Ubp-M serine 552 phosphorylation by cyclin-dependent kinase 1 regulates cell cycle progression

Abstract

In eukaryotic cells, genomic DNA is organized into a chromatin structure, which not only serves as the template for DNA-based nuclear processes, but also as a platform integrating intracellular and extracellular signals. Although much effort has been spent to characterize chromatin modifying/remodeling activities, little is known about cell signaling pathways targeting these chromatin modulators. Here, we report that cyclin-dependent kinase 1 (CDK1) phosphorylates the histone H2A deubiquitinase Ubp-M at serine 552 (S552P), and, importantly, this phosphorylation is required for cell cycle progression. Mass spectrometry analysis confirmed Ubp-M is phosphorylated at serine 552, and in vitro and in vivo assays demonstrated that CDK1/cyclin B kinase is responsible for Ubp-M S552P. Interestingly, Ubp-M S552P is not required for Ubp-M tetramer formation, deubiquitination activity, substrate specificity, or regulation of gene expression. However, Ubp-M S552P is required for cell proliferation and cell cycle G 2/M phase progression. Ubp-M S552P reduces Ubp-M interaction with nuclear export protein CRM1 and facilitates Ubp-M nuclear localization. Therefore, these studies confirm that Ubp-M is phosphorylated at S552 and identify CDK1 as the enzyme responsible for the phosphorylation. Importantly, this study specifically links Ubp-M S552P to cell cycle G 2/M phase progression.

Keywords: CDK1; CRM1; G2/M phase; H2A deubiquitination; Ubp-M; cell cycle; gene expression; phosphorylation.

Figure 1.

Ubp-M is phosphorylated at the serine 552 residue. (A) Ubp-M is phosphorylated in vivo. Coomassie blue staining of affinity purified Ubp-M from sf9 cells incubated with (lane 3) or without (lane 2) Calf intestinal alkaline phosphatase (CIP). CIP treatment decreases the intensity of the top band and increases the intensity of the bottom band. (B) The serine 552 residue of Ubp-M is phosphorylated. Mass spectrometry analysis indicated that Ubp-M is phosphorylation at serine 552. (C) Generation of the Ubp-M phosphorylated serine 552 (S552P) antibody. Top: peptide sequence used to generate the Ubp-M S552P antibody. Bottom: characterization of the Ubp-M S552P antibody. Antibodies used are labeled on the left side of the panel. (D) Ubp-M S552P occurs in cell cycle M phase. Western blot analysis of extracts from unsynchronized, interphase, and M phase cells. Antibodies used are labeled on the left side of the panels.

Figure 2.

Cyclin-dependent kinase 1 is responsible for Ubp-M serine 552 phosphorylation. (A) Cyclin-dependent kinase 1 (CDK1) phosphorylates Ubp-M serine 552 in vitro. Top: peptide sequence flanking serine 552 contains a fully conserved CDK1 binding motif. Bottom: in vitro kinase assay using Ubp-M fragments (529–599) as substrates. (B) Roscovitine treatment reduces Ubp-M S552P (middle panel) without affecting total Ubp-M levels (top panel). (C) CGP74514A treatment reduces Ubp-M S552P (middle panel) without affecting total Ubp-M levels (top panel). (D) CDK1 knockdown abolishes Ubp-M S552P. Top panel indicates that the level of CDK1 is significantly reduced by shRNA treatment. Middle two panels indicate that knockdown of CDK1 reduces Ubp-M S552P levels (third panel) without affecting total Ubp-M levels (second panel).

Figure 3.

S552P does not affect Ubp-M deubiquitination activity in vitro or in vivo. (A) Purification of wild-type and mutant (S552A and S552E) Ubp-M. S552P accounts for the majority of the Ubp-M mobility shift observed by western blot. Mutation of serine 552 to alanine caused a significant reduction in mobility shifted Ubp-M, while mutation of serine 552 to glutamic acid partially mimics the phosphorylation-mediated mobility shift. (B) S552P does not affect Ubp-M tetramer formation. Superose 6 analysis revealed that wild-type and mutant (S552A and S552E) elute at a molecular weight of 443 kDa. (C) Ubp-M S552A and S552E mutations do not affect Ubp-M H2A deubiquitination activity in vitro. Histone H2A deubiquitination assay with mononuclosomes (second and third panels) and core histone (fourth panel) as substrates. Wild-type and mutant (S552A and S552E) Ubp-M display similar uH2A deubiquitination activity in vitro. The amount of Ubp-M used and the H2A levels were similar in core histone (20 min), and mononucleosome (10 min) reaction. His. indicates histone substrates and Nucl. indicates nucleosome substrates. (D) Ubp-M S552A and S552E mutations do not affect the substrate specificity of Ubp-M. Histone deubiquitination assay with uH2B-containing mononucleosomes and core histones as substrates. His. indicates histone substrates and Nucl. indicates nucleosome substrates. (E) Ubp-M S552P is not required for regulation of global H2A ubiquitination levels. The indicated constructs were transfected into HeLa cell lines stably expressing Flag-H2A and HA-ubiquitin and the effects on global H2A ubiquitination levels were analyzed by western blot assay. (F) Ubp-M S552 phosphorylation is not required for Ubp-M regulation of gene expression. The constructs indicated on top of the panel were transfected into control and Ubp-M knockdown cells and the effects on the expression of HoxD10 were analyzed by RT-PCR assay.

Figure 4.

Ubp-M S552P regulates cell cycle G₂/M phase progression. (A) Ubp-M S552P occurs in cell cycle M phase. Western blot assay of extracts from cells at the indicated time points following release from double thymidine block. Antibodies used are labeled in the left side of the panel. (B) Ubp-M S552P stains M phase cells. Hela cells were stained with S552P antibody and PI, and cell cycle was analyzed by FACS. (C) Characterization of Ubp-M stable knockdown cell lines expressing Flag tagged wild-type, S552A, or S552E Ubp-M. Antibodies used are labeled in the left side of the panel. (D) Growth curve of control, Ubp-M knockdown, and Ubp-M knockdown cells expressing wild-type or mutant (S552A and S552E) Ubp-M. Wild-type Ubp-M, but not the S552A and S552E mutants, rescued the growth defects in Ubp-M knockdown cells. (E) Expression of wild-type, but not mutant (S552A and S552E) mutant Ubp-M, rescues the decrease in G₂/M phase cell population caused by Ubp-M knockdown.

Figure 5.

Ubp-M S552P regulates the interaction between Ubp-M and CRM1. (A) Ubp-M subcellular localization is regulated by CRM1 mediated nuclear export. LMB treatment increased the nuclear localization of RFP-Ubp-M. Two hundred RFP-positive cells were examined and representative images shown. (B) Fold change in the number of cells with nuclear wild-type or mutant (S552A or S552E) RFP-Ubp-M in control and LMB treated cells. 200 RFP-positive cells were examined. The percentage of cells with nuclear wild-type, S552A or S552E RFP-Ubp-M without LMB treatment was set to 1. (C) Fold change of nuclear wild-type and mutant (S552A and S552E) RFP-Ubp-M in control and CRM1-overexpressing cells. 200 RFP-positive cells were examined. The percentage of nuclear wild-type and mutant (S552A and S552E) Ubp-M in control cells was set to 1. (D) Ubp-M S552P regulates the interaction between Ubp-M and CRM1. CRM1 was only detected in anti-Flag immunoprecipitates of wild-type, but not S552A and S552E mutant, Ubp-M. (E) CRM1 interacts with non-phosphorylated Ubp-M. CRM1 specifically pulled down non-phosphorylated Ubp-M but not phosphorylated Ubp-M, as confirmed by anti-S552P antibody. (F) A proposed model for the function of Ubp-M S552P. Ubp-M subcellular localization is determined by the balance between export and import processes. As cells prepare to enter M phase, cytoplasmic cycle B/CDK1 kinase is activated and phosphorylates Ubp-M S552. S552P disrupts Ubp-M interaction with CRM1, allowing Ubp-M to be retained in the nucleus and deubiquitinate nucleosomal uH2A, which is required for chromosome condensation and cell cycle progression.