Control of Cell Proliferation by Peptide Growth Factors Autocrine Growth Factor Production Causes Malignant Transformation?
Transforming Activities From Condition Media from a Tumor Cell Line Condition Media from the Moloney MuSV transformed 3T3 cell line Normal NRK Cells Tumor NRK cells Todaro and De Larco PNAS 1978
Transforming Activities From Condition Media from a Tumor Cell Line SGF: sarcoma growth factor Todaro and De Larco PNAS 1978
Transforming Activities is Due to TGF-beta Moloney MuSV transformed 3T3 Cell Pellet Acid-ethanol extraction Gel filtration HPLC TGF-α EGF TGF-β + + EGF NO colony formation Stimulate colony formation Anzano, M. et al. (1982) Cancer Res. 42:4776
Paradoxical Activity of TGF-β Effects of TGF-ß on the anchorage-independent growth of Myc-1 cells. In the presence of PDGF, TGF-ß stimulates growth (A) and in the presence of EGF, TGF-ß inhibits growth (B). Roberts, A. et al. (1985) Proc. Natl. Acad. Sci. USA 82:119.
Purification of Bone Inducing Factors
Purification of Bone Inducing Factors CIF-A = TGF-beta 1 CIF-B = TGF-beta 2
Purification of Bone Inducing Factors SCIENCES VOL. 242
SCIENCES VOL. 242 Purification of BMPs
TGF-β Structure and Function furin cystine knot motif White balls cystine residues and disulfide bonds Daopin, 1992. Science 257: 369-373
TGF-β Maturation Khalil Microbes Infect. 1999
Massague Ann Rev Biochem 1998
Massague Ann Rev Biochem 1998
TGF-beta Binds to Cell Surface Receptors TGF-beta was labeled with radioactive I 125 Type III Type II Type I
TGF-beta Inhibits Normal Cell Proliferation 0pM 1pM 5pM 10pM 25pM 50pM
Isolation of TGF-beta Resistance Mutant Cells Mink lung epithelial Cells 25 pm TGF-beta 7 1.6X10 cells treated with EMS (ethyl methansulfonate) for 24 hr 40% killing rate TGF-beta Resistance Clonies
Cell Proliferation Assay in the Presence of TGF-β Laiho M, 1990 JBC
Some of the Mutant Cell Lines Show Abnormal TGF-b Receptor Expression Laiho M, 1990 JBC
Genetic Screening of the Cell lines Lacking of TGF-beta Receptors Three types of cell clones: -R cells: Loss of Type I receptor binding -DR Cells: loss of both Type I and Type II binding -S Cells: Receptor binding normal but no signaling Why there are no mutant cell clones in which the type I receptors are normal but lacking the type II receptors? Laiho M, 1990 JBC
TGF-beta Receptor Signaling Mechanism Laiho M, 1990 JBC
Expression Cloning of TGF-beta Receptors Transient transfection of COS cells Ligand binding Isolating positive cells Recovering plasmid DNA encoding gene of interest Lin and Lodish 1991 Cell 755-785
Cloning of TGF-beta I Receptor Franze et al. Cell 75, 681-92
TGF-beta Receptor Signaling Modeling
Type I and II TGF-beta receptor families Massague Ann Rev Biochem 1998
Functional Reconstitution of TGF-beta Signaling Wrana and Massague 1992 Cell
Reporter Gene Assay for Studying TGF-beta Signaling Wrana and Massague 1992 Cell
TβTRI Kinase Complexed with Inhibitor FKBP12
Decapentaplegic (dpp) Wild type Class I Class II Class III
Identification and Cloning of dpp Downstream Targets Wild Type dpp/maddpp dpp/mad+ Loss of function mutations of Mad are dominant maternal effect enhancers of dpp during early embryogenesis and dominant zygotic enhancers of dpp in imaginal discs Newfeld et al., 1995
Phosphorylation of Smad3 by TGF-beta RI at the C-terminus SSXS Motif Liu et al. PNAS 1997
TGF-β-dependent &Independent Smad2 Phosphorylation Mv1Lu wt R1B (TβRI Defective) TGFβ Nocodazole TGFβ Nocodazole - + 12 15 20 - + 12 15 20 Mps1
Phosphorylation of R-Smads by TGF-beta Receptor Kinase Smad1 Smad5 Smad8 Smad2 Smad3 Smad4 Smad6 Smad7 BMP Signaling TGF-β and Activin Signaling TGF-β/Activin/BMP Signaling TGF-β/Activin/BMP? R-Smads Co-Smad Inhibitory-Smad
Smads Are DNA Binding Proteins
Reporter Gene Assays for Studying TGF-beta Signaling SBE: Smad Binding Element 5 -GTCTAGAC-3
TβRI TβRII Cytoplasmic retention factor Smad2/3 P Phosphorylation Smad2/3? Smad2/3 P Smad4 Smad4 Smad2/3 Nuclear retention factor Smad2/3 P Smad4 Smad4 Smad2/3 P TFs Smad4 Transcription Smad2/3 Dephosphorylation
Transcription Activation and Repression by TGF-beta Derynck and Zhang Nature 2002
33% ovarian cancer TβRI TβRII 10-15% colon cancer Smad2 Smad2 P <2% colon cancer 50% pancreatic cancer 20-30% colon cancer Smad4 Smad3 Smad3 P Smad2 P Smad3 P Smad4 TFs Smad2 P Smad3 P Smad4 Transcription Massague et al. Cell 2000
Nucleocytoplasmic Shuttling of Smads 2, 3, and 4 Permits Sensing of TGF- Receptor Activity Gareth J. Inman, Francisco J. Nicola s and Caroline S. Hill
What is p-smad2? How to detect it? Why is cycloheximide used?
What was the inhibitor added at different time points?
TGF-beta receptors remain active for at least 3 4 hr in HaCaT cells
What does this experiment tell us about Smad2 and Smad4 in relationship to receptor activity?
What happened to P-Smad2 upon treatment with SB-431542?
Why there is less Smad4 in Smad2 IP samples treated with SB- 431542?
What accounts for low nuclear staining of Smad2/3 in the presence of SB-431542?
What is LMB? What does it do? What does this experiment tell us?
Domain Organization of Smad
Phosphorylation of Smad1
Protease Digestion of Labeled Smad1
Protease Digestion of Labeled Smad1
Phosphorylation of Smad1 Why the predicted digestion pattern of Smad1 allowed us to identify the phosphorylated region as part of the linker region of Smad1?
Chymotrypsin cleaves peptides at the carboxyl side of tyrosine, tryptophan, and phenylalanine
EGF induced Smad1 Phosphorylation
EGF induced Smad1 Phosphorylation at the Linker Region
HGF induced Smad1 Phosphorylation
In vitro Phosphorylation of Smad1 by Erk2 and BMPR1
Smad1 linker has potential MAPK sites Serines were mutated to alanine to prevent MAPK-induced phosphorylation
EGF-induced phosphorylation of the Smad1 linker region does not prevent Smad1/Smad4 complex formation RTK block in BMP signaling is downstream of Smad1/Smad4 complex formation
Phosphorylation of Linker Region Represses Smad1
BMP induces nuclear accumulation of Smad1 No treatment BMP BMP + EGF BMP/EGF + U0126 U0126 Smad-linker mutant EGF-induced phosphorylation of Smad inhibits nuclear accumulation induced by BMP
Phosphorylation of Linker Region Represses Smad1
MAPK phosphorylation of Smad1 prevents translocation of Smad1/Smad4 complexes BMP target gene expression is blocked due to a block in Smad1-Smad4 nuclear translocation
BMP signaling is regulated at multiple steps in the pathway Extracellular Intracellular