Phosphoproteome analysis of the crosstalk between sumoylation and phosphorylation in mouse spermatocytes

Abstract

Spermatogenesis is supported by various posttranslational modifications. There is growing evidence supporting a crosstalk between sumoylation and phosphorylation in different cell types. We have recently shown that inhibition of global sumoylation with a sumoylation inhibitor (Ginkgolic acid, GA) arrested purified mouse spermatocytes in vitro; the spermatocytes could not condense chromatin and disassemble the synaptonemal complex. Our data have also revealed that some kinases regulating the meiotic prophase (PLK1 and AURKB) were inhibited upon the inhibition of sumoylation. Nevertheless, specific phosphorylated targets affected by the inhibition of sumoylation have not been identified. To address this gap, in this study, we performed a comparative phospho-proteome analysis of the control spermatocytes and spermatocytes treated with the GA. Our analysis has narrowed down to several proteins implicated in the regulation of cell cycle and/or meiosis. Two of these targets, NPM1 and hnRNPH1, were studied further using western blotting in both cell lines and primary cells. Decrease in sumoylaion-dependend phosphorylation of NPM1 on Ser125 regulated by AURKB can be a contributing factor to the inability of spermatocytes to condense chromatin by the end of the prophase and should be studied further.

Figure 1.. Analysis of changes in the proteome of spermatocytes upon inhibition of sumoylation.

A. A representative 2-D gel image used for the analysis. B. Summary of proteins differently expressed upon an inhibition of sumoylation in spermatocytes. Changes in phosphorylation are shown as a phopho-ratio at 30 and 50 μM of GA as compared to the control (DMSO). Potential upstream kinases were identified using PhosphositePlus. Expression in spermatocytes, interaction with SUMO and roles in germ cells were assessed using a literature search.

Figure 2.. NPM1 is regulated by sumoylation.

A1) and A2) Treatment of GC-1 cells with GA (A1) and siRNAs (A2) against UBC9 (E1) and KAP1(E2) caused a decrease in high molecular weight (HMW) SUMO conjugates, and an increase in free SUMO, disappearance of the upper and a significant decrease in the lower NPM1 band. Immunoblots with anti-SUMO and NPM1 (Ser125) antibodies are shown. Anti-actin antibody was used as a loading control. B) Phosphatase treatment caused a decrease in the upper and increase in the lower NPM1 band, suggesting the presence of phosphorylated isoforms at the upper and non-phosphorylated isoform at the lower band. C) Spermatocyte-enriched fraction was treated with 50 and 70μM of GA. The 50μM treatment caused a significant decrease in the phosphorylation of NPM1 without changes in the total protein level. 70μM GA treatment caused a decrease in both isoforms of the protein.

Figure 3.. hnRNPH1 is regulated by sumoylation.

Treatment of GC-1 spermatogona with siRNAs (A1) against UBC9 (E1) and the primary cell with cell line with GA (A2) caused a decrease in all isoforms of hnRNPH1. Anti-tubulin antibody was used as a loading control; for A1, the tubulin antibody was incubated with the blot before the SUMO antibody, and so the tubulin signal is shown on the same image.