Identification of cell-specific targets of sumoylation during mouse spermatogenesis

Abstract

Recent findings suggest diverse and potentially multiple roles of SUMO in testicular function and spermatogenesis. However, SUMO targets remain uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from freshly purified spermatocytes and spermatids. One-hundred and twenty proteins were uniquely identified in the spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization and in-vitro sumoylation studies. GPS-SUMO software was used to identify consensus and non-consensus sumoylation sites within the amino acid sequences of the proteins. The analyses confirmed the cell-specific sumoylation and/or SUMO interaction of several novel, previously uncharacterized SUMO targets such as CDK1, RNAP II, CDC5, MILI, DDX4, TDP-43 and STK31. Furthermore, several proteins that were previously identified as SUMO targets in somatic cells (e.g., KAP1, MDC1) were identified as SUMO targets in germ cells. Many of these proteins have a unique role in spermatogenesis and during meiotic progression. This research opens a novel avenue for further studies of SUMO at the level of individual targets.

Inhibition of CDK1 activity by sumoylation

“…A sumoylation-dependent phosphorylation and phosphorylation-dependent sumoylation have been identified [23,24,25], and inhibition of sumoylation by the sumoylation inhibitor GA significantly decreased tyrosine phosphorylation of multiple proteins [19]. In accordance with these data, using immunoprecipitation followed by mass spectrometry identification, we have recently identified several kinases as targets of sumoylation in mouse germ cells (meiotic spermatocytes and spermatids) [26]. We have also observed significant changes in germ cell phosphorylation patterns (including specific phosphorylation events required for meiotic progression) upon inhibition of sumoylation with GA (unpublished data).…”

“…We have also observed significant changes in germ cell phosphorylation patterns (including specific phosphorylation events required for meiotic progression) upon inhibition of sumoylation with GA (unpublished data). One of the interesting SUMO targets identified at our published screen was CDK1 kinase, a crucial and indispensable regulator of both mitotic and meiotic G2/M progression [26,27]. An in vitro sumoylation assay supported possible sumoylation of CDK1; and co-immunoprecipitation experiments using mouse germ cell and human HEK cell lysates confirmed possible covalent and non-covalent interactions between CDK1 and SUMO [26,27].…”

“…One of the interesting SUMO targets identified at our published screen was CDK1 kinase, a crucial and indispensable regulator of both mitotic and meiotic G2/M progression [26,27]. An in vitro sumoylation assay supported possible sumoylation of CDK1; and co-immunoprecipitation experiments using mouse germ cell and human HEK cell lysates confirmed possible covalent and non-covalent interactions between CDK1 and SUMO [26,27]. A bioinformatics analysis revealed the presence of the consensus sumoylation site in the amino acid sequence of the mouse but not the human CDK1; However, the alignment of the two sequences revealed a difference in only one amino acid, with a possible target lysine still present at the same position [26,27].…”

“…An in vitro sumoylation assay supported possible sumoylation of CDK1; and co-immunoprecipitation experiments using mouse germ cell and human HEK cell lysates confirmed possible covalent and non-covalent interactions between CDK1 and SUMO [26,27]. A bioinformatics analysis revealed the presence of the consensus sumoylation site in the amino acid sequence of the mouse but not the human CDK1; However, the alignment of the two sequences revealed a difference in only one amino acid, with a possible target lysine still present at the same position [26,27]. Interestingly, another important cell cycle regulator, CDK2 (not identified by our screen), contained no such sequence.…”

Epigenetic: A new approach to etiology of infertility

“…In contrast, the cytoskeleton proteins is modified by SUMO in spermatid. 29 One of the proteins involved in epigenetic modifications in spermatogenesis is SUMO-1. SUMO-1 expression in testis showed another specific function, namely to help the inactivation of sex chromosomes during meiosis.…”