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.

Top 20 proteins were: (dSNAF = distributed normalized spectral abundance factor)

Table S2

one kinase!

What is the kinase?

Fig. 3B

Hypothesis: DYRK1A phosphorylates LIN52-Ser28

DYRKs are highly conserved

Yak1p is DYRK homolog in S. cerevisiae

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

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

GSTDYRK1A

GSH

GSH

GSH

GSH

GSH

GSH

GSH

GSH

GSH

GSH

GSHGSH

GSH

GSHGSH

GSH

GSH

GSH

GSHGSH

GSH

GSH

GSTDYRK1A

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 antibodyor anti-GST

What is the kinase?

Fig. 3E

Moving in vivo Two cell lines: BJ-hTERT and T98G

both endogenously express DYRK1A but only weakly express DYRK1BT98G cells have been used for previous analyses

What is the kinase?

Fig. S5A

Do LIN52 and DYRK1A associate in vivo?

Transiently transfect T98G cells with HA-tagged DYRK1A or DYRK1B

coIP

What is the kinase?

Fig. 4A

Does DYRK1A phosphorylate LIN52 in vivo?

Inhibit DYRK1A activity with Harmine (dissolved in DMSO)

What is the kinase?

Fig. 4BC

Does DYRK1A inhibition affect complex formation?

DYRK1A phenotypes?

Fig. 4D

If DYRK1A affects DREAM complex formation, thenit should affect the ability of cells to leave G0 and move through the cell cycle.

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

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

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

Fig. 5E, S6C

DYRK1A phenotypes?

What if LIN-52 is nonphosphorylatable?

DYRK1A phenotypes?

Fig. S6B

DYRK1A-wt expression shows

flattened, arrested cells.

Senescent?

DYRK1A phenotypes?

Fig. S6B

DYRK1A-wt expression shows

flattened, arrested cells.

Senescent?

A closer look:

DYRK1A-wt

DYRK1A-K188R

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)

DYRK1A, LIN52 and Senescence?

ChIP-chip

DYRK1A, LIN52 and Senescence?

ChIP-chip

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?

DYRK1A, LIN52 and Senescence?Is there a connection of DREAM and RAS?

Lots of overlap!

Fig. S7A

DYRK1A, LIN52 and Senescence?

Fig. 6B

Activated Ras is known to induce senescence in immortalized BJ-hTERT cellsStably transfected with V5-tagged LIN52-wt or LIN52-S28A

Does it still disrupt DREAM complex in this cell line?

DYRK1A, LIN52 and Senescence?

Fig. 6C

Add a retrovirus that expresses activated Ras (HRAS-G12V). Select with puromycin.

LIN52:

DYRK1A, LIN52 and Senescence?

Fig. 6D

Add a retrovirus that expresses activated Ras (HRAS-G12V). Select with puromycin.Two weeks later, measure senescence

DYRK1A, LIN52 and Senescence?

Fig. 6E

What if we knockdown DYRK1A expression instead?

DYRK1A, LIN52 and Senescence?

Fig. 6F

What if we knockdown DYRK1A expression instead?

Fig. 6G