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:
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. (DP, RB-like, E2F4, and MuvB)
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. OR Protein Complexes + = DREAMComplex + = Myb Protein MMB Complex
(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 DREAMComplex MMB Complex
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. 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
GST
DYRK1A
GSH
GSH
GSH
GSH
GST
DYRK1A
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

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

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

53. DYRK1A, LIN52 and Senescence?
Senescence is irreversible arrest in G0
Senescent cells express SA-b-gal (senescent associated b-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? What’s 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?
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?
Fig. 6B

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

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

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

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

63. Fig. 6G