Characterisation of several ankyrin repeat protein variant 2, a phosphoprotein phosphatase 1-interacting protein, in testis and spermatozoa

Abstract

Phosphoprotein phosphatase 1 (PPP1) catalytic subunit gamma 2 (PPP1CC2), a PPP1 isoform, is largely restricted to testicular germ cells and spermatozoa. The key to understanding PPP1 regulation in male germ cells lies in the identification and characterisation of its interacting partners. This study was undertaken to determine the expression patterns of the several ankyrin repeat protein variant 2 (SARP2), a PPP1-interacting protein, in testis and spermatozoa. SARP2 was found to be highly expressed in testis and spermatozoa, and its interaction with human spermatozoa endogenous PPP1CC2 was confirmed by immunoprecipitation. Expression analysis by RT-qPCR revealed that SARP2 and PPP1CC2 mRNA levels were significantly higher in the spermatocyte fraction. However, microscopy revealed that SARP2 protein was only present in the nucleus of elongating and mature spermatids and in spermatozoa. In spermatozoa, SARP2 was prominently expressed in the connecting piece and flagellum, as well as, to a lesser extent, in the acrosome. A yeast two-hybrid approach was used to detect SARP2-interacting proteins and a relevant interaction with a novel sperm-associated antigen 9 (SPAG9) variant, a testis and spermatozoa-specific c-Jun N-terminal kinase-binding protein, was validated in human spermatozoa. Given the expression pattern of SARP2 and its association with PPP1CC2 and SPAG9, it may play a role in spermiogenesis and sperm function, namely in sperm motility and the acrosome reaction.

Disruption of protein phosphatase 1 complexes with the use of bioportides as a novel approach to target sperm motility

“…The molecular difference between these two PP1γ isoforms resides in the carboxyl-terminus (C-terminus) (15). Regulation of PP1 catalytic activity is mediated by forming holoenzymes with regulatory interactors of protein phosphatase one (RIPPOs, previously called PP1-interacting proteins -PIPs) (16)(17)(18)(19)(20)(21). In mammalian sperm, protein phosphatase inhibitor 2 (PPP1R2), a PP1 inhibitor, is central in controlling PP1 activity and, consequently, motility (7).…”

“…In human mature sperm, part of the total PP1 population is bound to PPP1R2 and within these complexes PP1 is inactive (20). PPP1R2 is a highly intrinsically disordered protein that only acquires a defined structure when associated with PP1 (16,18,19). PPP1R2 contains two degenerated RVxF motifs (20,22).…”