Protein kinases A and C and phosphatidylinositol 3 kinase regulate glycogen synthase kinase‐3A serine 21 phosphorylation in boar spermatozoa

Abstract

The cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) pathways control most relevant functions in male germ cells including motility. Recently we demonstrated that phosphorylation state of glycogen synthase kinase-3alpha (GSK3A) is also a key event in the control of boar spermatozoa motility. However, the upstream regulators of GSK3A serine phosphorylation (inhibition) in male germ cells remain largely unknown. This work investigates the involvement of PKA, PKC and PI3K pathways in GSK3A phosphorylation in boar spermatozoa. A capacitating medium (TCM) or the phosphodiesterase-resistant cell permeable cAMP analogue 8Br-cAMP cause a significant increase in Ser21 GSK3A phosphorylation associated with a simultaneous significant increase in boar spermatozoa motility. These effects are blocked after preincubation of spermatozoa with PKA inhibitor H89 or PKC inhibitor Ro-32-0432. The PI3K inhibitor LY294002 increases both spermatozoa motility parameters and the basal GSK3A phosphorylation, but does not affect either TCM- or 8Br-cAMP-stimulated GSK3A phosphorylation. PI3K inhibition effects are mediated by an increase in intracellular cAMP levels in boar spermatozoa and are suppressed by PKA inhibitor H89. In summary, we demonstrate that PKA, PKC and PI3K pathways crosstalk in porcine male germ cells to crucially regulate GSK3A phosphorylation which subsequently controls cell motility. In addition, our results suggest that PI3K is upstream of PKA which lies upstream of PKC in this regulatory cascade(s). Our findings contribute to elucidate the molecular mechanisms underlying the regulation of one of the most relevant male germ cell functions, motility.

Protein kinase C activity in boar sperm

“…However, this effect was not observed in calcium depleted media. This result suggests that PKA lies upstream of PKC in the boar sperm intracellular pathway, supporting a previous work by us (Bragado et al, 2010), and that it is also involved in lipid organization of the plasma membrane. Interestingly, PKC-stimulated membrane fluidity is higher when calcium is present in the medium, showing that the canonical calcium pathway involved in membrane fluidity is also present and functional (Fig.…”

“…As expected, membrane acrosome integrity is also affected by those components (Purohit et al, 1999;Visconti et al, 1999;Breitbart, 2002;Ickowicz et al, 2012), but enhanced in each medium by PMA treatment. As an interconnection between PKA and PKC in boar sperm signaling pathway has been suggested (Bragado et al, 2010), we also studied the effect of H89, a PKA inhibitor, on plasma membrane fluidity. Noteworthy, the obtained results suggest the presence of a PKA-dependent and calcium-independent mechanism/pathway involved in the control of the fluidity of boar sperm plasma membrane (see Fig.…”

“…In boar sperm, PKC activity has been studied using different PKC inhibitors, such as Ro-32-0432, assigning a role for PKC in the intracellular signaling involved in motility (Bragado et al, 2010); and staurosporine, bismaleimide I and bismaleimide X, suggesting PKC involvement in the control of sperm cell's volume (Petrunkina et al, 2007). Further, PMA effects studied in spermatozoa from this species showed that PMA effectively increases the Ser/Thr phosphorylation of substrates downstream of PKC (Teijeiro & Marini, 2012) and one of these substrates was identified as AMPK, which is phosphorylated at threonine 172 (Hurtado de Llera et al, 2014).…”

Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways

“…For GSK3a, it was demonstrated that phosphorylation of Ser21 by PKA can lead to its enzymatic inhibition. 40 With regard to Ca 21 signaling, pleckstrin phosphorylation (Thr123: ↑30 5nM ; Ser117: ↑60 5nM , a major PKC target in platelets 41 ) is regulated, whereas CalDAG-GEF1 phosphorylation is regulated at Ser576 (↑10 2/5nM ) and potentially PKA phosphorylated at Ser116 and Ser117 (both: ↑10 2/5nM ) as well as Ser587 (↑30/10 2/5nM ), which is in accordance with our own recent data 20 demonstrating that phosphorylation of CalDAG-GEF1 inhibits Rap1b activation in human platelets. Moreover, cPLA2 phosphorylation is upregulated at Ser727 (↑30 2/5nM ).…”

Insulin Increases Epiblast Cell Number of In Vitro Cultured Mouse Embryos via the PI3K/GSK3/p53 Pathway

“…CT99021 at 1 mM was shown to reduce GSK3b activity to 1% in an in vitro assay in which the inhibitor was shown to be highly specific [32]. H-89 at 10 mM was used to activate GSK3, as it is a robust activator of GSK3 whose inclusion in culture medium has been shown to prevent or inhibit the phosphorylation of GSK3 (which would inactivate GSK3) in embryonic kidney cells at 10 mM [31,50], muscle cells at 50 mM [51], spermatozoa at 100 mM [52], and glioma cells at 10 mM [53]. Pifithrin-a was used to inhibit p53 at 30 mM [38], as it has been established as an effective inhibitor of p53 that blocks the activation of p53 responsive LacZ in ConA cells, and inhibits p53-mediated apoptosis at 10-20 mM [54].…”