Presentation on theme: "SIGNALING FROM THE CELL SURFACE TO THE NUCLEUS"— Presentation transcript:
1 SIGNALING FROM THE CELL SURFACE TO THE NUCLEUS • 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 ACTIVATETRANSCRIPTION OF SPECIFIC GENES• PROTEIN- TYROSINE KINASE RECEPTORS• RECEPTORS LINKED TO PROTEIN- TYROSINEKINASES - THE CYTOKINE RECEPTOR SUPERFAMILY• RECEPTORS LINKED TO PROTEOSOME- MEDIATEDDEGRADATION OF INHIBITORS OF SPECIFICTRANSCRIPTION FACTORS
2 SIGNALING PATHWAYS LEADING TO ACTIVATION OF TRANSCRIPTION FACTORS AND MODULATION OF GENE EXPRESSION FOLLOWING LIGAND BINDING TO CERTAIN GS PROTEIN–LINKED RECEPTORS
3 TGF-b: Key Roles in Controlling Cell Proliferation and Synthesis of theExtracellular MatrixTGF-b1Hinck et al., (1996) BiochemistryTHE TGF- ß SUPERFAMILY INCLUDESTGF- ß1, TGF- ß2, TGF- ß3, ACTIVIN, INHIBIN, MULLERIAN INHIBITINGSUBSTANCE,AND AT LEAST 16BONE MORPHOGENETIC PROTEINSImportant Points:I would like to begin by first thanking the Program in Cell Biology, and in particular, members of the search committee for inviting me here today. I’ve enjoyed myself immensely on this visit, and have had my interesting discussions, both with the faculty and the students. And for that, I’m very grateful. I’m also thankful to have the opportunity to describe the findings of my studies which are aimed at elucidating the molecular mechanisms underlying TGF-b signaling in normal and diseased cells.
4 TGF-b: 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 OFEXTRACELLULAR PROTEASES• PROMOTION OF CELL MATRIX AND CELL- CELL ATTACHMENT
5 Schematic diagram of formation of mature dimeric TGFb proteins from secreted monomeric TGFb precursors.
6 THE TGFb SIGNALING PATHWAY TGFb SIGNALS THROUGHHETEROMERICCOMPLEXES OFTYPES I AND IISERINE/THREONINEKINASE RECEPTORS,LEADING TOPHOSPHORYLATION OFEITHER SMAD2 OR SMAD3.A COMPLEX OF ONEOF THESE PHOSPHORYLATEDSMAD PROTEINS AND SMAD4THEN TRANSLOCATESTO THE NUCLEUS,WHERE IT BINDSTO OTHER TRANSCRIPTIONFACTORS TO ACTIVATETRANSCRIPTIONOF A VARIETY OF GENES
8 COMBINATORIAL ACTIVATION OF TRANSCRIPTION BY SMAD PROTEINS 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.
10 Role of TGF-b in Human Cancer Important Points:So what then is the role of TGF-b in Human Cancer???The biological activities of TGF-b can be divided into three major categories, namely its ability to (1) inhibit the proliferation of epithelial and endothelial lineages; (2) regulate ECM remodeling; and (3) govern immunosuppression by inhibiting lymphocyte activation and proliferation, or by stimulating their apoptosis.Under normal conditions, TGF-b is one of the body’s primary defenses against uncontrolled cell growth. Ligation of its receptors prevents cell cycle progression by applying the brakes during mid-to-late G1 of the cell cycle. Thus, TGF-b and its signaling components function as tumor suppressor genes. However, following mutation or loss of signaling components within the TGF-b signaling pathway, ligation of TGF-b receptors promotes, rather than suppresses, carcinogenesis. This paradoxical situation is depicted here:Panel 1: Under normal circumstances, cells in tissues will not divide unless ordered to do so. These orders are communicated through extracellular signals that direct their entry into the cell cycle -- these signals can be mediated and communicated to a cell through soluble growth factors, alterations in the ECM, or through direct cell-cell contact. Just describe what TGF-b does to cells in G1 -- Basically, TGF-b induces either growth arrest or can stimulate cellular differentiation. With respect to growth arrest, TGF-b prominently downregulates MYC; inhibits synthesis of CDK4 and CDK6 and inhibits their activities via upregulation of CDKIs, most notably p15/p16. The net effect of these events result in the hypophosphosphorylation of Rb and the sequestration of E2F family members.Mutations in this pathway allow cells to escape the negative growth constraints imposed upon cells by TGF-b, thereby allowing them to proliferate even in its presence.Finally, additional mutations allow cells to acquire the ability to invade the surrounding stroma and metastasize to distant locales.It is the later two events where TGF-b will actually drive the process of carcinogenesis. Although no longer sensitive to growth inhibition, many tumor cells retain their ability to respond to TGF-b, and as such, these cells actively secrete TGF-b-inducible growth factors, proteins that stimulate invasion and metastasis, angiogenic factors, and suppress the activities of infiltrating immune cells.
11 Human Diseases with Alterations in the TGF-b Signaling Pathway Important Points:The list of diseases mediated by TGF-b is quite extensive, and unfortunately promises to grow as the molecular mechanisms underlying more and more human pathologies are uncovered.Point out:A) involvement of TGF-b in fibrosis, hypertension, osteoporosis, and atherosclerosis.B) mutation of type II receptor has been detected in atherosclerotic lesions.C) point out that even after extensive surveying of a variety of human tumors, no mutations in Smad3 have been detected to date.While a number of studies have clearly shown that the genes for type II receptor and Smads 2 and 4 are tumor suppressor genes, no such data was available regarding the type I receptor and its role in human cancers. Several papers provided descriptive data suggesting that cells obtained from pancreatic, cervical, breast, biliary, and B-cll might house alterations or mutations within the type I receptor gene; however, no mutations were actually described in these reports.
12 SIGNALING FROM THE CELL SURFACE TO THE NUCLEUS • RECEPTORS LINKED TO PROTEIN- TYROSINEKINASES - 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 PROTEINTYROSINE PHOSPHATASES• INHIBTION OF SIGNALING BY PROTEINS CONTAINING ONLY SH2 DOMAINS• RECEPTORS LINKED TO PROTEOSOME- MEDIATEDDEGRADATION OF INHIBITORS OFCERTAIN TRANSCRIPTION FACTORS
13 HEMATOPOIESIS EPO ACTS TO STIMULATE THE PROLIFERATION AND DIFFERENTIATIONOF ERYTHROIDPROGENITORCELLS TO MATURERED CELLS
19 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
20 STRUCTURE OF THE EXTERNAL SEGMENT OF THE HUMAN GROWTH HORMONE RECEPTOR THE PLASMA MEMBRANE IS ATTHE BOTTOM OF THE FIGUREAMINO ACIDS THAT BINDGROWTH HORMONE ARE IN BLUEAMINO ACIDS THAT BIND THESECOND MOLECULE OFGROWTH HORMONE RECEPTORARE IN GREEN
21 THREE- DIMENSIONAL STRUCTURE OF THE COMPLEX OF ONE MOLECULE OF HUMAN GROWTH HORMONE AND TWO GROWTH HORMONE RECEPTORSPLASMA MEMBRANEIS AT THE BOTTOMOF THE FIGURE
22 SIGNAL TRANSDUCTION PROTEINS THAT BIND TO THE CYTOSOLIC DOMAIN OF THE ERYTHROPOIETIN RECEPTOR
23 TWO POSSIBLE MECHANISMS BY WHICH EPO ACTIVATES THE EPO RECEPTOR
24 SIGNAL TRANSDUCTION BY THE EPO RECEPTOR ACTIVATED JAK2 PHOSPHORYLATES UP TO 8TYROSINE RESIDUES ON THE CYTOSOLOC DOMAINOF THE EPO RECEPTOR. EACH PHOSPHOTYROSINECAN FORM THE “DOCKING SITE” FOR THE SH2DOMAIN OF A SIGNAL TRANSDUCTION PROTEIN
25 MODEL OF AN SH2 DOMAIN BOUND TO A SHORT TARGET PEPTIDE. IN THIS TARGET PEPTIDE,THE PHOSPHOTYROSINE(P-TYR) AND ISOLEUCINE(+3ILE) FIT INTO A TWO-PRONGED SOCKET ON THE SURFACE OF THE SH2DOMAIN. THE PHOSPHATEGROUP COVALENTLYATTACHED TO THETYROSINE RESIDUEIS LIGHT BLUE.
26 DIMERIZATION OF STAT PROTEINS LEADS TO FORMATION OF A FUNCTIONALLY ACTIVE TRANSCRIPTION FACTOR
27 TERMINATION OF SIGNAL TRANSDUCTION BY THE EPO RECEPTOR
28 TERMINATION OF SIGNAL TRANSDUCTION BY THE EPO RECEPTOR #2
29 GENERAL STRUCTURE AND ACTIVATION OF RECEPTOR TYROSINE KINASES (RTKS) AS WITH THE EPO RECEPTOR,LIGAND BINDING INDUCES ACONFORMATIONAL CHANGETHAT PROMOTES OR STABILIZESRECEPTOR DIMERS.THE KINASE ACTIVITY OF EACHSUBUNIT OF THE DIMERICRECEPTOR INITIALLYPHOSPHORYLATES TYROSINERESIDUES NEAR THE CATALYTICSITE IN THE OTHER SUBUNIT,CAUSING ITS ACTIVATION.SUBSEQUENTLY, TYROSINERESIDUES IN OTHER PARTS OF THECYTOSOLIC DOMAIN BECOMEPHOSPHORYLATED AND SERVEAS DOCKING SITES FOR SH2DOMAINS OF SIGNALING PROTEINS
34 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).
35 Cycling of the Ras protein between the inactive form with bound GDP and the active form with bound GTP
36 Activation of Ras following binding of a ligand to a RTK
37 Model of SH3 domain bound to a short target peptide
38 Kinase cascade that transmits signals downstream from activated Ras protein
39 Signaling pathways leading to activation of transcription factors and modulation of gene expression following ligand binding to RTKs
40 Activation of protein kinase B by the PI- 3’ kinase signaling pathway (part 1).
41 Activation of protein kinase B by the PI- 3’ kinase signaling pathway (part 2).
42 Cleavage of PIP2 by phospholipase C (PLC) yields DAG and IP3.
43 Elevation of cytosolic Ca2+ via the inositol-lipid signaling pathway
44 UBIQUITIN-MEDIATED PATHWAY FOR DEGRADATION OF CELLULAR PROTEINS A CONJUGATING ENZYMECATALYZES FORMATIONOF A PEPTIDE BONDBETWEEN THE SMALLPROTEIN UBIQUITIN (UB)AND THE SIDE-CHAIN –NH2OF A LYSINE RESIDUE INA TARGET PROTEIN.ADDITIONAL UB MOLECULESARE ADDED, FORMING AMULTIUBIQUITIN CHAIN.THIS CHAIN DIRECTS THETAGGED PROTEIN TO APROTEASOME, WHICHCLEAVES THE PROTEIN INTONUMEROUS SMALL PEPTIDEFRAGMENTS.PROTEOLYSIS OF UBIQUITIN-TAGGED PROTEINS OCCURSALONG THE INNER WALLOF THE CORE.
45 ACTIVATION OF THE TRANSCRIPTION FACTOR NF-kB MANY DIFFERENT EXTRACELLULAR SIGNALS CAN INDUCE ACTIVATION OF NF-kB; THESE SIGNALS ACTIVATE AN I-kB KINASE COMPLEX.THIS COMPLEX PHOSPHORYLATES TWO N-TERMINAL SERINE RESIDUES IN I-kB. PHOSPHORYLATED I-kB IS UBIQUITINATED AND SUBSEQUENTLY DEGRADED BY THE PROTEOSOME. REMOVAL OF I-kB UNMASKS THE NUCLEAR LOCALIZATION SITES IN BOTH THE P50 AND P65 SUBUNITS OF NF-kB. NF-kB ENTERS THE NUCLEUS, BINDS TO SPECIFIC SEQUENCES IN DNA AND REGULATES TRANSCRIPTION.