Identification of goat sperm ecto‐cyclic AMP independent protein kinase substrate localized on sperm outer surface

Abstract

We have demonstrated the location of a cyclic AMP independent serine/threonine protein kinase (ecto-CIK) on the outer surface of mature goat spermatozoa. We purified and characterized the major physiological protein substrate (MPS) of ecto-CIK. 32P-labeled membrane proteins phosphorylated by endogenous ecto-CIK of intact cauda-epididymal spermatozoa was solubilized with 1% Triton X-100 and then fractionated by following several chromatographic techniques like Sephacryl S-300 molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography and chromatofocussing. The MPS of ecto-CIK has been purified to apparent homogeneity and it was found to be a monomeric protein of 100 kDa. Three isoforms of MPS have been found with pI of 6.37, 6.05, and 5.14 and all these isoforms served as the specific substrate of ecto-CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate. Addition of MPS (pI 5.14) antibody caused head-to-head sperm agglutination. The Fv/Fab fragment of anti-MPS caused significant inhibition of sperm motility. The data show that MPS is an ecto-protein localized on the sperm head. MPS may thus play an important role for the regulation of sperm-egg interaction.

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“…In another report, two 36 kDa proteins (36 k-A and 36 k-B) were phosphorylated in a cAMP-dependant manner at serine residues, in hamster sperm flagella during an increase in the hyperactivated motility [69]. A ser/thr protein kinase, ecto-cyclic AMP-independent protein kinase (ecto-CIK) has been localized on the outer surface of mature goat spermatozoa [70]. Its substrate MPS (major physiological substrate) has been characterized and localized in sperm cell and is speculated to play an important role in sperm-egg interaction in this species [70].…”

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

“…Likewise, a cAMP-independent serine/threonine phosphorylation has been described. Thus, an ecto-cAMPindependent protein kinase has been located on the outer surface of mature goat spermatozoa (Maiti et al 2004) and a proline-directed serine/threonine phosphorylation associated to mouse sperm capacitation has been reported (Jha et al 2006).…”

Identification and characterization of a sperm motility promoting glycoprotein from buffalo blood serum

“…The serum FMSF is clearly different from FMP because the molecular mass of the former is markedly higher than the latter. Sperm-bound proteins such as 34 KDa hyaluronic acid binding protein (Ghosh et al, 2002), 36 KDa ectophosphoprotein phosphatase (Barua et al, 2001), and 100 KDa ecto-phosphoprotein: the substrate of sperm outer surface cyclic AMP-dependent protein kinase (Maiti et al, 2004) have been implicated to activate sperm flagellar motility. A 52 KDa glycoprotein from porcine follicular fluid and 58 KDa porcine blood serum antithrombin III also enhance sperm motility (Lee et al, 1992(Lee et al, , 1994.…”