Structural and functional characterization of a cell cycle associated HDAC1/2 complex reveals the structural basis for complex assembly and nucleosome targeting

Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1. The amino-terminal region forms a tight dimerization domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxy-terminal domain of DNTTIP1 has a structure related to the SKI/SNO/DAC domain, despite lacking obvious sequence homology. We show that this domain in DNTTIP1 mediates interaction with both DNA and nucleosomes. Thus, DNTTIP1 acts as a dimeric chromatin binding module in the HDAC1:MIDEAS corepressor complex.     

KIBRA Regulates Aurora Kinase Activity and Is Required for Precise Chromosome Alignment During Mitosis

The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser⁸³ by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser⁵³⁹, probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.     

Phosphorylation of right open reading frame 2 (Rio2) protein kinase by polo-like kinase 1 regulates mitotic progression

Polo-like kinase 1 (Plk1) plays essential roles during multiple stages of mitosis by phosphorylating a number of substrates. Here, we report that the atypical protein kinase Rio2 is a novel substrate of Plk1 and can be phosphorylated by Plk1 at Ser-335, Ser-380, and Ser-548. Overexpression of Rio2 causes a prolonged mitotic exit whereas knockdown of Rio2 accelerates mitotic progression, suggesting that Rio2 is required for the proper mitotic progression. Overexpression of phospho-mimicking mutant Rio2 S3D but not the nonphosphorylatable mutant Rio2 S3A displays a profile similar to that of wild-type Rio2. These results indicate that the phosphorylation status of Rio2 correlates with its function in mitosis. Furthermore, time-lapse imaging data show that overexpression of Rio2 but not Rio2 S3A results in a slowed metaphase-anaphase transition. Collectively, these findings strongly indicate that the Plk1-mediated phosphorylation of Rio2 regulates metaphase-anaphase transition during mitotic progression.     

Vaccinia-related kinase 1 (VRK1) is an upstream nucleosomal kinase required for the assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage

Cellular responses to DNA damage require the formation of protein complexes in a highly organized fashion. The complete molecular components that participate in the sequential signaling response to DNA damage remain unknown. Here we demonstrate that vaccinia-related kinase 1 (VRK1) in resting cells plays an important role in the formation of ionizing radiation-induced foci that assemble on the 53BP1 scaffold protein during the DNA damage response. The kinase VRK1 is activated by DNA double strand breaks induced by ionizing radiation (IR) and specifically phosphorylates 53BP1 in serum-starved cells. VRK1 knockdown resulted in the defective formation of 53BP1 foci in response to IR both in number and size. This observed effect on 53BP1 foci is p53- and ataxia-telangiectasia mutated (ATM)-independent and can be rescued with VRK1 mutants resistant to siRNA. VRK1 knockdown also prevented the activating phosphorylation of ATM, CHK2, and DNA-dependent protein kinase in response to IR. VRK1 activation in response to DNA damage is a novel and early step in the signaling of mammalian DNA damage responses.     

The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression

Regulation of NF-kappaB transactivation function is controlled at several levels, including interactions with coactivator proteins. Here we show that the transactivation function of NF-kappaB is also regulated through interaction of the p65 (RelA) subunit with histone deacetylase (HDAC) corepressor proteins. Our results show that inhibition of HDAC activity with trichostatin A (TSA) results in an increase in both basal and induced expression of an integrated NF-kappaB-dependent reporter gene. Chromatin immunoprecipitation (ChIP) assays show that TSA treatment causes hyperacetylation of the wild-type integrated NF-kappaB-dependent reporter but not of a mutant version in which the NF-kappaB binding sites were mutated. Expression of HDAC1 and HDAC2 repressed tumor necrosis factor (TNF)-induced NF-kappaB-dependent gene expression. Consistent with this, we show that HDAC1 and HDAC2 target NF-kappaB through a direct association of HDAC1 with the Rel homology domain of p65. HDAC2 does not interact with NF-kappaB directly but can regulate NF-kappaB activity through its association with HDAC1. Finally, we show that inhibition of HDAC activity with TSA causes an increase in both basal and TNF-induced expression of the NF-kappaB-regulated interleukin-8 (IL-8) gene. Similar to the wild-type integrated NF-kappaB-dependent reporter, ChIP assays showed that TSA treatment resulted in hyperacetylation of the IL-8 promoter. These data indicate that the transactivation function of NF-kappaB is regulated in part through its association with HDAC corepressor proteins. Moreover, it suggests that the association of NF-kappaB with the HDAC1 and HDAC2 corepressor proteins functions to repress expression of NF-kappaB-regulated genes as well as to control the induced level of expression of these genes.     

CRM1 mediates nuclear export of HDAC7 independently of HDAC7 phosphorylation and association with 14-3-3s

CRM1, 14-3-3 proteins, and CaMK play important roles in trafficking of HDAC7, but the interplay between these proteins in this process is not clearly understood. Here, we show that CRM1 is capable of promoting cytoplasmic localization of wild-type and mutant HDAC7 (S178A/S344A/S479A), which is normally found in the nucleus. Using phospho-specific antibodies to HDAC7, we demonstrate that CaMK I promotes phosphorylation of S178, S344, and S479 of HDAC7. We also show that endogenous S178-phosphorylated HDAC7 is localized in both the nucleus and the cytoplasm, whereas S344- and S479-phosphorylated HDAC7 are exclusively localized in the nucleus. An HDAC7 mutant, S178E/S344E/S479E, which lost the ability to bind 14-3-3s, is localized in both the nucleus and the cytoplasm. Furthermore, the nuclear export of S178E/S344E/S479E is inhibited by LMB, but is enhanced by the CRM1. Taken together, these results strongly suggest that CRM1 mediated-nuclear export of HDAC7 is independent of HDAC7 phosphorylation and its association with 14-3-3s.     

Phosphorylation of Ran-binding protein-1 by Polo-like kinase-1 is required for interaction with Ran and early mitotic progression

Polo-like kinase-1 (Plk1) is essential for progression of mitosis and localizes to centrosomes, central spindles, midbody, and kinetochore. Ran, a small GTPase of the Ras superfamily, plays a role in microtubule dynamics and chromosome segregation during mitosis. Although Ran-binding protein-1 (RanBP1) has been reported as a regulator of RanGTPase for its mitotic functions, the action mechanism between Ran and RanBP1 during mitosis is still unknown. Here, we demonstrated in vitro and in vivo phosphorylation of RanBP1 by Plk1 as well as the importance of phosphorylation of RanBP1 in the interaction between Plk1 and Ran during early mitosis. Both phosphorylation-defective and N-terminal deletion mutant constructs of RanBP1 disrupted the interaction with Ran, and depletion of Plk1 also disrupted the formation of a complex between Ran and RanBP1. In addition, the results from both ectopic expression of phosphorylation-defective mutant construct and a functional complementation on RanBP1 deficiency with this mutant indicated that phosphorylation of RanBP1 by Plk1 might be crucial to microtubule nucleation and spindle assembly during mitosis.     

Phosphorylation of MCM3 protein by cyclin E/cyclin-dependent kinase 2 (Cdk2) regulates its function in cell cycle

MCM2-7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2-7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2-7 proteins contributes to the formation of the MCM2-7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2-7 complex.     

Acetylation of core histones in response to HDAC inhibitors is diminished in mitotic HeLa cells

Histone acetylation is a key modification that regulates chromatin accessibility. Here we show that treatment with butyrate or other histone deacetylase (HDAC) inhibitors does not induce histone hyperacetylation in metaphase-arrested HeLa cells. When compared to similarly treated interphase cells, acetylation levels are significantly decreased in all four core histones and at all individual sites examined. However, the extent of the decrease varies, ranging from only slight reduction at H3K23 and H4K12 to no acetylation at H3K27 and barely detectable acetylation at H4K16. Our results show that the bulk effect is not due to increased or butyrate-insensitive HDAC activity, though these factors may play a role with some individual sites. We conclude that the lack of histone acetylation during mitosis is primarily due to changes in histone acetyltransferases (HATs) or changes in chromatin. The effects of protein phosphatase inhibitors on histone acetylation in cell lysates suggest that the reduced ability of histones to become acetylated in mitotic cells depends on protein phosphorylation.     

ATM regulates ionizing radiation-induced disruption of HDAC1:PP1:Rb complexes

Ionizing radiation elicits signaling events that coordinate DNA repair and interruption of cell cycle progression. We previously demonstrated that ionizing radiation (IR) of cells activates nuclear protein phosphatase-1 (PP1) by promoting dephosphorylation of Thr320, an inhibitory site in the enzyme and that the ATM kinase is required for this response. We sought to identify potential targets of IR-activated PP1. Untreated and IR-treated Jurkat cells were labeled with (32)P orthophosphate, and nuclear extracts were subjected to microcystin affinity chromatography to recover phosphatase complexes that were analyzed by 2D-PAGE and mass spectrometry. Several proteins associated with protein phosphatases demonstrated a significant decrease in (32)P intensity following IR, and one of these was identified as HDAC1. Co-immunoprecipitation revealed complexes containing PP1 with HDAC1 and Rb in cell extracts. In response to IR, there was an ATM-dependent activation of PP1, dephosphorylation of HDAC1, dissociation of HDAC1-PP1-Rb complexes and increased HDAC1 activity. These results suggest that IR regulates HDAC1 phosphorylation and activity through ATM-dependent activation of PP1.