1. SIGNALING FROM THE CELL SURFACE TO THE NUCLEUS
• PROTEIN KINASE A
• PHOSPHORYLATION AND ACTIVATION OF CREB TRANSCRIPTION FACTOR
• RECEPTOR SERINE KINASES - RECEPTORS
FOR 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- MEDIATED
DEGRADATION OF INHIBITORS OF SPECIFIC
TRANSCRIPTION 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 the
Extracellular Matrix
TGF-b1
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
Important 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 OF
EXTRACELLULAR 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 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

7. THE TGFb SIGNALING PATHWAY

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- 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- MEDIATED
DEGRADATION OF INHIBITORS OF
CERTAIN TRANSCRIPTION FACTORS

13. HEMATOPOIESIS EPO ACTS TO STIMULATE THE PROLIFERATION
AND DIFFERENTIATION
OF ERYTHROID
PROGENITOR
CELLS TO MATURE
RED CELLS

16. EPO “GENE KNOCK- OUT” MICE ARE NORMAL EXCEPT THEY HAVE NO ADULT- TYPE RED BLOOD CELLS AND DIE AT EMBRYONIC DAY 14

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 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

21. 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

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 8
TYROSINE RESIDUES ON THE CYTOSOLOC DOMAIN
OF THE EPO RECEPTOR. EACH PHOSPHOTYROSINE
CAN FORM THE “DOCKING SITE” FOR THE SH2
DOMAIN 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 SH2
DOMAIN. THE PHOSPHATE
GROUP COVALENTLY
ATTACHED TO THE
TYROSINE RESIDUE
IS 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 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

30. Structure of the FGF - FGR Receptor Complex

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 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.

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.