DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly

Abstract

In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence.

Figure 1.

Analysis of DREAM phosphorylation reveals a critical role of LIN52-S28. (A–C) Detection of phosphorylated amino acid residues in the LIN proteins by the immunoprecipitation/MS/MS of p130, BMYB, and merged data set of all samples. The graphs show spectral counts of peptides where phosphorylated amino acids were detected (gray bars), and the total spectral counts for the corresponding peptides (white bars). Boxes indicate sites that were always phosphorylated in p130 immunoprecipitation samples. (D) Mutational analysis of LIN52-S28 and LIN37-S182 residues. Ectopically expressed V5-tagged wild-type or the indicated mutants of LIN52 and LIN37 were immunoprecipitated using anti-V5 antibody, and the binding of p130 and BMYB was detected by immunoblot. (E) Immunoprecipitation/Western blot assay shows that endogenous p130 interacts with the wild type but not the S28A-LIN52 mutant. Expression of LIN52 constructs was detected by immunoprecipitation/Western blot with the V5 tag antibody. (F) Immunoprecipitation/Western blot reveals that the immunoprecipitates for p130 and BMYB contain different species of LIN52. (G) Western blot shows that the upper form of LIN52 coprecipitated with p130, LIN9, or LIN37 collapses to the lower form with phosphatase treatment. (H) T98G cells were synchronized at various stages of the cell cycle by serum starvation and readdition, and LIN52 was detected in the extracts by immunoblot.

Figure 2.

Intact LIN52 is required for the DREAM complex assembly. (A) Immunoprecipitation/Western blot assays show that only the wild-type LIN52 allele can rescue the binding of p130 to LIN9 and LIN37 in cells with reduced expression of LIN52. LIN52 was depleted in T98G cells by stable expression of LIN52-shRNA and rescued by wild-type LIN52 or the S28A mutant. The DREAM complex was assayed by immunoprecipitation/Western blot. Input panels show the levels of the proteins in cell extracts. (B) The immunopreciptiation/Western blot assays show that both wild-type and the S28A mutant of LIN52 bind to BMYB and up-regulate the levels of LIN37 in LIN52-depleted cells. (C) The interaction between p130 and E2F4 is independent of LIN52, as shown by immunoprecipitation/Western blot.

Figure 3.

DYRK1A interacts with LIN52 in vivo and phosphorylates LIN52-S28. (A) Positions and amino acid sequences of DYRK1A peptides detected in two independent LIN52 immunoprecipitation/MS-MS experiments. Dots indicate tryptic cleavage sites. (B) Clustal W alignment of DYRK1A orthologs from different species reveals a protein kinase family that is highly conserved in evolution. (C) Amino acid sequence around LIN52-S28 (black arrow), including the DYRK1A consensus motif (white arrows), is evolutionarily conserved. (D) In vitro kinase assay shows that both recombinant DYRK1A and DYRK1B can phosphorylate LIN52. Phosphorylated or total GST-LIN52 was detected by immunoblots with phospho-S28-LIN52 antibody and anti-GST antibody, respectively. (E) DYRK1A and DYRK1B were detected in BJ-hTERT and T98G cell extracts by immunoblot using recombinant purified GST-tagged proteins (rDYRK1A or rDYRK1B, 30 ng per lane) as a reference. The rDYRK1A and rDYRK1B samples were also blotted for GST to ensure equal loading.

Figure 4.

DYRK1A contributes to the DREAM complex assembly and entry into quiescence. (A) Immunoblot of LIN52 in the extracts from T98G cells incubated with DYRK1A-specific siRNA (48 h) or 10 μM harmine (20 h). (B) Depletion of DYRK1A in T98G cells by RNAi reduces the binding of LIN52 to p130 but not BMYB, as shown by immunoprecipitation/Western assay. (C) Depletion of DYRK1A but not DYRK1B in T98G cells by RNAi results in a decreased binding of p130 to LIN37, as shown by immunoprecipitation/Western blot. (D,E) Treatment of T98G cells by DYRK1A siRNA (a pool of four oligos) or treatment with 10 μM harmine interferes with G0/G1 growth arrest upon serum deprivation, as shown by FACS analysis of BRDU-labeled DNA. Cells were incubated with 10% FBS (10 FBS) or 0% FBS (0 FBS) for 24 h before the assay. (F) The graph shows the average percentage of BrDU-positive cells ± SD measured in three independent experiments shown in D and E. Two-tailed t-test: 0 FBS control versus siDYRK1A single and pool, P = 0.00008 and 0.014, respectively.

Figure 5.

Overexpression of DYRK1A suppresses cell proliferation. (A) Overexpression of DYRK1A causes growth suppression. The indicated cell lines were transduced with retroviruses to express DYRK1A or GFP and were used for cell proliferation assay after antibiotic selection. The graph shows the density of the cultures determined by crystal violet staining relative to day 1 (average of the triplicate samples ± SD). The normalized OD values at day 7 were significantly different for all pairs of DYRK1A- and GFP-expressing cell lines (two-tailed t-test, P < 0.005). (B) Active but not kinase-dead DYRK1A suppresses colony growth of U2OS cells. Tet-on U-2 OS cells were treated as described in the Materials and Methods. Colony counts of induced samples relative to uninduced (taken as 100%) are shown above the images. (C) Overexpression of active but not kinase-dead DYRK1A suppresses proliferation of U2OS cells. Two clones of each type of cell line were grown ±doxycycline and counted. Induced cell counts are shown as percentage of uninduced (average values ± SD of two experiments, each done in triplicate). (D) Overexpression of active but not kinase-dead DYRK1A in U-2 OS cells affects the DREAM and BMYB–MuvB complexes, as shown by immunoprecipitation/Western blots. DYRK1A alleles were expressed in U-2 OS cells by retroviral infection and by antibiotic selection. Two wild-type-expressing samples (wt1 and wt2) with different levels of DYRK1A are shown to emphasize a potent effect of DYRK1A on the DREAM and BMYB–MuvB complexes in U-2 OS cells. (E) Overexpression of LIN52-S28A but not the wild type can override the growth suppression effect of DYRK1A in NIH 3T3 cells. GFP, DYRK1A, or the DYRK1A-K188R mutant were introduced into NIH 3T3 cell lines expressing the LIN52 alleles or vector. After antibiotic selection, the cell lines were equally plated, grown for 7 d, and stained by crystal violet dye. Images of two representative wells for each condition are shown.

Figure 6.

DYRK1A and intact LIN52-S28A contribute to oncogenic Ras-induced senescence. (A) Western blot shows that overexpression of either the wild type or LIN52-S28A mutant in BJ-hTERT cells results in down-regulation of the endogenous LIN52 protein. (B) The LIN52-S28A mutant but not wild-type LIN52 disrupts the DREAM complex in BJ-hTERT fibroblasts, as shown by immunoprecipitation/Western blot assay. (C) The indicated BJ-hTERT cell lines were infected with retroviruses to express HRAS-G12V or empty vector, and the expression of HRAS-G12V was assayed by Western blot. Vinculin was used as a loading control. (D) BJ-hTERT cells expressing LIN52 alleles or vector were infected with HRAS-G12V and stained for SA-β-gal on day 14 post-infection. Representative images of the cells are shown as well as average values ± SD of three independent experiments, each counting >100 cells per condition. Two-tailed t-test: vector versus LIN52 wild type (LIN52-WT) and S28A mutant (LIN52-S28A), P = 0.24 and 0.01, respectively. Bar, 100 μM. (E) BJ-hTERT cells were transduced with either control or two different DYRK1A-specific shRNA lentiviruses, followed by infection with retrovirus encoding HRAS-G12V. Western blot shows the levels of DYRK1A and HRAS-G12V in these cells. Vinculin was used as a loading control. (F) Ras-induced senescence assay in BJ-hTERT cell lines treated with DYRK1A-shRNA. The cells were infected and processed as in D. Two-tailed t-test: vector versus shDYRK1A-1 and shDYRK1A-2, P = 0.09 and 0.05, respectively. Bar, 100 μM. (G) The model shows how DYRK1A promotes the DREAM complex assembly, G0/G1 arrest, and senescence. DYRK1A phosphorylation of the Ser 28 residue in the LIN52 subunit of the MuvB core promotes the DREAM complex assembly and repression of E2F target genes such as BMYB. MuvB core subunits are shaded in blue.