Cell surface phosphorylation by a novel ecto-protein kinase: A key regulator of cellular functions in spermatozoa

Abstract

Since 1976 many studies have been reported on the occurrence and functional significance of ecto-protein kinases in a variety of cell types although their precise biochemical identity is largely unknown. This study reports for the first time purification to apparent homogeneity of an ecto-protein kinase (ecto-CIK) and some of its characteristics using caprine sperm as the cell model. The ecto-CIK is a unique membrane-specific serine/threonine protein kinase. It is a strongly basic 115 kDa protein made up of two subunits: 63 and 55 kDa. The ecto-kinase undergoes a remarkable lateral movement on the outer cell surface culminating in capping on the sperm acrosomal tip. MPS, its major protein substrate is also located on the acrosomal tip. Both ecto CIK and MPS serve as potential regulators of flagellar motility. This novel enzyme appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane-bound proteins.

Ecto-Cyclic AMP Independent Protein Kinase-A Potent Regulator of L-type Calcium Channel and Forward Motility of Goat (Capra indicus) Epididymal Spermatozoa

“…The membrane preparation was finally dispersed in 25 mM potassium phosphate buffer, pH 7.0, containing 1 mM PMSF, 2 mM β-mercaptoethanol, 1 mM EDTA 30% (v/v) glycerol and were stored at -20°C. The protein content of the plasma membrane was estimated using BSA standard [40] Purification of membrane-bound Ecto-CIK The ecto-CIK was purified to apparent homogeneity from the plasma membrane of mature cauda epididymal spermatozoa [41]. Isolated plasma membrane was treated with 1% Triton X-100 at 4°C for 1 hour for the solubilization of proteins.…”

“…Antiserum against the purified CIK was raised in rabbit by 4 successive injections at the 1 st , 7 th , 15 th , and 21 st days as described earlier [41]. First injection was given subcutaneously using 500 μg of CIK in complete Freund's adjuvant.…”

“…The incubation was carried out at 37°C for 5 min. When casein was used as substrate, the reaction was stopped by adding 0.1 ml 0.5% casein as carrier protein containing 250 mM Kphosphate-10 mM ATP and 2 ml 10% TCA [41]. The radiolabelled protein was recovered by filtration through whatman no 1 filter paper washed with 5% TCA dissolved in scintillation fluid and counted for radioactivity.…”

“…Thereby, it demonstrates that CIK is a unique membrane protein-specific kinase, which specifically phosphorylates serine and threonine residues of the sperm outer cellsurface phosphoproteins [35]. It was further demonstrated that ecto-CIK plays a vital role in the regulation of sperm forward progression and velocity through its substrate protein (MPS) [33][34][35][36]. The aim of this present study is to investigate the role of the phosphorylation event mediated by the CIK on the activation of forward motility in the epididymis through plasma membrane bound voltage dependent L type calcium channel.…”

“…Studies from our laboratory have provided several lines of evidences for the occurrence of a cyclic AMP-independent protein kinase (ecto-CIK) on the external surface of caprine (Capra indicus) cauda epididymal mature spermatozoa that causes phosphorylation of the membrane-bound phosphoproteins [30][31][32]. Our recent studies using caprine sperm as the model have described for the first time the purification to apparent homogeneity of an ectoprotein kinase [33] as well as its major phosphoprotein substrate: MPS [34] located on the sperm external surface. Ecto-CIK is a 115 kDa protein made up of two subunits: 63 kDa and 55 kDa.…”