Lecture 4 February 11, 2008

1. Lecture 4 February 11, 2008 Phosphorylation and nuclear translocation of signal transducers by ligand-activated receptor kinases Cell Structure, Signaling and Differentiation Winter Quarter, 2008 Gerry Weinmaster 390A Basic Science Research Building gweinmaster@mednet.ucla.edu

2. TGF-b superfamily signaling is regulated at multiple levels and activates multiple genes

3. TGF-b binds to receptor serine kinases that phosphorylate Smad signal transducers Phosphorylated Smads translocate to the nucleus to directly activate TGF target genes

4. TGF-b is synthesized and secreted as a inactive precursor that requires both proteolytic cleavage and removal of the latency associated peptide (LAP) for active dimer formation RXXR The active TGF-b dimer contains monomers with 7 highly conserved cysteine residues, 6 of which form the characteristic cysteine knot and the 7th in each monomer disulfide bond to form the dimer

5. Ligand traps directly bind BMPs to block specific ligand-receptor interactions required for downstream signaling

6. Soluble extracellular proteins that directly bind ligand and prevent receptor activation

7. Signaling by TGF-b can also be modulated by interactions with the Type III receptors Betaglycan and Endoglin Signaling

8. The decoy receptor BAMBI incorporates into ligand-receptor complexes to prevent activation of type I receptors

9. FKBP12 maintains the receptor in an inactive state in the absence of ligand Type 1 receptor Smad Ligand-induced receptor phosphorylation antagonizes FKBP binding and creates a Smad binding site

10. TGF-b binding to kinase active R-II recruits and phosphorylates the kinase inactive R-I Phosphorylation activates the R-I kinase activity

11. TGF-b-induced phosphorylation of R-I creates a binding site for Smads Phosphorylation of Smads leads to release from the activated receptor and subsequent unfolding and oligomerization of phosphorylated Smads

12. R-Smads only transiently interact with type I receptors following activation by type II receptors P KD I P Flag co-IP KD No Flag co-IP No Flag co-IP Smads only associate with R-I that has been phosphorylated by R-II R-I/Smad interaction produces phosphorylated Smad Phosphorylated Smad is released from the activated R-I

13. Pathway-restricted Smads are phosphorylated on conserved C-terminal serine residues

14. In response to ligand binding phosphorylated R-Smad complexes form heteromeric complexes with the common partner Smad4

15. Smad2/Smad4 complexes translocate to the nucleus to effect gene transcription

16. Activated Smads in the nucleus associate with DNA binding cofactors as well as activators or repressors of transcription

17. I-Smads compete with R-Smads for binding to activated type I receptors I-Smads cannot be phosphorylated and thus remain associated with type I receptors

18. I-Smads block R-Smad interactions with activated type I receptors and Co-Smads

19. FGF8 and IGF2 also expand the neural plate as found for Chordin and Noggin Suggestive of a block in BMP signaling

20. How could FGF8 and IGF2 block BMP signaling? Putative MAPK sites within the linker region of Smad1 could be phosphorylated by activation of FGF signaling

21. Smad1 linker has potential MAPK sites Serines were mutated to alanine to prevent FGF-induced phosphorylation

22. Mutation of linker phosphorylation sites prevents FGF-induced phosphorylation of Smad1 But Erks are still phosphorylated in response to FGF treatment

23. The MEK inhibitor U0126 prevents FGF8-induced phosphorylation of Erk and Smad1 Identifies MEK as the kinase responsible for phosphorylation of the Smad1 linker

24. Regulation of neural differentiation through RTK and BMP induced Smad phosphorylation How does MAPK phosphorylation of the linker region block Smad1 signaling?

25. 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

26. BMP induces nuclear accumulation of Smad No treatment BMP BMP + EGF Smad-linker mutant U0126 BMP/EGF + U0126 EGF-induced phosphorylation of Smad inhibits nuclear accumulation induced by BMP

27. 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

28. Fuentealba et al, Cell 131, 2007 (Eddy De Robertis’ Lab)

29. BMP signaling is regulated at multiple steps in the pathway Extracellular Intracellular