• PROTEIN KINASE A

• PHOSPHORYLATION AND ACTIVATION OF CREB TRANSCRIPTION FACTOR

• RECEPTOR SERINE KINASES - RECEPTORSFOR THE TGF-ß SUPERFAMILY

• PHOSPHORYLATION AND ACTIVATION OF SMAD TRANSCRIPTION FACTORS• PARTNERING WITH OTHER TRANSCRIPTION FACTORS TO ACTIVATE

TRANSCRIPTION OF SPECIFIC GENES

• PROTEIN- TYROSINE KINASE RECEPTORS

• RECEPTORS LINKED TO PROTEIN- TYROSINE KINASES - THE CYTOKINE RECEPTOR SUPERFAMILY

• RECEPTORS LINKED TO PROTEOSOME- MEDIATEDDEGRADATION OF INHIBITORS OF SPECIFIC TRANSCRIPTION FACTORS

SIGNALING FROM THE CELL SURFACE TO THE NUCLEUS

SIGNALING PATHWAYS LEADING TO ACTIVATION OF TRANSCRIPTION FACTORS AND MODULATION OF GENE EXPRESSION

FOLLOWING LIGAND BINDING TO CERTAIN GS PROTEIN–LINKED RECEPTORS

TGF-: Key Roles in Controlling Cell Proliferation and Synthesis of the

Extracellular Matrix

TGF-1

Hinck et al., (1996) Biochemistry

THE TGF- ß SUPERFAMILY INCLUDES TGF- ß1, TGF- ß2, TGF- ß3, ACTIVIN, INHIBIN, MULLERIAN INHIBITING SUBSTANCE,

AND AT LEAST 16 BONE MORPHOGENETIC PROTEINS

TGF-: Key Roles in Controlling Cell Proliferation and Synthesis of the

Extracellular Matrix

BIOLOGICAL FUNCTIONS OF TGF-ß INCLUDE:

• INHIBITION OF CELL PROLIFERATION• INDUCES INHIBITORS OF CYCLIN - DEPENDENT KINASES• TYPE II RECEPTOR FREQUENTLY LOST OR MUTATED IN CANCERS

• INDUCTION OF SYNTHESIS OF EXTRACELLULAR MATRIX PROTEINS: FIBRONECTIN, COLLAGENS, PROTEOGLYCANS

• INHIBITION OF SYNTHESIS OF EXTRACELLULAR PROTEASES:COLLAGENASE, PLASMINOGEN ACTIVATOR

• INDUCTION OF SYNTHESIS OF INHIBITORS OF EXTRACELLULAR PROTEASES

• PROMOTION OF CELL MATRIX AND CELL- CELL ATTACHMENT

Schematic diagram of formation of mature dimeric TGF proteins from secreted

monomeric TGF precursors.

TGF SIGNALS THROUGH HETEROMERIC COMPLEXES OF TYPES I AND II SERINE/THREONINE KINASE RECEPTORS, LEADING TO PHOSPHORYLATION OF EITHER SMAD2 OR SMAD3.

A COMPLEX OF ONE OF THESE PHOSPHORYLATED SMAD PROTEINS AND SMAD4 THEN TRANSLOCATES TO THE NUCLEUS, WHERE IT BINDS TO OTHER TRANSCRIPTION FACTORS TO ACTIVATE TRANSCRIPTION OF A VARIETY OF GENES

PPType IIReceptorsType IReceptorsTGF- CellMembraneNuclearMembrane

3Smad 4SmadPPP Gene activationTHE TGF

SIGNALING PATHWAY

PPType IIReceptorsType IReceptorsTGF- CellMembraneNuclearMembrane

3Smad 4SmadPPPmRNA3TFEP -1 PAIpromotermRNA( - Gene encoding cell cycle

)inhibitorP 15 p gene unknown transcription factors( Gene encoding inhibitor of protease

that degrades extracellular matrix)proteins

THE TGF SIGNALING PATHWAY

SMAD3 PROTEINS BIND ONLY TO 4 BASE PAIRS OF DNA: 5’ AGAC 3’

EACH TFE3 TRANSCRIPTION FACTOR BINDS TO A 3 BASE PAIR SEQUENCE 5’ CAC 3’

A DIMER OF TWO TFE3s BINDS TO A 6 BASE PAIR SEQUENCE 5’ CACGTG 3’ (GTG IS THE COMPLEMENT OF CAC)

THUS A SEQUENCE 5’ AGACxxxCACGTG 3’ BINDS ONE SMAD3 PROTEIN AND ONE TFE3 DIMER IN A PRECISE ARRANGEMENT, ALLOWING FOR TRANSCRIPTION ACTIVATION, IN THIS CASE OF THE PAI-1 GENE.

COMBINATORIAL ACTIVATION OF TRANSCRIPTION BY SMAD PROTEINS

QuickTime™ and aAnimation decompressor

are needed to see this picture.

Role of TGF- in Human Cancer

Human Diseases with Alterations in the TGF- Signaling Pathway

• RECEPTORS LINKED TO PROTEIN- TYROSINE KINASES - THE CYTOKINE RECEPTOR SUPERFAMILY

• PHOSPHOTYROSINE RESIDUES BINDING TO SPECIFIC SH2 DOMAINS• ACTIVATION OF STAT TRANSCRIPTION FACTORS• PARTNERING OF STATs WITH OTHER TRANSCRIPTION FACTORS• TERMINATION OF SIGNALING BY ACTIVATION OF PROTEIN

TYROSINE PHOSPHATASES • INHIBTION OF SIGNALING BY PROTEINS CONTAINING ONLY SH2 DOMAINS

• RECEPTORS LINKED TO PROTEOSOME- MEDIATEDDEGRADATION OF INHIBITORS OF CERTAIN TRANSCRIPTION FACTORS

SIGNALING FROM THE CELL SURFACE TO THE NUCLEUS

CFU-GEMMCFU-GMGranulocytesMonocytesPlateletsEosinophilsLymphoid ProgenitorCFU-MEGBFU-ECFU-ECFU-EoPluripotent Stem CellEpoEpo SCFGM-CSFIL-3IL-3, GM-CSF, SCFIL-6G-CSFM-CSFIL-3, GM-CSFIL-3, GM-CSFTPOErythrocytesSCF

EPO ACTS TO STIMULATETHE PROLIFERATION AND DIFFERENTIATION OF ERYTHROID PROGENITOR CELLS TO MATURERED CELLS

HEMATOPOIESIS

ERYTHROPOIETIN (EPO)THE PROTEIN THAT CONTROLS RED BLOOD CELL PRODUCTION

165 AMINO ACIDS

~ 40% CARBOHYDRATE

• PRODUCED BY THE KIDNEY IN RESPONSE TO LOW O2 PRESSURE IN THE BLOOD

• BINDS TO EPO RECEPTORS ON THE SURFACE OF ERYTHROCYTE PROGENITOR CELLS IN THE BONE MARROW

• STIMULATES THESE CELLS TO DIVIDE 5 TO 7 TIMES; EACH OF THE ~30 TO 100 DAUGHTERS THEN DIFFERENTIATES INTO A RED BLOOD CELL

• USED CLINICALLY TO TREAT ANEMIA CAUSED BY KIDNEY FAILURE OR BY DISEASES SUCH AS AIDS

Tissue pO2 Epo + - RED CELL MASS EPO REGULATES RED CELL MASS IN RESPONSE TO TISSUE HYPOXIA

EPO “GENE KNOCK- OUT” MICE ARE NORMAL EXCEPT THEY HAVE NO ADULT- TYPE RED

BLOOD CELLS AND DIE AT EMBRYONIC DAY 14

ExtracellularIntracellularCYTOKINE RECEPTOR SUPERFAMILYTWO CONSERVED DISULFIDE BONDSCONSERVED Trp- Ser- Xaa- Trp -Ser SEQUENCE

BOX 1 BINDING SITE FOR JAK NO ENZYMIC ACTIVITYIN CYTOSOLIC DOMAIN

CYTOKINE RECEPTORS THAT FORM HORMONE- INDUCED OR HORMONE-

STABILIZED HOMO-DIMERS:

• ERYTHROPOIETIN (EPO)

• GRANULOCYTE COLONY STIMULATING FACTOR • THROMBOPOIETIN (TPO)

• PROLACTIN (PRL)

• GROWTH HORMONE (GH)

SALES OF EPO AND G-CSF ARE IN EXCESS OF $4 AND $2 BILLION/

YEAR, RESPECTIVELY

STRUCTURE OF HUMAN GROWTH

HORMONE

LIKE EPO AND OTHER CYTIOKINES, GROWTH HORMONE FORMS A 4- ALPHA HELIX BUNDLE.

AMINO ACIDS THAT BIND TO THE FIRST GROWTH HORMONE RECEPTOR ARE IN GREEN; THOSE THAT BIND TO THE SECOND GROWTH HORMONE RECEPTOR ARE IN BLUE

STRUCTURE OF THE EXTERNAL SEGMENT

OF THE HUMAN GROWTH HORMONE

RECEPTOR

THE PLASMA MEMBRANE IS AT THE BOTTOM OF THE FIGURE

AMINO ACIDS THAT BIND GROWTH HORMONE ARE IN BLUE

AMINO ACIDS THAT BIND THE SECOND MOLECULE OF GROWTH HORMONE RECEPTOR ARE IN GREEN

THREE- DIMENSIONAL STRUCTURE OF THE COMPLEX OF ONE MOLECULE OF HUMAN GROWTH HORMONE

AND TWO GROWTH HORMONE RECEPTORS

PLASMA MEMBRANE IS AT THE BOTTOM OF THE FIGURE

KINASE DOMAINN-TERMINALCONSERVEDMODULATORYTYROSINE KINASE130 kDa CYTOSOLIC PROTEIN TYROSINE KINASE

HOMOLOGOUS TO JAK1 AND TYK2

WIDELY EXPRESSED IN HEMATOPOIETIC CELLS AND FIBROBLASTS

NO SH2 OR SH3 DOMAINS

JAK2PSEUDO-KINASE DOMAIN

68 kDa CYTOSOLIC PROTEIN TYROSINE PHOSPHATASE

EXPRESSED ONLY IN HEMATOPOIETIC CELLS

MUTATED IN Motheaten MICE

SH2SHP1SH2CATALYTIC DOMAIN

SIGNAL TRANSDUCTION PROTEINS THAT BIND TO THE CYTOSOLIC DOMAIN

OF THE ERYTHROPOIETIN RECEPTOR

JAK 2JAK 2EPOJAK 2PP EPO- INDUCED RECEPTOR DIMERIZATION;

TRANS-PHOSPHORYLATION OF JAK2

JAK 2JAK 2EPOJAK 2PP EPO- INDUCED CONFORMATIONAL CHANGE OF

DIMERIC RECEPTOR; TRANS-PHOSPHORYLATION

OF JAK2

JAK 2

TWO POSSIBLE MECHANISMS

BY WHICH EPO ACTIVATES

THE EPO RECEPTOR

ACTIVATED JAK2 PHOSPHORYLATES UP TO 8TYROSINE RESIDUES ON THE CYTOSOLOC DOMAINOF THE EPO RECEPTOR. EACH PHOSPHOTYROSINE

CAN FORM THE “DOCKING SITE” FOR THE SH2DOMAIN OF A SIGNAL TRANSDUCTION PROTEIN

SIGNAL TRANSDUCTION BY THE EPO RECEPTOR

IN THIS TARGET PEPTIDE, THE PHOSPHOTYROSINE (P-TYR) AND ISOLEUCINE (3ILE) FIT INTO A TWO-PRONGED SOCKET ON THE SURFACE OF THE SH2 DOMAIN. THE PHOSPHATE GROUP COVALENTLY ATTACHED TO THE TYROSINE RESIDUE

IS LIGHT BLUE.

MODEL OF AN SH2 DOMAIN BOUND TO A

SHORT TARGET PEPTIDE. 

DIMERIZATION OF STAT PROTEINS LEADS TO FORMATION OF A FUNCTIONALLY

ACTIVE TRANSCRIPTION FACTOR

TERMINATION OF SIGNAL TRANSDUCTION

BY THE EPO RECEPTOR

TERMINATION OF SIGNAL TRANSDUCTION BY THE EPO RECEPTOR #2

AS WITH THE EPO RECEPTOR, LIGAND BINDING INDUCES A CONFORMATIONAL CHANGE THAT PROMOTES OR STABILIZES RECEPTOR DIMERS.

THE KINASE ACTIVITY OF EACH SUBUNIT OF THE DIMERIC RECEPTOR INITIALLY PHOSPHORYLATES TYROSINE RESIDUES NEAR THE CATALYTIC SITE IN THE OTHER SUBUNIT, CAUSING ITS ACTIVATION.

SUBSEQUENTLY, TYROSINE RESIDUES IN OTHER PARTS OF THE CYTOSOLIC DOMAIN BECOME PHOSPHORYLATED AND SERVE AS DOCKING SITES FOR SH2 DOMAINS OF SIGNALING PROTEINS

GENERAL STRUCTURE AND ACTIVATION OF RECEPTOR TYROSINE KINASES (RTKS)

Structure of the FGF - FGR Receptor Complex

Structures of MAP kinase in its inactive, unphosphorylated form and active,

phosphorylated form Phosphorylation of MAP kinase by MEK at tyrosine 185 (pY185) and threonine 183

(pT183) leads to a marked conformational change in the phosphorylation lip (red).

Cycling of the Ras protein between the inactive form

with bound GDP and the active form with bound GTP  

Activation of Ras following binding

of a ligand to a RTK

Model of SH3 domain bound to a short target peptide 

 Kinase cascade that transmits

signals downstream

from activated Ras protein

Signaling pathways leading to activation of transcription factors and modulation of gene

expression following ligand binding to RTKs

Activation of protein kinase B

by the PI- 3’ kinase signaling

pathway (part 1). 

Activation of protein kinase B by the PI- 3’ kinase signaling pathway (part 2). 

Cleavage of PIP2 by phospholipase C (PLC)

yields DAG and IP3.

Elevation of cytosolic Ca2 via the inositol-lipid signaling pathway

A CONJUGATING ENZYME CATALYZES FORMATION OF A PEPTIDE BOND BETWEEN THE SMALL PROTEIN UBIQUITIN (UB) AND THE SIDE-CHAIN –NH2 OF A LYSINE RESIDUE IN A TARGET PROTEIN.

ADDITIONAL UB MOLECULES ARE ADDED, FORMING A MULTIUBIQUITIN CHAIN.

THIS CHAIN DIRECTS THE TAGGED PROTEIN TO A PROTEASOME, WHICH CLEAVES THE PROTEIN INTO NUMEROUS SMALL PEPTIDE FRAGMENTS.

PROTEOLYSIS OF UBIQUITIN-TAGGED PROTEINS OCCURS ALONG THE INNER WALL

OF THE CORE.

UBIQUITIN-MEDIATED PATHWAY FOR DEGRADATION

OF CELLULAR PROTEINS

MANY DIFFERENT EXTRACELLULAR SIGNALS CAN INDUCE ACTIVATION OF NF-B; THESE SIGNALS ACTIVATE AN I-B KINASE COMPLEX.

THIS COMPLEX PHOSPHORYLATES TWO N-TERMINAL SERINE RESIDUES IN I-B. PHOSPHORYLATED I-B IS UBIQUITINATED AND SUBSEQUENTLY DEGRADED BY THE PROTEOSOME. REMOVAL OF I-B UNMASKS THE NUCLEAR LOCALIZATION SITES IN BOTH THE P50 AND P65 SUBUNITS OF NF-B. NF-B ENTERS THE NUCLEUS, BINDS TO SPECIFIC SEQUENCES IN DNA AND REGULATES TRANSCRIPTION.

ACTIVATION OF THE TRANSCRIPTION

FACTOR NF-B