Identification and characterization of two distinct PPP1R2 isoforms in human spermatozoa

Abstract

BackgroundProtein Ser/Thr Phosphatase PPP1CC2 is an alternatively spliced isoform of PPP1C that is highly enriched in testis and selectively expressed in sperm. Addition of the phosphatase inhibitor toxins okadaic acid or calyculin A to caput and caudal sperm triggers and stimulates motility, respectively. Thus, the endogenous mechanisms of phosphatase inhibition are fundamental for controlling sperm function and should be characterized. Preliminary results have shown a protein phosphatase inhibitor activity resembling PPP1R2 in bovine and primate spermatozoa.ResultsHere we show conclusively, for the first time, that PPP1R2 is present in sperm. In addition, we have also identified a novel protein, PPP1R2P3. The latter was previously thought to be an intron-less pseudogene. We show that the protein corresponding to the pseudogene is expressed. It has PPP1 inhibitory potency similar to PPP1R2. The potential phosphosites in PPP1R2 are substituted by non-phosphorylable residues, T73P and S87R, in PPP1R2P3. We also confirm that PPP1R2/PPP1R2P3 are phosphorylated at Ser121 and Ser122, and report a novel phosphorylation site, Ser127. Subfractionation of sperm structures show that PPP1CC2, PPP1R2/PPP1R2P3 are located in the head and tail structures.ConclusionsThe conclusive identification and localization of sperm PPP1R2 and PPP1R2P3 lays the basis for future studies on their roles in acrosome reaction, sperm motility and hyperactivation. An intriguing possibility is that a switch in PPP1CC2 inhibitory subunits could be the trigger for sperm motility in the epididymis and/or sperm hyperactivation in the female reproductive tract.

Not so pseudo: the evolutionary history of protein phosphatase 1 regulatory subunit 2 and related pseudogenes

“…Moreover, the finding of positive and negative selection signatures suggests that it could be functionally relevant. Indeed, we confirmed that two PPP1R2-related proteins are translated in human sperm (PPP1R2P3 and PPP1R2P9), and are heat stable in their native form [48]. The importance of these PPP1R2-related proteins in physiological conditions, such as spermatogenesis and sperm physiology, should be assessed in future studies.…”

“…Gels were stained with Coomassie blue colloidal (Sigma-Aldrich Química, S.A., Sintra, Portugal) using standard procedures [48]. Bands were then excised from the gel using commercial PPP1R2 band as control and destained.…”

Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

“…In these species, PP1γ is located in the postacrosomal region, in the equatorial segment of the head, and in the middle and principal piece of the tail. A recent report by Korrodi-Gregório et al showed a similar pattern [53]. However, our result with OA suggest that this phosphatase may not play a role in sperm motility, as different concentrations of OA did not change the percentage of motile sperm or their motility pattern (data not shown).…”

Post‐Translational Modifications of Histones in Human Sperm

“…The separation of sperm head and tail fractions was performed as described in “PREPARATION OF SAMPLES FOR WESTERN BLOTTING.” We utilized a fraction separation methodology modified according to Lee at al. [1995], Kuretake et al [], and Korrodi‐Gregório et al []. Isolation and the characterization of histones H3 and H4 by MALDI‐TOF MS was performed as previously described [Cincarova et al, ; Legartova et al, ], with minor modifications.…”