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.
Highlights
Protein Ser/Thr Phosphatase PPP1CC2 is an alternatively spliced isoform of Catalytic subunit of protein phosphatase 1 (PPP1) (PPP1C) that is highly enriched in testis and selectively expressed in sperm
Identification of PPP1R2P3 - a novel PPP1 regulator 2 (PPP1R2) isoform in testis The catalytic subunit isoforms PPP1CC1 and PPP1CC2 were used as baits to screen a human testis cDNA library by yeast two-hybrid [2,15]
The PPP1R2P3 mRNA is present in human testis cDNA library repository of the Mammalian Gene Collection (MGC) program [24]
Summary
Protein 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. Protein phosphorylation is a post-translational mechanism that plays a crucial role in sperm physiology, controlling motility, capacitation, hyperactivated motility and the acrosome reaction [1,2]. Low sperm motility is one of the main causes of male role in the acrosome reaction [2,6,7]. Inhibition of PPP1CC2 activity by the toxins okadaic acid or calyculin A induced and stimulated motility in caput and caudal sperm, respectively [3,5]. The evolutionary conservation and the importance of Ser/Thr phosphatases in regulating flagellar motility, is highlighted by the involvement of a PPP1 homolog in the regulation of rooster sperm motility [10] and by the involvement of a Ser/Thr phosphatase in the regulation of microtubule sliding velocity in Paramecium and Chlamydomonas [11,12]
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