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
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DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly Larisa Litovchick, Laurence A. Florens, Selene K. Swanson, Michael.
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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, BostonBrigham and Women’s Hospital, Harvard Medical School, Boston
Stowers Institute, Kansas CityUniversity of Kansas Medical Center, Kansas City
Genes and Development
25:801-813
April 15, 2011
RB and E2F Families
Rb, p107 and p130 with E2Fs control the Restriction Point
RB and E2F Families
Rb, p107 and p130 with E2Fs control the Restriction Point
RB and E2F Families
Rb, p107 and p130 with E2Fs control the Restriction Point
Protein Complexes
MuvB CoreComplex
Essentially aCo-repressor
+ =
DREAM Complex(DP, RB-like, E2F4, and MuvB)
Represses transcription to hold cells in G0
Protein Complexes
+ =
MMB Complex
S phase
Myb Protein
(transcriptionfactor)
+ =
OR DREAMComplex
MuvB CoreComplex
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
Myb
of B-Myb
Protein Complexes
What controls which complex MuvB core ends up in?
Model:
Protein Complexes
What controls which complex MuvB core ends up in?
Isolate the complexes by immunoprecipitationsa-p130HA-tagged p130a-LIN52V5-tagged LIN52a-BMYB
Some cells were cycling, some in G0
some in S
Identify co-precipitating proteins MMB Complex
DREAMComplex
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
Protein Complexes
Who binds whom?
Fig. S1B
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
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
Phosphorylation in the Protein Complexes
Mass Spec can also show phosphorylations! Big mass differences
Serine:
Phosphoserine:
Phosphorylation in the Protein Complexes
Many phosphoserine, phosphothreonine and phosphotyrosines detected
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)
Fig. 1C
Phosphorylation in the Protein Complexes
Phosphorylation in the Protein Complexes
Fig. S2
Phosphorylation in the Protein Complexes
Fig. S2
Fig. 1A-C
Phosphorylation in the Protein Complexes
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 epitopeStably transfectedIP with aV5
Fig. S3
Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly?
Do the mutants still assemble in the MuvB core? coIP
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?
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
Fig. 1F
Is phosphorylation of LIN52-Ser28 or LIN37-Ser182 Required for DREAM Assembly?
Co-IP’s using endogenous LIN52
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
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 synchronizeRe-feed and extract proteins at various timepoints for western blotsConfirm cell-cycle enrichment with FACS
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
Is LIN52 is required for DREAM complex formation?
Knockdown LIN52 expression with transient transfection of siRNAcoIP 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
Is LIN52 is required for DREAM complex formation?
Fig. S4B
LIN52 knockdown also reduces LIN9 and LIN37 accumulation
Knockdown with one of two siRNAsWestern blot of total lysates
Is LIN52 is required for DREAM complex formation?
Fig. 2A
Stably transfect cells with a gene to express shRNA targeted to endogenous LIN52 (5’UTR)
Is LIN52 is required for DREAM complex formation?
Fig. 2A
Stably transfect cells with a gene to express shRNA targeted to endogenous LIN52 (5’UTR)
coIP
Is LIN52 is required for DREAM complex formation?
Fig. 2A
Stably transfect cells with a gene to express shRNA targeted to endogenous LIN52 (5’UTR)Transfect with wt LIN52 or LIN52-S28A mutant
coIP
Is LIN52 is required for DREAM complex formation?
Extend the analysis to the MMB complex and other DREAM complex members
Fig. 2
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?
What is the kinase?
The kinase transiently associates with LIN52, so it might show up in the MudPIT data.