Multiple Roles of Protamine Kinase SRPK1 and Phosphatase PP1γ in Sperm Development

Protein phosphorylation regulates key events of male germ cell differentiation. In the testis, SRPK1 and PP1γ are involved in protamine phosphorylation and dephosphorylation, respectively, and their deregulation is linked to defective chromatin structure and male infertility. The aim of this study was to expand our knowledge on the molecular role of these phosphorylation-modulating proteins through the identification of novel partners in the testis, potentially supporting additional roles in sperm development. Using immunoprecipitation followed by LC-MS/MS, we identified yet undescribed interactions of SRPK1 and PP1γ with proteins involved in sperm formation and regulation of spermatogenic gene expression. We reported that SRPK1 interacts with the key splicing regulator ASF/SF2, as well as with other proteins involved in posttranscriptional regulation such as PABP1 or IGF2BP3. We showed that the testis-enriched isoform 2 of PP1γ (PP1γ2) interacts with PIHI1D1, part of the R2TP chaperone complex, and predicted that ZFR, NDUFB10 and ILF2 interactions with PP1γ2 happen within the C-terminal sequence which is specific of PP1γ2 proteoform. Both SRPK1 and PP1γ interactomes include ILF2 and TBLX1R1, with roles in gene expression regulation. Most of the identified proteins are known phosphoproteins in male germ cells, supporting a functional interaction. Overall, our results suggest additional roles for SRPK1 and PP1γ during spermatogenesis, shedding light into the orchestrated regulation of the complex mechanisms driving sperm formation. Data are available via ProteomeXchange with identifiers PXD054959 and PXD054960. SIGNIFICANCE OF THE STUDY: In the present work, we report the first testicular protein interactome of two broad phosphorylation-modulating proteins, the kinase SRPK1 and the phosphatase PP1γ, known to be essential regulators of chromatin remodeling during male germ cell development. Our novel protein-protein interactions reveal additional roles for SRPK1 and PP1γ in male germ cells, in key processes including splicing, gene expression, protein homeostasis, or formation of sperm-specific structures. Our data will therefore contribute to a better understanding of the complex and orchestrated molecular mechanisms controlling testicular activity and proper sperm formation. The lack of male germ cell-specific protein-protein interaction datasets currently challenges the research on the still largely unknown molecular mechanisms driving male germ cell differentiation. This jeopardizes exploratory approaches and in silico validations in a testis-specific manner, as most available datasets have been obtained in somatic cells. Thus, our protein-protein interaction data will be a useful resource to those interested in identifying the origins of male reproductive health problems related to protein phosphorylation. Furthermore, our analyses suggesting that some of these interactions may also occur in other organs extend the relevance of our findings to the broader scientific community.