Evidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase A activation during human sperm capacitation.

TitleEvidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase A activation during human sperm capacitation.
Publication TypeJournal Article
Year of Publication2014
AuthorsBattistone MA, Alvau A, Salicioni AM, Visconti PE, Da Ros VG, Cuasnicú PS
JournalMol Hum Reprod
Date Published2014 Sep 1
ISSN1460-2407
Abstract

Sperm capacitation involves an increase in intracellular Ca(2+) concentration as well as in protein kinase A (PKA)-dependent protein tyrosine (Tyr) phosphorylation. Interestingly, in humans, a decrease in extracellular Ca(2+) concentration ([Ca(2+)]e) during capacitation induces an increase in Tyr phosphorylation indicating the complexity of Ca(2+) signaling during this process. In view of this, in the present study we further investigated the Ca(2+)-mediated signaling pathways implicated in Tyr phosphorylation during human sperm capacitation. Results revealed that sperm incubation in a medium without added Ca(2+) (Ө Ca(2+)) increased Tyr phosphorylation but did not modify PKA-mediated phosphorylation. Moreover, inhibition of either PKA or Src family kinase signaling cascades in Ө Ca(2+) downregulated both PKA substrate and Tyr phosphorylations indicating that the [Ca(2+)]e effects on Tyr phosphorylation depend on PKA targets. Inhibition of calmodulin or Ser/Thr protein phosphatase 2B (PP2B) also increased Tyr phosphorylation without affecting PKA-mediated phosphorylation, supporting the potential role of these Ca(2+) downstream effectors in the increase in Tyr phosphorylation observed in Ө Ca(2+). Experiments aimed to identify the kinase responsible for these observations revealed the presence of Proline-rich Tyrosine Kinase 2 (PYK2), a Focal Adhesion Kinase (FAK) family member, in human sperm, and the use of PF431396, a FAK inhibitor, supported the involvement of PYK2 in Tyr phosphorylation downstream of PKA activation. Results also showed that PYK2 was activated in Ө Ca(2+) as well as during capacitation and that PF431396 affected capacitated sperm motility, acrosome reaction, and ability to penetrate both mouse cumulus matrix and zona-free hamster eggs. Together, our observations support PYK2 as an intermediary component of Ca(2+) signaling between PKA-mediated and Tyr phosphorylation that is required for achieving functional human sperm capacitation.

DOI10.1093/molehr/gau073
Alternate JournalMol. Hum. Reprod.
PubMed ID25180269