Presentation is loading. Please wait.

Presentation is loading. Please wait.

DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly Larisa Litovchick, Laurence A. Florens, Selene K. Swanson, Michael.

Similar presentations


Presentation on theme: "DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly Larisa Litovchick, Laurence A. Florens, Selene K. Swanson, Michael."— Presentation transcript:

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 Womens Hospital, Harvard Medical School, Boston Stowers Institute, Kansas City University of Kansas Medical Center, Kansas City Genes and Development 25: 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 G 0

6 Protein Complexes += MMB Complex S phase Myb Protein (transcription factor) += OR DREAMComplex 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 -p130 HA-tagged p130 -LIN52 V5-tagged LIN52 -BMYB Some cells were cycling, some in G 0 some in S Identify co-precipitating proteins MMB Complex DREAMComplex

11 Protein Complexes MudPIT – multidimensional protein identification technology IPd 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 IPd. 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 Fig. 1C Phosphorylation in the Protein Complexes

19 Fig. S2

20 Phosphorylation in the Protein Complexes Fig. S2

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

22 Fig. 1D 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 V5

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

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

25 Fig. 1E 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

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

27 Fig. 1G 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

28 Fig. 1H 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

29 Fig. 1E 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

30 Is LIN52 is required for DREAM complex formation? Knockdown LIN52 expression with transient transfection of siRNA coIP endogenous proteins Wheres 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? Fig. S4B LIN52 knockdown also reduces LIN9 and LIN37 accumulation Knockdown with one of two siRNAs Western blot of total lysates

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

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

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

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 Fig. 2 Can the unphosphorylated LIN52 make the MuvB complex? DREAM complex? MMB complex? Does it affect the p130/E2F4/DP1 complex?

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) Table S2 one kinase!

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

39 What is the kinase? Fig. 3C 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

40 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 GST DYRK1A GSH GST DYRK1A

41 What is the kinase? Fig. 3D 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

42 What is the kinase? Fig. 3E 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

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

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

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

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

47 DYRK1A phenotypes? Fig. S6A 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

48 DYRK1A phenotypes? Fig. 5A 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

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

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

51 DYRK1A phenotypes? Fig. S6B DYRK1A-wt expression shows flattened, arrested cells. Senescent?

52 DYRK1A phenotypes? Fig. S6B DYRK1A-wt expression shows flattened, arrested cells. Senescent? A closer look: DYRK1A-wt DYRK1A-K188R

53 DYRK1A, LIN52 and Senescence? Senescence is irreversible arrest in G 0 Senescent cells express SA- -gal (senescent associated -galactosidase) Can be induced by activated Ras (and other oncogenes) Is there a connection? Activated Ras has been shown to down-regulate 355 genes by microarray (Mason et al., 2004) DREAM complex binds to 588 genetic loci (Litovchick et al., 2007) How did they find those 588 loci? ChIP-chip (or ChIP on chip)

54 DYRK1A, LIN52 and Senescence? ChIP-chip

55 DYRK1A, LIN52 and Senescence? ChIP-chip

56 DYRK1A, LIN52 and Senescence? ChIP-chip shows 588 sites where DREAM binds. What do they have in common? Whats the consensus sequence for DREAM binding?

57 DYRK1A, LIN52 and Senescence? Is there a connection of DREAM and RAS? Lots of overlap! Fig. S7A

58 DYRK1A, LIN52 and Senescence? Fig. 6B Activated Ras is known to induce senescence in immortalized BJ-hTERT cells Stably transfected with V5-tagged LIN52-wt or LIN52-S28A Does it still disrupt DREAM complex in this cell line?

59 DYRK1A, LIN52 and Senescence? Fig. 6C Add a retrovirus that expresses activated Ras (HRAS-G12V). Select with puromycin. LIN52:

60 DYRK1A, LIN52 and Senescence? Fig. 6D Add a retrovirus that expresses activated Ras (HRAS-G12V). Select with puromycin. Two weeks later, measure senescence

61 DYRK1A, LIN52 and Senescence? Fig. 6E What if we knockdown DYRK1A expression instead?

62 DYRK1A, LIN52 and Senescence? Fig. 6F What if we knockdown DYRK1A expression instead?

63 Fig. 6G


Download ppt "DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly Larisa Litovchick, Laurence A. Florens, Selene K. Swanson, Michael."

Similar presentations


Ads by Google