invasiveness (48)(92).

Overexpressing PRL-1 and 3 cells exhibit an EMT change in cell

morphology (69)(96). Studies later showed that PRL-3 mediates EMT

interference of cell-cell adhesion, focal adhesion complexes and promotes

cytoskeleton rearrangement (48).

Cells adhere to each other by adherences junctions and a large group of

cell adhesion molecules called cadherins. PRL-3 can mediate cell-cell

adhesion either directly by suppressing expression of CDH22/E-cadherin

or indirectly through the PTEN-PI3K-Akt signaling network (47). PRL-3

overexpression up-regulates PTEN/PI3K and activates AKT. phosphoART

phosphorylate and inactivates GSK-3β, therefore increasing Snail activity.

Raised Snail subsequently down-regulates critical components of the

adherence junction complex, such as E-cadherin, y-catenin and

cytokeratin (92).

A focal adhesion is a protein complex that attaches cells to the

extracellular matrix (ECM) (7). PRLs increase cell motility and invasion by

disrupting focal adhesion complexes (49). PRL-3 increases Src activity by

reducing the expression of Src kinase (Csk), a kinase that inhibits Src

acitivity by phosphorylation. Enhanced Src phosphorylation then activates

focal adhesion kinase (FAK) and p130Cas; together, they delay focal

adhesion turnover (46). PRL-3 also directly reduces the number of cellular

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focal adhesion complexes by down-regulating components of focal

adhesion, such as paxillin and vinculin (92). PRLs increase invasiveness

through common pathways, as they all up-regulate p130Cas (1)(49)(97).

However unlike in PRL-1 and 3 c-Src activity is unchanged in PRL-2. This

suggests that PRL-2 regulates p130Cas activity by an alternative pathway

(49).

PRL-1 and 3 also mediates cytoskeleton rearrangement through Rho

family GTPase, an important regulator of actin cytoskeleton reorganization

(26)(48). PRL-1 and 3 decrease the levels of RhoA GTP and Rac1 GTP,

which results in an increase in cell spreading (1)(92).

Extracellular signal-regulated protein kinase ½ (ERK½) is a major

signaling pathway involved in several fundamental cellular processes (46).

PRLs overexpression increases the phosphorylation level and the activity

of ERK½ activity (48)(67)(33). Activation of ERK½ by PRL-3 drives up

regulation of MMP2 and MMP9 by Ap1 and Sp1 transcription factors (48).

Matrix metalloproteinases (MMPs) are major hydrolytic enzymes that

break up the ECM, increased expression of MMPs allows cell invasion into

the surrounding tissue (48). In our previous study, we showed that PRL-2

overexpressing tumors generated from MMTV PRL-2 mice crossed with

ErB2 mice displayed increase phosphorylation of ERK1/2 (33).

The PRLs family participates in a variety of cellular pathways and some of

which are shared among all PRLs. However, studies to date show PRL-2

is distinct from other PRLs as it is not involved in some major pathways,

such as the PTEN/PI3K/AKT, cSrc and p53 pathways. PRL-2 does not

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activate p53, Akt and c-Src suggesting that PRL-2 functions mediate a

pathway that is different from PRL-1 and 3 (97). Thus the identification of

PRL-2 substrates will be critical for elucidating its role in signaling

pathways.

PRL inhibitors

PRLs are very attractive targets for anti-cancer therapy because of their

unquestionable involvement in tumorigenesis. Several inhibitors have

been identified to date, including pentamidine, prenylation inhibitor,

rhodanine, natural compound and thienopyridone. Pentamidine was the

first PRLs inhibitor to be identified, it is known to inhibit growth of several

cancer cell lines. However, pentamidine also has an inhibitory effect on

several other PTPs. Therefore, it is not conclusive as to whether the

inhibitory effect is mediated by PRL or another phosphatase family (7).

Given that PRLs are the only members in the PTP family that undergo

prenylation, a lot of interest was placed on the Farnesyltransferase

(FTase) inhibitors (TIFs) and Geranylgeranyltransferase inhibitos

(GGTTs). However, these inhibitors proved ineffective in clinical trials

because they have an effect on a wide spectrum of targets outside of the

PTPs (7). High throughtput screening of chemical libraries discovered

several inhibitors, such as rhodanine and the natural compounds

biflavinoids ginkgetin and sciadopitysin (3). However, their specificity,

toxicity and mechanism of action have not been investigated.

Thienopyridone is a selective small molecule PRL inhibitors and it

suppresses tumor cell anchorage independent growth through p130Case

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cleavage and anoikis (20). In addition, it also has a good selectivity for

PRLs but not for other PTPs (49). To date, however clinically useful and

specific inhibitors of PRLs have not been reported (64). Given the high

homology between PRL members, it is difficult to identify specific

inhibitors for each PRLs member. PRLs might share similar functions

therefore knockdown of one PRL may be compensated by other PRLs

member. The identification of a PRL-2 downstream substrate might

provide an easier downstream target for inhibitor design and a better

substrate for an in vitro phosphatase assay to evaluate PRL inhibitors

(97).

Project Goals

Although mounting evidence has established the role of PRLs in tumor

progression and metastasis, the mechanism of their regulation and the

basis of their transforming activity are not yet understood. We have

previously identified PRL-2 as a PTP that is overexpressed during breast

cancer development (33). In multiple breast cancer cellular models, we

have confirmed the role of PRL-2 in cell migration and transformation.

Also, PRL-2 promoted tumor formation in vivo in both a xenograft model

and in breast-cancer prone MMTV-ErbB2 transgenic mice. Thus, the

identification of bona fide physiological substrates or interacting partners

of PRL-2 is the first step to understanding its mechanism of action and its

downstream signalling pathway. In addition, we generated a PRL-2

knockout mouse model to provide insight into the physiological function of

PRL-2.

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Chapter II: Methods and materials

Cell culture and transfection

Cell lines were cultured in DMEM (Hyclone) supplemented with 10%

FBS and 10µg/ml gentamicin at 37°C with 5% CO2. Transfections were

performed using lipofetamine 2000 (Invitrogen) following the

manufacturer’s instructions and media were replaced 4hr after

transfection. Experiments were generally carried out 24hr post-

transfection.

Stable Transfection

HEK293 cells were cultured to 80% confluence and transfected with

pcDNA3.1 Flag vector, Flag-PRL-2 or Flag-PRL-2CSDA vector. To

select for stable transfectants, 200ug/ml of G418 was added to the

medium 48hr after transfection.

Affinity Purification of Mass Spectrometry (AP-MS)

AP-MS was performed as described (17). Five plates (p150) of 80%

confluent stable cells were harvested for each AP-MS. Cells were scraped

and washed in ice cold PBS 3x before the addition of 1mL lysis buffer

(50mM Hepes-KOH, pH8.0, 100mM KCl, 2mM EDTA, 0.1% NP-40, 10%

glycerol and 10mM NaF), freshly supplemented with 1x protease inhibitor

and 1mM DDT. Lysate was incubated on ice for 20 min to facilitate the

lysis process before undergoing two freeze-thaw cycles. Cell debris was

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cleared by centrifugation at 13,000g for 15min. Lysate concentrations

were measured by Bradford assays and equal amounts of lysate were

incubated with 100ul washed Sepharose beads to reduce non specific

interactions. After 1hr of incubation at 4oC, the beads were pelleted and

removed. 7ul (per 10mg of pre-cleared lysate) of pre-washed packed

FLAG-M2 agarose (Sigma) beads were added to the lysate and together

they rotated with gentle end over end agitation overnight at 4oC. Beads

were washed extensively with lysis buffer and FLAG rinsing buffer (50mM,

NH4HCO3, pH8.0, 75mM KCL and 2mM EDTA), followed by addition of

500ul of freshly prepared elution buffer (0.5M NH4OH, pH~11.0, 0.5mM

EDTA). Eluted products were then lyophilized by speed vacuum, trypsin

digested and analyzed with LC/MS/MS at the McGill Mass Spectrometry

Facility.

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Silver staining

Polyacrylamide gels were fixed in 12% acetic acid, 50% ethanol and

0.05% formaldehyde overnight. The next day, they were washed in 20%

ethanol, sensitized for 2 min in 0.02% sodium thiosulfate, and rinsed with

water twice. Gels were incubated for 20min in the staining solution (0.2%

silver nitrate, 0.076% formaldehyde), rinsed with large volumes of water

and developed in freshly prepared developing solution (6% sodium

carbonate, 0.0004% sodium thiosulfate and 0.05% formaldehyde). The

reaction was stopped by adding 12% acetic acid.

Plasmid construction

CNNM3 fragment was amplified from CNNM3 cDNA (Openbiosystem)

using KAPA HiFi DNA Polymerase (Kappa Biosystem). The PCR mixture

contained a final concentration of 1xKAPA GC HiFi buffer, 2mM dNTP,

50ng of CNNM3 vector, and 0.4ul of Kapa Hifi polymerase. The PCR

program was 95oC for 3mins, followed by 30 cycles of 98oC for 20s, 60oC

for 15s, and 72oC for 2mins. Purified PCR products were cut and inserted

into a pcDNA3 Flag vector. CNNM3 domain truncated mutants cDNA was

amplified by specific primers flanking the sequence of interest (Table 2).

The point deletion mutants of CNNM3 were generated by site

mutagenesis using Kapa Hifi DNA pol. The same PCR mixture as above

was used, except that Flag 2xCBS or Flag CNNM3 vectors were used as

the template. PCR condition changes included increasing the annealing

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temperature to 60oC for 1min and the extension time to 72oC for 10min.

PCR products were digested overnight with DpnI, and transformed into

DH5α.

Immunoprecipitation and pull down

Cells were lysed with Triton buffer (1% Triton, 20mM Tris and 150mM

NaCl pH7.5) supplemented with protease inhibitor (Sigma), cleared by

centrifuging at 13,000×g for 10mins and quantified using BCA Protein

Assay (Thermoscience). For immunoprecipitation (IP), equal amounts of

proteins (~3mg) were incubated with 2ul of PRL-2 antibody (Milipore) or

CNNM3 antibody (Protecintech) overnight. The next day, 24ul of pre-

washed agaroseA/B bead (Bioshop) were added to the lysate for an

additional 2hr incubation. In the GST pull down assay, approximately

400ug of proteins were incubated with 16ul of pre-washed glutathione

sepharose (GE Healthcare) for 2hr at 4oC. In Flag IP 5ul of M2-Flag resin

(beads) were added and incubated overnight. In all cases, beads were

washed several times with lysis buffer and protein were eluted by boiling

the beads in 2xSDS loading buffer.

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PCR mouse genotyping

Genomic DNA was isolated from mouse tails using standard pheno-

chloroform extraction. Multiplex PCR used three primers F1, R2, and R3

(Table 2) (Fig. 20). The PCR mixture contained a final concentration of 1x

reaction buffer, 2mM dNTP, 1ul DFS Taq Pol (Bioron), 0.4uM of each

primer, and 1ul of ~50ug/ul of purified genomic DNA in a total volume of

20ul. PCR conditions were 95oC for 2mins, followed by 30 cycles of 95oC

for 45s, 60oC for 1 min, and 72oC for 80s.

Western blot of thymus tissues

Thymus tissues were isolated, snap freezed in liquid nitrogen and

stored at -80oC. To prepare protein lysates, thymus tissues were

homogenized in Ripa lysis buffer (150mM NaCl, 1% NP-40, 0.5% sodium

deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with freshly added

complete protease inhibitors (Roche biochemicals) on ice. The

homogenates were centrifuged at 13,000g for 10min at 4oC, and the

concentration quantified by Bradford assay. Equal amounts of cell lysates

were resolved by 12% SDS-polyacrylamide gel electrophoresis (PAGE)

and transferred to PVDF. Membranes were blotted in milk for 1hr followed

by incubation of PRL-2 antibody (Millipore) in PBS-T (Phosphate buffered

saline with 0.1% Tween-20) overnight at 4oC. The next day, membranes

were blotted with anti-mouse secondary antibody (1:10000).

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Immunoreactive proteins were detected with enhanced

chemiluminescence detection reagents (Western Lightning Plus)

according to the manufacturer’s instructions.

Murine embryonic fibroblasts (MEF)

The PRL-2 KO mouse was generated by gene trapped ES cell (Sanger

Institute). Heterozygote mice were set up for mating, and embryos were

isolated at E14.5. Harvested embryos were washed multiple times in PBS,

and red tissues were removed and finely minced. Each embryo was

digested with 6ml 0.25% Trypsin/EDTA containing 10mM HEPES at 37oC

for 30min. Large chunks of tissues were removed by centrifugation at

1000rpm for 5min. The cells in the supernatants were plated in 150cm

dishes, and incubated at 37oC with 5%CO2.

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Chapter III: Results

Identification of PRL-2 interacting partners and substrates

Several techniques can be used to identify interacting partners/substrates

of PRLs, including yeast two hybrid (47)(107), affinity purification (78) and

differential protein expression profiling (57)(62). In our study, we combined

these techniques with the PTP trapping system to identify physiological

substrates. The use of trapping mutants increases the likelihood of

isolating substrate complexes as it reduces the transient nature of this

complex seen in wildtype PTPs (46). We have previously attempted

PRL-2 yeast two hybrid experiments. However, we were unable to identify

any potential candidates (data not shown). We switched our strategies to

a FLAG tag affinity purification system as previously described (17)(25). In

order to generate enough protein for affinity purification, we created a pool

of stable HEK293 cell lines overexpressing Flag empty, Flag wildtype, and

Flag PRL-2CSDA substrate trapping mutants. PRL-2CSDA mutants

contains two mutations, D69S and C110S, within the conserved catalytic

domain of PTPs (Fig. 26). They block the ongoing PTP catalysis,

stabilizing the enzyme-substrate interaction, and therefore trapping the

substrate within the PTP catalytic pocket (9). We have successfully

generated a stable HEK293 cell lines expressing moderate amounts of

Flag PRL-2 and PRL-2CSDA (Fig. 6).

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We performed a large-scale affinity purification and western blot to

validate the efficiency of the IP and protein elution (Fig. 7). Although some

Flag PRL-2 still remains on the beads fraction, we detected flag PRL-2 in

the eluted fraction, validating successful IP and elution of PRL-2 and its

putative interacting partners.

Silver-staining was performed on the eluted proteins to identify the

difference in banding patterns between Flag empty control, Flag PRL-2

and Flag PRL-2CSDA (Fig.8). Several bands appeared in Flag PRL-2 and

PRL-2CSDA, but not in the flag empty, suggesting these are interacting

partners that associate with PRL-2.

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However, these bands could not be directly identified from the gel by mass

spectrometry because of their low intensity. Therefore we adapted a

technique that combined the affinity purification step directly to mass

spectrometry (AP-MS). In total, three independent AP-MS were performed

under similar conditions. A list of background/non-specific binding present

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in the IP of flag empty samples are listed in Table 3. In addition, we also

cross referenced them with previously published common background

contaminants using the same technique and cell line (17).

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Table 4 and 5 contain candidate interacting partners/substrates

immunoprecipited using PRL-2 wildtype or PRL-2 CSDA but not present

in the Flag control. In our results, several candidates interacted with both

PRL-2 wildtype and trapping mutant. They include nucleolin, Y box

binding protein 1 (YBX1), translocated in Liposarcoma Protein (TLS), and

PRL-1. Analysis of peptide sequence coverage has isolated peptide

sequences unique to PRL-1, indicating PRL-2 interact with PRL-1 in vivo.

A couple of candidates also interact only with PRL-2CSDA trapping

mutants, including 14-3-3 (14-3-3ε, ζ/σ), casein kinase II (CKII), Gem

associated protein (Gemin3,6), serine/threonine kinase receptor

associated protein (STRAP), SET translocation (SET) and survival of

motor neuron (SMN). Table 6 summarizes results from a large scale

mapping of human protein-protein interactions by mass spectrometry

(25). In this study, the researchers included PRL1,2,3 using similar

methods to look at the interacting partners of human bait proteins (25).

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CNNM family members interact with PRL-2

The Ancient Conserved Domain Protein (ACDP) family consists of 4

members, CNNM1-4 (also referred to as ACPD1-4 or cyclinM1-4). Here

we showed that wildtype PRL-2 pulled down all member(s) of ACDP in

three separate AP-MS experiments (Table 4). Analysis of the peptide

coverage mapped sequences that are unique to each CNNM members

(Table 7). Furthermore, CNNM3 and 4 also interact with PRL-2 and 3 in

the large scale IP (Table 6). The consistent IP results and redundant

interaction with all members of the CNNM family suggests a conserved

and important function of this interaction. However, we decided to focus

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our interest on CNNM3 because it has been IPed in all AP-MS

experiments.

PRL-1,2,3 all interact with CNNM3

We validated the interaction of PRL-2 with CNNM3 in by a co-transfected

Myc-CNNM3 and Flag PRL-2 in HEK293 cell followed by IP with a Flag

antibody (Fig. 9). In accordance with the AP-MS, PRL-2 interacted with

CNNM3, but the interaction was lost with the PRL-2CSDA trapping

mutant.

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We want to know if all PRLs interact with CNNM3 since they share high

sequence similarity, similar structure, and possibly functional homology.

Also, previous studies identified common interacting partners between

PRLs (Table 1). We showed in a GST pull down that all members of PRLs

interact with CNNM3 (Fig. 10).

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Endogenous interaction of PRL-2 and CNNM3 in several cell lines

In order to eliminate the possibility that the interaction is an artefact of the

AP-MS system and/or protein overexpression, we performed a co-

immunoprecipitation (co-IP) experiment in several cell lines. Protein

lysates from different cell lines were immunoprecipitated with PRL-2 and

CNNM3 antibody separately. We observed that not only PRL-2 antibody

IP CNNM3, but reversibly CNNM3 antibody is also able to IP PRL-2 in all

four cell lines, These results confirms the physiological interaction

between endogenous PRL-2 and CNNM3 in vivo (Figure 7).

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CNNM3 ACD domain is required for interaction

Knowing which CNNM3 functional domain interacts with PRL-2 can give

insight into the mechanism of interaction. All CNNMs possess four

transmembrane domains and an ancient conserved domain (ACD)

domain (Fig. 12).

Figure 12: Schematic of ACDP family conserved structure domains

As the name implies, the ACD domain is highly conserved within ACDP, it

consists of two cystathionine-beta-synthase (CBS) domains, a cyclin box

and a cyclic nucleotide-monophosphate-binding domain (cNMP) binding

domain (91). Six CNNM3 truncated mutants fused to Flag were created

based on the predicted domain structure of CNNM3. A GST PD assay

was performed on cells co-transfected with Flag fused truncated mutants

and GST-PRL-2 (Fig.13). According to an SDS-PAGE of total cell lysis

(TCL), all constructs were expressed at the expected size. Interaction was

lost in the ∆282 mutant that contains just the transmembrane domain

suggesting that the ACD domain is critical in mediating PRL-2 interaction.

∆380 is another mutant that lost the interaction. It contains a

transmembrane domain and one CBS suggesting that both CBS domains

are essential for the interaction.

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All PRLs interact with CNNM3 via the CBS domains

We produced two additional deletion mutants spanning the ACD region:

the 2xCBS included both CBS domains and ccbox composed of the cyclic

motif and cNMP binding domain. PRL-1 and PRL-3 were also included in

the co-IP experiment because we want to know if the interaction occurs at

the same domain for all the PRLs. Flag IP is performed on Flag

2xCBS/ccbox and its interaction with GST-PRL-1,2,3 (Fig. 14). PRL-1,2,3

bind to the 2xCBS domain, but not the cyclin box and cNMP binding

domain.

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We have demonstrated in ACD domain that CBS domains alone are

sufficient for PRLs interaction and that this binding shared a common

region within the PRL family.

Point mutations within the CBS domain disrupted PRL-2 binding

CBS domains are found in a variety of proteins with diverse functions

and mutations within these CBS domain are associated with several

inheritable diseases (8)(76). Lists of pathogenic CBS domain mutations

were generated from an extensive literature search and we performed

sequence alignment of these CBS domains to translate these mutations

into the equivalent residues in CNNM3 CBS domains (Table 8).

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We made nine point mutants in total and their locations are marked in the

sequence alignment of CNNMs CBS domains (Fig. 15).

Figure 15: Point mutations within the CNNM3 domains. We aligned the CBS domains of all CNNM family. As shown CBS domains are highly conserved and the point mutations we selected is conserve din all members.

We first performed the site mutagenesis in the CNNM3 2XCBS domain

instead of in the full length CNNM3 because we know that CBS domains

retain their structure and binding properties when separated from their

bulk protein (38). Seven point mutants were generated with Flag tagged

2xCBS constructs including Y301F, S334G, K360Q, D396A, E424A,

G433D and I445R (Table 8). Flag tagged CBS mutants were

co-transfected with GST-PRL-2 into HEK293 cells and IP with Flag (Fig.

16). TCL show expression of each CBS point mutant and GST PRL-2.

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Since D396A and G433D mutations disrupted the interaction of PRL-2

with CNNM3 CBS domains. Subsequently, we generated CNNM3 full

length D396A, and G433D mutants to warrant the deleterious effect of

these mutations. We also included a CNNM3 full length T437N and

D439A mutant because of the conserved pathogenic role of these

residues in different CBS containing proteins (Table 8). Two pull down

assays were used. First, Flag bead was used to IP Flag tagged CNNM3

full length point mutants and GST PRL-2 (Fig. 17a). Consistent with

previous experiments, G433D disrupts CNNM3 interactions with PRL-2.

Surprisingly D396A mutants reduce the level of interaction but did not

abolish it. We repeated the experiment using a GST PD assay to verify if

this discrepancy results from an IP artifact. GST pull down was performed

on cells transfected with GST PRL-2 and Myc tagged CNNM3 full length

point mutants (Figure 17b). Similar results were obtained, confirming that

the G433D mutation completely abolished the interaction with PRL-2 while

D396A dramatically reduced the interaction. All together, these results

identified two key residues located in CNNM3 CBS domains that are

important for PRL-2 interaction.

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Global phosphorylation did not promote CNNM3 interaction with PRL-2CSDA trapping mutant

The fact that PRL-2CSDA trapping mutant loses interaction with CNNM3

was surprising. Since a substrate must be phosphorylated in order to bind

to the catalytic pocket of the PTP, we want to know if the phosphorylation

of CNNM3 might promote its interaction with the trapping mutant. Thus,

we increased the global phosphorylation of all proteins by increasing the

activity of upstream kinase. We co-transfected the Hela cell with three

constructs, Flag CNNM3, GST PRL-2 or GST PRL-2CSDA and a

constitutively activated Src mutant. Although Src kinase increased overall

expression of Flag CNNM3, it did not affect its binding to PRL-2 and was

not trapped by PRL-2CSDA (Fig.18).

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PRLs mutations disrupting their interaction with CNNM3

To investigate the reason why the PRL-2 C101SD49A trapping mutant

failed to interact with CNNM3, we tested the following PRL-2 mutants.

PRL-2 C101S (CS), D69A (DA), C101SD49A (CSDA), A108SC101S

(ASCS) and V110TC101S (VTCS) are mutants that inactivate PRL-2

phosphatase activity; A108S (AS) and V110T (VT) are mutants that

increase PRL-2 in vitro activity; the PRL-2 mutant with all tyrosine

phosphorylation sites mutated (6YF) has no known function. To ensure

disruption of these mutants was not the artifact of procedure, we

performed the IP in two separate systems. In the first method, GST pull

down was performed on cells transfected with GST-PRL-2 mutants and

Flag CNNM3 (Fig.19A). In the second method, Flag IP was performed on

a Flag PRL-2 mutant and Myc CNNM3 (Fig.19B). The results are

consistent in both assays, and show that CS alone completely eliminates

the interaction while the DA mutation impaired the binding greatly. In

addition, AS and VT mutations show the same binding as the wildtype

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PRL-2. However, with addition of CS mutations these interactions were

completely abolished. These results demonstrate the important role of the

PRL-2 C101 and D69 residues play in for the interaction with CNNM3.

PRL-2 6YF mutants also demonstrated a dramatic decrease in interaction

compared to wildtype, suggesting that tyrosine phosphorylation of PRL-2

might regulate its interaction with CNNM3. Looking at other PRLs, we

performed a Flag IP assay on PRL-1 and PRL-3 mutants with equivalent

mutations. PRL-3 results are identical to PRL-2: CS and CSDA mutations

abrogate all interactions, while DA weakens the interaction. Unexpectedly,

PRL-1 CS, and DA mutants display almost equal binding as wildtype while

CSDA abruptly ends all interaction. C101 and D69 residues are important

for the PRLs-CNNM3 interaction and the structural difference between the

PRL-1 and PRL-3 active sites might account for the difference of CS and

DA mutations on interaction (39).

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PRL-2 knockout mouse model

The oncogenic role of PRLs has been extensively researched; however

their exact biological functions remain unknown. In order to characterize

the physiological function of PRL-2, we generated a PRL-2 knockout

(KO) mouse model using PRL-2 gene trapped embryonic stem (ES)

cells purchased from the Sanger Institute. In PRL-2 gene trapped,

several elements are inserted into intron1 of PRL-2, including a splice

acceptor site (SA), the betaGeo gene and a transcription termination

sequence PolyA (Fig.20). With the insertion, the PRL-2 KO mouse

produces a truncated PRL-2 protein fused with betaGeo. A multiplex

PCR was performed as described in order to identify the different PRL-2

genotypes (Fig.20).

A western blot was performed on the genotyped mouse to confirm the

knockdown of PRL-2 at protein level (Fig.21). Results show a gradual

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reduction of band intensity from wildtype mouse to knockout.

Interestingly, CNNM3 levels were not affected by the status of PRL-2.

We have previously tested the specificity of the PRL-2 antibody

(Fig.21B). The PRL-2 antibody is found to recognize both PRL-1 and

PRL-2, therefore the upper band in the PRL-2 blot belongs to PRL-1.

Immortalized MEF cell line

Murine embryonic fibroblasts (MEFs) are a great source of homogenous

stable cell lines that can be used in several cell based assays. MEFs

isolated from the PRL-2 knockout mouse provide a much more stable

and consistent PRL-2 knockdown compared to siRNA. Embryos from

heterozygote parents are harvested at E14.5 and trypsin digested to

isolate the primary MEF. Aliquots of cells were frozen and are currently

in the process of immortalization. We have successfully isolated

wildtype, heterozygote and knockout PRL-2 primary MEF lines as

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shown by PCR genotype and western blot (Fig.22).

Reduced body weight of knockout PRL-2 mouse

We piloted a study on the body weight of the PRL mouse because we

observed KO mice are smaller in size compared to the wildtype mouse.

Several heterozygote crosses were designed to ensure a fair distribution

of wildtype, heterozygote and knockout in the offspring for comparison.

At 4 weeks old, we noticed a gradual decrease of the body weight from

WT to HET to KO (Fig.23). Moreover, the body weights of knockout

PRL-2 mice are significantly lower than their wildtype counterpart. This

result suggests a possible role of PRL-2 in metabolic signaling.

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Chapter 4: Discussion

The PRL family belongs to the DSPs family of phosphatases, whose

expression is elevated in both metastatic cancer and primary tumors

(46). PRLs promote cell proliferation, migration and invasion. These

oncogenic properties have made PRLs a highly attractive target for

novel anticancer therapeutics (64). Although the involvement of PRL-2

in cancer has been investigated, little is known about its precise

physiological function, regulation, substrates and downstream signal

transduction pathways (93). Identification of PRLs substrates will be the

first step towards answering some of these questions, as well as to the

development of useful clinical specific inhibitors for PRL-2.

Identification of PRL-2 interacting partners using AP-MS

AP-MS is a newly developed technique used to identify protein

interacting partners. AP-MS coupled the mass spectrometry technique

directly after the Flag IP. The elimination of gel purification step allows

AP-MS to detect less abundant proteins, reduces sample loss from gel

extraction and avoids bias introduced from selecting stain bands for

analysis (17). Tables 4 and 5 contain all the potential candidates

identified in the AP-MS experiments; however the biological relevance of

these binding partners should be examined carefully for several

reasons. First, a previously reported PRL-2 binding partner RabGGTII is

not present in the results. The discrepancy can be accounted for by the

fact that RabGGTII’s interaction had only been found in an

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overexpressed system (79). Second, in comparison to gel extraction

techniques, direct LC MS/MS analysis generates more non-specific

contaminants.The higher background can potentially mask the identity of

low abundance proteins (17). Third, although mass spectrometry can

determine the presence/absence of a peptide, it cannot detect the

relative difference in peptide binding. Several PRLs interacting partners

(i.e. actin and keratin) were identified in the PRL-2 IP results, however,

they were excluded as potential candidates because they are also found

in the control Flag empty IP (Table 3).

A literature search revealed an interesting relationships between several

of these proteins, including PRL-1, CKII, 14-3-3, TLS and YBX1. PRL-1

trimerizes in vivo and trimerization is essential for its ability to increase

proliferation and migration (84). The sequence and structure

configuration required for trimerization in PRL-1 is conserved in PRL-2

and -3, suggesting that trimerization is a shared regulatory mechanism

for all PRLs (84). Our AP-MS results showed an interaction of PRL-2

and PRL-2CSDA with PRL-1. We propose that PRL-1,-2, and -3 form

hetero-oligomers in vivo, and that the oligomerization of different PRL

members may dictate diverse regulatory mechanisms or substrate

specificities. CKII is a serine/threonine kinase, and the phosphorylation

of PTEN by CKII lead’s to its proteasome-mediated degradation. All

PRLs contain consensus CKII phosphorylation sites, suggesting that

CKII can regulate PRL-2 by phosphorylation (2). PRL-2 interacts with

three (ε, ζ/σ) out of seven isoforms of 14-3-3. 14-3-3 binds to

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phosphorylated serine/threonine motifs on their target proteins (55). In

CDC25c, 14-3-3 binds and silences its NLS resulting in its cytoplasmic

retention (60). PRLs contain a polybasic region that functions as an

NLS, therefore, a 14-3-3 interaction might regulate the nuclear

localization of PRL-2 (84). YBX1 might be a downstream effector of

PRL-2. Similar to PRL-2, it regulates cell cycle progression, undergoes

nuclear localization and has elevated expression in breast and prostate

cancer (85). Interestingly, YBX1 also interacts with another PRL-2

interacting partner called TLS (also called FUS). YBX1 recruits the TLS

protein to pre-mRNA spliceosome machinery. Identification of both

proteins as PRL-2 interacting partners suggests PRL-2 is involved in the

RNA spliceosome complex (75). Pre-mRNA splicing is an essential

post-transcriptional RNA modification catalyzed by a large protein

complex spliceosome (12). snRNPs are important components of the

spliceosome; its biogenesis is mediated by the SMN complex containing

SMN, six Gemin proteins (Gemin2-7), and unrips (STRAP) (14)(15).

PRL-2 can be a component of the SMN complex, as PRL-2 was found

to interact with several components of this SMN complex, including

SMN, Gemin (3&6), and STRAP in our AP-MS results. In support of this,

previously published large scale mass spectrometry data also identified

STRAP and Gemin (3&4) as PRL-3 interacting partners (25) (Table 6).

SMN and Gemin3 subcellular localization is regulated by

dephosphorylation, suggesting that PRL-2 can be involved in the

regulation of the SMN complex (66).

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CNNM3 interaction with PRL-2 and the PRL family

PRL-2 interacts with all members of the CNNM family (Table 7). We

validated the interaction of PRL-2 with one of its members, CNNM3, in

both endogenous and overexpression systems. CNNM3 was also found

to interact with all PRLs, suggesting that this interaction can be an

important regulatory mechanism conserved for all PRLs. CNNM3

belongs to a family of ancient conserved domain proteins (ACDPs or

CNNMs). CNNMs are closely located to one another on chromosomes 2

and 10, they are also similar in length and size (Fig.24).

CNNMs are expressed ubiquitously in all tissues. The highest

expression for CNNM2 is in the brain, kidney and placenta, in the heart

and spleen for CNNM3, and in the heart for CNNM4 (91). Their

ubiquitous expression patterns, conservation in divergent species, and

presence of multiple members in one species warrants the functional

importance of the CNNM family (90). CNNM members are involved in

divalent metal transport. Their homologue in bacteria, Corc, is

implicated in Mg2+ and Co2+ homeostasis. In yeast, Amip3 is implicated

in resistance to copper toxicity and, in S. cerevisiae, MAM3 is a

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manganese resistant factor (80)(90)(98). CNMM members often

transport more than one type of metal; however, they all share their

greatest affinity for magnesium transport (5)(30)(80)(83)(98). CNNMs

physiological function was studied in different animal models. A

morpholino based expression knockdown of CNNM4 in zebrafish

showed defects in both heart and retinal ganglion cell development (72).

Also, mice placed on a low magnesium diet showed an up-regulation of

CNNM2 expression (30). The CNNMs family is clinically associated with

several diseases. All CNNMs were found by a genome-wide association

study to be involved in the regulation of magnesium homeostasis in

humans (53). CNNM1 is linked to urofacial syndrome (UFS) while

CNNM2 mutations result in dominant hypomagnesimia in humans

(5)(83). Lastly, mutations in CNNM4 cause Jalili syndrome, an

autosomal recessive disease of cone rod dystrophy (CRD) and

amelogenesis imeprfecta (AI) (32). The CNNMs family also has been

reported to be linked to cancer, CNNM1 is found to be hypermethylated

in melanoma cell lines (28). Although CNNM3 has been proposed to

share similar functions with other members of the family, their function

has not been studied. Our results therefore present the first insight into

the molecular function of CNNM3.

PRL-2 binds to CNNM3 at its CBS domains

All CNNM proteins contain four transmembrane and ACD domains. The

ACD domain is highly conserved within ACDPs, with approximately 92%

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amino acid similarity (Fig. 15). Its sequence is also well conserved

among species, from bacteria, yeast, C. elegans, Drosophila to

mammals (91). The ACD domain contains two cysthathionine-beta-

synthase (CBS) domains, a cyclin box and a cylic nucleotide-

monophosphate-binding domain (cNMP binding domain) (Fig.12). The

presence of the cyclin box motif is the reason ACDP was first thought to

belong to a family of cyclin proteins (91). Cyclin proteins use this 31

conserved sequence motif to interact with CDKs (Ser/Thr protein

kinase) (61). The cNMP binding domain containing proteins includes

cGMP/cAMP dependent protein kinase (PKG/PKA), and cyclic

nucleotide gated channels (CNG). We have shown that PRL-2 interacts

with CNNM3 at its CBS domains. Since its discovery in 1997, more than

4,000 CBS domains have been found from archaebacteria to

eukaryotes (8). CBS domains are found in a wide range of proteins, and

they carry out very different roles (Table 9). CBS domains are regulatory

domains and sensors of cellular energy status. They also play a role in

oligomerization and subcellular trafficking (36). CBS domains act as

regulatory subunits in AMPK. The binding of AMP to the CBS domain

releases autoinhibitory regions in the kinase domain, allowing AMPK to

bind and phosphorylate its substrate (76). In many cases, CBSs are

thought to be sensors of cellular energy status, binding AMP/ATP/ADP

(76). Binding of an adenine nucleotide to the CBS domain activates both

IMDPH and mtCBS PPase (36)(38). Interestingly, CBS domains are

important for subcellular localization of chloride channels (CLC).

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Truncation of CLC-5 CBS domains results in its retention in perinuclear

compartments instead of the acidic endosomes (36).

According to bioinformatics analysis, CBS dimers can interact with other

proteins. CLC5 containing a ‘PY’ motif between CBS dimmers

potentially interact with WW-domains of HECT-ubiquitin ligase (24). The

region between the CBS domains of IMPDH was proposed to bind

regulatory proteins (24). PRLs are the first protein identified to bind the

CBS domains.

Mutations in CBS domains result in several hereditary diseases,

including homocytstinuria (CBS protein), retinitis pigmentosa (IMPDH),

congenital myotonia (CLC-5), Wolf-Parkinson White syndrome (AMPKy)

and many more (Table 9). Interestingly, a single point mutation in

CNNM3 CBS domains completely abolished its binding to PRL-2

(Fig.17). G433D is a conserved residue in chloride channels (CLC). The

G433D mutation impedes CLC-2 ATP binding in vitro (76). Missense

mutations of this residue in CLC-1 results in muscular myotonia and

Albers-Schonberg disease in CLC-7, suggesting that this residue in

CLC CBS domains is critical for CLC function (73)(76). D396A is

another point mutation that dramatically affects the binding of PRL-2 to

CNNM3 CBS domains. D396A is a highly conserved residue within the

MgET CBS domain, a magnesium transporter closely related to the

CNNM family (34).

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Several crystal CBS crystal structures are available, which includes

IMPDH (35), MgET(37), OpuA(51), CBS protein(54) and CLC (52).

Although CBS domain alignment shared very low sequence identity,

they shared common structural properties and conserved tertiary

structures (32). Each CBS domain contains 3 β-sheets and 2 α-helices

(76). Two CBS domains interact with their beta strand to form a globular

structure with a deep hydrophobic cleft (76). Alternatively, the two

amphipathic alpha helices contribute to the binding surface within this

hydrophobic cleft (24). We modeled the CNNM3 CBS domain with the

published structure of CLC-5 CBS domains using Multiple Sequence

Alignment by CLUSTALW (52) (Fig.25). The alignment sequence shows

conservation of some amino acid sequences, in particular in the second

and third beta strand. The CNNM3 G433 mutation is located in the third

beta strand and it is conserved in all of the CBS domains compared

(Fig.25).

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PRL-2 catalytic domain mutations disrupts CNNM3 interaction

In the hope of better identifying PRL-2 substrates, we used the substrate

trapping mutant PRL-2CSDA in the AP-MS. The PRL-2 C101S mutation

allows binding of a substrate but blocks the catalysis, such that the

substrate is not released, thus enhancing the enzyme substrate

interaction (9). The D96A mutation stabilizes the WPD loop that comes

over the catalytic pocket to prevent the release of the substrate (9).

Considering the function of these mutants, if CNNM3 is a true substrate, a

stronger interaction should be detected in trapping mutants compared to

wildtype as illustrated in the interaction of PRL-3CS with NCL and KRT8

(57)(78). Alternatively, if CNNM3 is an interacting partner, binding of

CNNM3 to PRL-2CSDA should remain at the same level as wild type

PRL-2. Surprisingly, not only did the CSDA mutation not enhance the

CNNM3 interaction with PRL-2, it abolished the interaction completely.

The cysteine and aspartic acid are the two most important residues

required for PRL-2 catalysis, they are conserved in all PTPase and are

critical for phosphatase activity (9). C101 acts as a nucleophile to attack

the phosphorus centre of the substrate, and D96 acts as a general acid to

protonate the release of substrate (9). Additional PRL mutations showed

that the CS mutation alone completely abolished PRL-2 interaction and

that the DA mutation decreased the strength of the interaction. On the

other hand, AS and VT mutations did not affect CNNM3 binding at all.

PRL-2 A108 and V110 are two residues located close to the catalytic

motif, but they are not critical for PRLs catalytic function (Fig. 26). In fact,

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the presence of the A108 and V110 in place of S108 and T110

respectively is the reason why PRLs have much lower catalytic activity

compared to other PTPs (84). Our results suggest specific interactions of

CNNM3 to PRL-2 D96 and C101 residues, the two residues that are

critical for PRL phosphatase activity. Additional mutations within the WPD

and PTP catalytic motif will be used to confirm the specificity of this

interaction.

Model for CNNM3 PRL-2 interaction

The observation that CNNM3 binds only to wildtype PRL-2, but not the

trapping mutants, suggests that CNNM3 is not a substrate but a PRL-2

interacting partner. The lack of a regulatory domain in PRLs and their

remarkably low intrinsic phosphatase activity suggests that the PRLs are

regulated by their interacting proteins. Interacting proteins can act as

regulatory subunits to enhance PRLs phosphatase activity (22). Also,

PRLs activity may not depend on its phosphatase activity at all but rather

on its interaction with other proteins (59). The fact that CNNM3 is not a

substrate but binds to the catalytic domain of PRL-2 suggests its role as a

regulatory protein inhibiting PRL-2 phosphatase activity. I propose that

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CNNM3 CBS domains (G433) bind to the catalytic pocket of PRL-2 via

C101 and D69, preventing the binding of PRL-2 substrates, therefore

inhibiting PRL-2 phosphatase activity. To validate our model, we need to

confirm the direct interaction of PRL-2 with CNNM3 at its catalytic domain.

Several experiments can be carried out. First, direct interaction of PRL-2

and CNNM3 can be demonstrated in an in vitro binding system consisting

only of bacteria purified PRL-2 and CNNM3. Second, phosphatase activity

assays can measure the dosage dependent inhibition of PRL-2

dephosphorylation activity by CNNM3 binding or just CBS binding. Third,

we want to generate a crystal structure of the CBS domain in complex

with PRL-2. Finally, we will perform immunofluorescence studies to

confirm the co-localization of these proteins.

Although we have shown that PRL-2 only interacts with CBS domains of

CNNM3, several studies suggest that other domains of CNNM3 might play

role in regulating this interaction. First, CNNM4 missense mutations

outside the CBS domain are implicated in Jalili syndrome, suggesting that

other domains are essential for CNNM4 function (63). Second, the CBS

domain alone lacks substrate specificity. CBS domains of CLC channels

are able to functionally substitute one another as well as with the CBS

domain of IMPH2, suggesting that other domains might contribute to the

regulatory and/or substrate recognition function of the CBS domain (24).

The cyclic nucleotide binding domain is another interesting domain found

in CNNM3. cNMP binding domains in cyclic nucleotide-gated (CNG) ion

channels are modulated by cellular cAMP levels. Binding of cAMP to a

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cNMP binding site changes the conformation of the CBS domain,

activating the ion channels (77). It will be interesting to test if the addition

of cNMP in vitro will affect CNNM3 and PRL-2 interactions.

Physiological role of PRL-2

In this study, we successfully generated a knockout mouse model of PRL-

2. A significant reduction in body weight was detected in the PRL-2

knockout mouse compared to its wildtype counterpart, suggesting that

PRL-2 might play an important physiological role. Further analysis of the

PRL-2 KO mouse model will reveal the exact cause of this weight loss.

This model can also be used to study the mechanism of action of the PRL-

2 and CNNM3 interaction. Knockdown of PRL-2 in thymic tissues and

MEF did not affect the level of CNNM3, suggesting that PRL-2 does not

regulate CNNM3 protein levels. We will perform western blot analysis to

investigate the effect of PRL-2 knockdown on CNNM3 level in other tissue

samples. CNNM3 and PRL-2 interaction may be involved in the

phototransduction cascade in cone photoreceptors. CNNM4 mutations

cause cone-rod dystrophy (CRD), a disease associated with loss of cone

compared to rod photoreceptors (63). On the other hand, PRL-1 is found

to be a molecular component of the cone photoreceptor’s response to

oxidative stress. Its expression is preferentially localized to the cone

photoreceptor cell outer segment (100)(102). PRLs are normally

associated with the cytoplasmic face of plasma membranes therefore they

may function to modulate membrane channels or participate in the

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regulation of the phototransduction cascade (100). I anticipate that PRL-2

interacts with CNNM3, a metal transporter in the plasma membrane of

cone photoreceptors and that this interaction modulates the

phototransduction cascade. As mentioned above, the cNMP binding

domains are found in CNNM3 and CNG. CNGs are photoreceptors found

in the outer segment of rod photoreceptors, they are essential for the

generation of primary electrical signals in response to light. Binding of

cAMP to cNMP binding sites change the conformation of the CBS domain,

mediates the influx of calcium and sodium (77). Thus, we hypothesize that

cNMP might also regulate the interaction of PRL-2 and CNNM3 in cone

photoreceptors. Light elevates the cNMP level in the cell, increasing the

binding of cGMP which changes the conformation of the CBS domain,

releasing it from the catalytic domain of PRL-2 and activating its

phosphatase activity downstream (63). The first step to validate our

hypothesis will be investigate our knockout mouse models to see if there is

retinal degeneration, in particular any abnormality or loss of cone

photoreceptors. Second, we want to perform immunostaining of both PRL-

2 and CNNM3 to see if they co-localize to the same region in the

membrane of cone receptors. In addition, the CNNMs family also plays a

role in magnesium transport. CNNM2 mutations are associated with

dominant hypomangesmia, an abnormal urinary Mg2+ excretion. It would

will be interesting to measure the urinary content of Mg2+ in the PRL-2 KO

mouse. Mice kept at low magnesium diet have shown up-regulation of the

CNNM2 transcript, therefore we can also challenge our mouse with a low

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magnesium diet (94).

Conclusion

We have identified several putative candidates proteins that with PRL-2

using AP-MS. Furthermore, the interaction of PRL-2 with one of these

candidates: CNNM3 was validated using co-IP. We identified CNNM3 as

the first interacting partner shared by all PRL members and PRLs as the

first proteins that interacts with CBS domains. We characterized and

proposed a model for the function of the CNNM3 and PRL-2 interaction.

We also suggested their possible physiological function in cone

photoreceptors. In addition, we generated a PRL-2 knockout mouse

model and found significant reduction in their body weight, suggesting an

important physiological role of PRL-2. The identification of PRL-2 partners

will lead to a better understanding of its cellular mechanisms and the PRL-

2 KO mouse model will provide us with an excellent tool to study its

physiological function.

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