Protein phosphorylation is a central regulatory mechanism in countless cellular processes. Deletion of the PP1 serine/threonine phosphatase gene Ppp1cc in mice results in male infertility due to a severe impairment in spermatogenesis. This disruption in spermatogenesis is hypothesized to arise due to a deficiency of the testis specific Ppp1cc isoform PPP1CC2. To learn more about the function of PPP1CC2 in spermatogenesis, we have employed several proteomic approaches aimed at identifying both regulatory proteins and substrates that interact with PPP1CC2 in the testis. First, we created transgenic mouse embryonic stem cell lines expressing a tandem affinity tagged version of PPP1CC2. Tandem affinity purification using these cell lines identified a number of known PP1 interacting proteins, and one novel interactor DDOST (dolichyl-di-phosphooligosaccharide-protein glycotransferase) which we hypothesize to have a role in spermatogenesis. In a second approach, we conducted GST pull down assays from mouse testis lysates to identify PPP1CC2 interacting proteins. TSSK1 (testis-specific serine kinase 1) was identified as a novel PPP1CC2 interacting protein. We then demonstrated that TSSK1 interacts with PPP1CC2 in an indirect manner via a common interacting protein TSKS (testis-specific serine kinase substrate). Binding of TSKS to PPP1CC2 is regulated via phosphorylation of a PP1 docking motif on the TSKS surface, and localization of TSSK1 and TSKS in the testis is disrupted in Ppp1cc mutants. Finally, to identify candidate substrates of PPP1CC2 in the testis, we conducted a comparative phosphoproteomic analysis and identified 33 different peptides that were hyperphosphorylated in the testis of 3 week old Ppp1cc knockout mice. Amongst these candidate substrates are several proteins essential for mouse spermatogenesis—HMGA1 (high mobility group AT-hook 1), HSPA4 (heat shock protein 4), YBX2 (Y box protein 2) and SYCP2 (synaptonemal complex protein 2). Taken together, our results suggest that PPP1CC2 interacts with a number of different proteins in the testis, and is likely to play a role at several different stages of spermatogenesis, in both meiotic and post-meiotic spermatogenic cells.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/43653 |
| Date | 13 January 2014 |
| Creators | MacLeod, George Graham |
| Contributors | Varmuza, Susannah |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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