Rapid PKA‐catalysed phosphorylation of boar sperm proteins induced by the capacitating agent bicarbonate

Abstract

In boar spermatozoa, the capacitating agent bicarbonate has been shown to induce rapid changes both in plasma membrane lipid architecture and in motility; in each case, a PKA-dependent pathway is involved. Early bicarbonate-induced changes in protein phosphorylation were probed using a commercial antibody against the phosphorylated form of the consensus substrate site for cyclic AMP-dependent protein kinase. The antibody detected relatively few bands in sperm extracts, of which only a small number showed incubation-dependent changes. While the quantitative response varied between boar ejaculates, in general terms bicarbonate induced phosphorylation increases in bands of 96, 64, and 59 kDa within 80 sec. The changes reached a maximum after about 160 sec, declined somewhat thereafter, and then increased again slowly as incubation progressed further (up to 21 min). The bicarbonate-induced increases were strongly dependent on the presence of BSA in the incubation medium. They were inhibited by H89 (PKA inhibitor) but not by GF (PKC inhibitor), and were enhanced by papaverine (phosphodiesterase inhibitor) and by calyculin (protein phosphatase inhibitor). The cyclic AMP analogue cBIMPS was able to mimic bicarbonate action though its effect was less dramatic. Stearated Ht31, a permeable inhibitor of PKA's binding to A-kinase anchoring protein, did not affect either the intensity or the specificity of the bicarbonate-induced phosphorylation changes, though it blocked motility entirely. Immunocytochemical studies revealed marked bicarbonate-dependent phosphorylation changes in the post-acrosomal region of the head and in the neck, midpiece, and anterior regions of the tail. Fractionation of stimulated spermatozoa showed that all bands detectable with the antibody were bound to heads and to midpieces and associated large tail fragments; no bands were detected in either small tail or membrane fragments or in the cytoplasmic fraction. Differential extraction of the midpiece/large tail fraction revealed two protein bands with closely similar electrophoretic mobilities to the 96- and 59-kDa phosphorylated bands; MALDI-TOF analyses of these bands revealed both to be members of the Odf2 family.

Calyculin A-Sensitive Protein Phosphatases are Involved in Maintenance of Progressive Movement in Mouse Spermatozoa In Vitro by Suppression of Autophosphorylation of Protein Kinase A

“…However, there are only limited data regarding cAMP-dependent Ser/Thr phosphorylation in sperm proteins (chum salmon [42], boar [43,44], sea urchin [45], human [46] and mouse [47]). In this study, incubation with cBiMPS (0.1 mM) induced Ser/Thr phosphorylation of the sperm flagellar proteins, including the >250-kDa, 170-kDa, 155-kDa, 140-kDa and 42-kDa substrate proteins of AGC family kinases (Fig.…”

Changes in content and localization of proteins phosphorylated at tyrosine, serine and threonine residues during ram sperm capacitation and acrosome reaction

“…Besides the well-characterized protein kinase A (PKA), other Ser/Thr kinases and the ERKs have been involved in sperm capacitation and acrosome reaction (Luconi et al 1998, du Plessis et al 2001, Liguori et al 2005. More recently, specific targets to PKA phosphorylation has been established in human (O'Flaherty et al 2004, Moseley et al 2005) and boar sperm (Harrison 2004). Likewise, a cAMP-independent serine/threonine phosphorylation has been described.…”

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

“…Moreover, these mice demonstrated no increase in protein tyrosine phosphorylation during capacitation. Recently, using antibodies against the phosphorylated form of the consensus target sequence, the role of PKA-dependent phosphorylation has further been established in human (16,17) and boar sperm (18). Increased phosphorylation is a prerequisite for the initiation of hyperactivated movement, a key attribute of the capacitated state (19,20).…”