Identification PMS1 and PMS2 as potential meiotic substrates of CDK2 activity

Abstract

Cyclin dependent-kinase 2 (CDK2) plays important functions during the mitotic cell cycle and also facilitates several key events during germ cell development. The majority of CDK2's known meiotic functions occur during prophase of the first meiotic division. Here, CDK2 is involved in the regulation of meiotic transcription, the pairing of homologous chromosomes, and the maturation of meiotic crossover sites. Despite that some of the CDK2 substrates are known, few of them display functions in meiosis. Here, we investigate potential meiotic CDK2 substrates using in silico and in vitro approaches. We find that CDK2 phosphorylates PMS2 at Thr337, PMS1 at Thr331, and MLH1 in vitro. Phosphorylation of PMS2 affects its interaction with MLH1 to some degree. In testis extracts from mice lacking Cdk2, there are changes in expression of PMS2, MSH2, and HEI10, which may be reflective of the loss of CDK2 phosphorylation. Our work has uncovered a few CDK2 substrates with meiotic functions, which will have to be verified in vivo. A better understanding of the CDK2 substrates will help us to gain deeper insight into the functions of this universal kinase.

Fig 1. Potential CDK2 substrates; PMS1 and PMS2.

Graphic representation of the evolutionary conservation of Thr331 and Thr337 in PMS1 (A) and PMS2 (B), respectively. Amino acid positions are labelled relative to the transcripts from Mus musculus. The CDK consensus site in each sequence is highlighted in red. (C) CDK2/cyclin A2 kinase assays directed against GST-tagged fusion peptides of PMS1 Thr331 (lane 2) or PMS2 Thr337 (lane 5) or their threonine-alanine point mutants (lanes 3 and 6, respectively). GST tag without fusion peptide are shown in lanes 1 and 4 as negative controls. Upper panel shows the phospho-image of each peptide and represents the amount of radiolabelling occurring following phosphorylation by CDK2/cyclin A2. The lower panel shows the Coomassie staining of radiolabelled proteins to indicate equal loading in each lane. (D) Phospho-image quantification of peptides from panel A. Data is presented as the absolute level of phosphorylation observed in each peptide as determined by densitometry analysis. Significance is determined by the difference in quantified phospho-signal by unpaired-T test. This experiment was repeated at least three times with similar results observed in each case.

Fig 2. CDK2 phosphorylation of full-length PMS2 and its effect on the complex with MLH1.

(A) CDK2/cyclin A2 kinase assay directed against GST-tagged fusion PMS2 (lanes 1,3,5) or PMS2^T337A (lanes 2,4,6) full length proteins. Upper panel shows the phospho-image of each protein and represents the amount of radiolabelling occurring following phosphorylation by CDK2/cyclin A2. The lower panel shows the Coomassie staining of radiolabelled proteins to indicate equal loading in each lane. (B) IP-western blot analysis of transfected MLH1-V5 or PMS2-MYC proteins from HEK293T cells. Antibodies used for immunoprecipitation are shown at the left hand side whilst antibodies used for detection are shown on the right hand side of the blot. Correct expression of tagged proteins is detected by the V5 or Myc tag following their respective immunoprecipitation (2^nd and 3^th panels from top, respectively). Co-immunoprecipitation of MLH1-V5 with PMS2-myc or PMS2^T337A-myc is shown in lanes 5 and 6. (C) Input western blot of lysates used for panel A. Here 10% of the total protein used for immunoprecipitation in panel A was probed for proteins relevant for the assay. CDK2, GAPDH and gamma-TUBULIN are shown as loading controls. Molecular weight markers in kDa are indicated on the left.

Fig 3. Protein analysis in WT, Cdk2KO, and Cdk2^T160A testis.

Western blot analysis of testis lysates extracted from p56 wild type (lanes 1–3), Cdk2KO (lanes 4–6) or Cdk2^T160A (lanes 7–9) mice. Different biological replicates are used for each lane shown. gamma-TUBULIN, GAPDH, and HSP90 are shown to indicate equal loading of each lane. Molecular weight markers in kDa are shown on the left.

Fig 4. Endonuclease activity of PMS2.

The endonuclease activity of PMS was measured in vitro using nuclear extracts from p53^ko/ko;Cdk2^ko/ko, p53^ko/ko, Pms2^ko/ko, and wild type (WT) MEFs. Heteroduplexes with either a 5’ or a 3’ nick were digested with AseI and PstI after incubation with nuclear extracts. Products of the digestions were run on agarose gels. The endonuclease activity of PMS2 leads to repair at the PstI site resulting in a 0.8kb and 1.2kb fragment. The far left lane shows a molecular weight marker. Controls included “plasmid only” and “no protein”.

Fig 5. CDK2 phosphorylates full-length MLH1.

(A) CDK2/cyclin A2 kinase assay directed against GST-tagged fusion MLH1 (all lanes). Increasing amounts of GST-MLH1 are used in lanes 1–3 as indicated. Upper panel shows the phospho-image of MLH1 and represents the amount of radiolabelling occurring following phosphorylation by CDK2/cyclin A2. The lower panel shows the Coomassie staining of radiolabelled proteins to indicate equal loading in each lane. (B) Sequence alignment of the evolutionary conservation of Thr495 in human MLH1. Amino acid positions are labelled relative to the transcripts from H. sapiens. The CDK consensus site is highlighted using the green box but is not conserved in the M. musculus, B. taurus, and G. gallus sequences.

Fig 6. HEI10 as a potential CDK2 substrate.

CDK2/cyclin A2 kinase assay directed against GST-tagged fusion peptides of HEI10 Ser244 (lane 3) or Ser257 (lane 5) or their serine-alanine point mutants (lanes 4 and 6, respectively). Histone H1 is shown as a positive control to indicate kinase activity of CDK2/cyclin A2 (lane 1) whereas GST tag without fusion peptide is shown in lane 2 as a negative control. Panel A shows the Coomassie staining of radiolabelled proteins to indicate equal loading in each lane. Also shown is the detection of recombinant GST-HEI10 protein using anti-HEI10 antibodies (lane 8). Panel B shows the phospho-image of each peptide (lanes 1–6) and full length HEI10 protein (lane 7) and represents the amount of radiolabelling occurring following phosphorylation by CDK2/cyclin A2.

Fig 7. Other potential meiotic substrates of CDK2.

Sequence alignment of the evolutionary conservation of various potential CDK consensus sites in meiotic proteins: BLM Thr777/Ser725 (A), BRCA2 Thr64/Ser93/Thr3111 (B), Exo1 Ser806 (C), RAD1 Ser182 (D), and MER3 Thr383 (E). Amino acid positions are labelled relative to the transcripts from Mus musculus. The CDK consensus site in each sequence is highlighted by a green box.