DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly

1. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly Larisa Litovchick, Laurence A. Florens, Selene K. Swanson, Michael P. Washburn and James A. DeCaprio Dana-Farber Cancer Institute, Boston Brigham and Women’s Hospital, Harvard Medical School, Boston Stowers Institute, Kansas City University of Kansas Medical Center, Kansas City Genes and Development 25:801-813 April 15, 2011

2. RB and E2F Families Rb, p107 and p130 with E2Fs control the Restriction Point

3. RB and E2F Families Rb, p107 and p130 with E2Fs control the Restriction Point

4. RB and E2F Families Rb, p107 and p130 with E2Fs control the Restriction Point

5. Protein Complexes + = MuvB Core Complex Essentially a Co-repressor DREAM Complex (DP, RB-like, E2F4, and MuvB) Represses transcription to hold cells in G0

6. Protein Complexes + = OR DREAMComplex + = Myb Protein (transcription factor) MMB Complex S phase MuvB Core Complex

7. Myb Family of Leucine Zipper transcription factors c-myb (and v-myb), A-Myb and B-Myb Consensus enhancer is 5’-YAACGC-3’ Targets include: Cyclin D1 Cdk1 Myc Bcl-2 B-Myb

8. Myb of B-Myb

9. Protein Complexes What controls which complex MuvB core ends up in? Model:

10. Protein Complexes What controls which complex MuvB core ends up in? Isolate the complexes by immunoprecipitations a-p130 HA-tagged p130 a-LIN52 V5-tagged LIN52 a-BMYB Some cells were cycling, some in G0 some in S Identify co-precipitating proteins DREAMComplex MMB Complex

11. Protein Complexes MudPIT – multidimensional protein identification technology IP’d proteins denatured and eluted from the beads Separated by HPLC using C18 column Digested with Lys-C and Trypsin proteases Peptides identified by tandem Mass Spec Identifies many proteins in the IP

12. Protein Complexes Who binds whom? Fig. S1B

13. Protein Complexes Who binds whom? Solid lines are interactions detected in both directions. Dashed lines were detected in one direction only. Red circles are proteins that were directly IP’d. Fig. S1A

14. Protein Complexes Who binds whom? MuvB detected with BMYB or E2F4/p130/DP1 but BMYB is never detected with E2F4/p130/DP1 DREAM and MMB are mutually exclusive Fig. S1A

15. Phosphorylation in the Protein Complexes Mass Spec can also show phosphorylations! Big mass differences Serine: Phosphoserine:

16. Phosphorylation in the Protein Complexes Many phosphoserine, phosphothreonine and phosphotyrosines detected

17. Phosphorylation in the Protein Complexes Is there any correlation of phosphorylation and MMB vs. DREAM complex? How often was this amino acid (residue) isolated in the phosphorylated form? How often was this amino acid (residue) isolated in the dephosphorylated form? Specifically looking at the 11 residues on four MuvB proteins (LIN9, LIN37, LIN52, LIN54)

18. Phosphorylation in the Protein Complexes Fig. 1C

19. Phosphorylation in the Protein Complexes Fig. S2

20. Phosphorylation in the Protein Complexes Fig. S2

21. Phosphorylation in the Protein Complexes Fig. 1A-C

22. Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly? Do these phosphorylations allow DREAM assembly? Or are they are result of DREAM assembly? Build point mutations: Serine to Alanine (unphosphorylatable) or Serine to Glutamate (phosphomimic) Tagged with V5 epitope Stably transfected IP with aV5 Fig. 1D

23. Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly? Do the mutants still assemble in the MuvB core? coIP Fig. S3

24. Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly? Do the mutants assemble into DREAM or MMB Complexes? coIP Conclusion? Fig. 1D

25. Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly? Do the mutants assemble into DREAM or MMB Complexes? Reverse coIP – IP for p130 and western blot for LIN52 Fig. 1E

26. Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly? Co-IP’s using endogenous LIN52 Fig. 1F

27. Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly? But is the doublet really due to phosphorylation? Prove it! After IP, treat with a mostly nonspecific phosphatase Fig. 1G

28. Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly? Is the LIN52 phosphorylation cell cycle-dependent?. Serum-starve cells to synchronize Re-feed and extract proteins at various timepoints for western blots Confirm cell-cycle enrichment with FACS Fig. 1H

29. Notice that the LIN52-S28A mutant decreases amount of LIN9 complexed with p130. Consistent with the idea that phosphorylation of LIN52 Ser28 is required for MuvB to bind p130/E2F4/DP1 DREAMComplex Fig. 1E

30. Is LIN52 is required for DREAM complex formation? Knockdown LIN52 expression with transient transfection of siRNA coIP endogenous proteins Where’s the data for: 1. did the knockdown work? 2. do LIN9 and p130 coIP with LIN52? 3. does the LIN52 knockdown affect the LIN9-p130 coIP? Fig. S4A

31. Is LIN52 is required for DREAM complex formation? LIN52 knockdown also reduces LIN9 and LIN37 accumulation Knockdown with one of two siRNAs Western blot of total lysates Fig. S4B

32. Is LIN52 is required for DREAM complex formation? Stably transfect cells with a gene to express shRNA targeted to endogenous LIN52 (5’UTR) Fig. 2A

33. Is LIN52 is required for DREAM complex formation? Stably transfect cells with a gene to express shRNA targeted to endogenous LIN52 (5’UTR) coIP Fig. 2A

34. Is LIN52 is required for DREAM complex formation? Stably transfect cells with a gene to express shRNA targeted to endogenous LIN52 (5’UTR) Transfect with wt LIN52 or LIN52-S28A mutant coIP Fig. 2A

35. Is LIN52 is required for DREAM complex formation? Extend the analysis to the MMB complex and other DREAM complex members Fig. 2

36. Is LIN52 is required for DREAM complex formation? Extend the analysis to the MMB complex and other DREAM complex members • Can the unphosphorylated LIN52 make • the MuvB complex? • DREAM complex? • MMB complex? • Does it affect the p130/E2F4/DP1 complex? Fig. 2

37. What is the kinase? The kinase transiently associates with LIN52, so it might show up in the MudPIT data. Top 20 proteins were: (dSNAF = distributed normalized spectral abundance factor) one kinase! Table S2

38. What is the kinase? Hypothesis: DYRK1A phosphorylates LIN52-Ser28 DYRKs are highly conserved Yak1p is DYRK homolog in S. cerevisiae Fig. 3B

39. What is the kinase? Hypothesis: DYRK1A phosphorylates LIN52-Ser28 DYRK1A consensus substrate site is R-x-x-S/T-P Matches the Ser-28 site and is evolutionarily conserved Fig. 3C

40. DYRK1A DYRK1A GST GST What is the kinase? Tested in vitro first, with recombinant proteins Express proteins in E. coli and purify GST-DYRK1A purified on glutathione-agarose Eluted with excess, soluble glutathione GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH GSH

41. What is the kinase? • in vitro kinase assay • recombinant GST-DYRK1A or GST-DYRK1B • recombinant GST-LIN52 • buffer with ATP • western blot with anti-phospho-Ser28 LIN52 antibody • or anti-GST Fig. 3D

42. What is the kinase? Moving in vivo Two cell lines: BJ-hTERT and T98G both endogenously express DYRK1A but only weakly express DYRK1B T98G cells have been used for previous analyses Fig. 3E

43. What is the kinase? Do LIN52 and DYRK1A associate in vivo? Transiently transfect T98G cells with HA-tagged DYRK1A or DYRK1B coIP Fig. S5A

44. What is the kinase? Does DYRK1A phosphorylate LIN52 in vivo? Inhibit DYRK1A activity with Harmine (dissolved in DMSO) Fig. 4A

45. What is the kinase? Does DYRK1A inhibition affect complex formation? Fig. 4BC

46. DYRK1A phenotypes? If DYRK1A affects DREAM complex formation, then it should affect the ability of cells to leave G0 and move through the cell cycle. Fig. 4D

47. DYRK1A phenotypes? Cell lines received retroviruses to express GFP or DYRK1A, along with puromycin resistance gene Transduced cells were selected with puromycin so all cells received the transgene. Viable cells were stained with crystal violet Fig. S6A

48. DYRK1A phenotypes? Cell lines received retroviruses to express GFP or DYRK1A, along with puromycin resistance gene Transduced cells were selected with puromycin. Viable cells were stained with crystal violet Fig. 5A

49. DYRK1A phenotypes? U2 OS cells (most sensitive) Stably transfected with DYRK1A wt or DYRK1A K188R (kinase dead) Under the control of a tetracycline/doxycycline inducible promoter Fig. 5B

50. DYRK1A phenotypes? What if LIN-52 is nonphosphorylatable? Fig. 5E, S6C