Unsaturated fatty acid-activated protein kinase (PKx) from goat testis cytosol.
The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca(2+). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diacylglycerol have no effect either alone or in combination. The membrane fraction does not show any appreciable kinase activity even after detergent treatment. PKx migrates as a single band of apparent molecular mass of 116 kDa on 10% SDS-PAGE after sequential chromatographic separation on DEAE-cellulose, phenyl-Sepharose, high-Q anion exchange and protamine-agarose affinity column. PKx phosphorylates histone H1, histone IIIs and protamine sulfate, but not casein. However, the best phosphorylation was obtained with a substrate based on PKC pseudosubstrate sequence (RFARKGSLRQKNV). The kinase phosphorylates two endogenous cytosolic proteins of 60 and 68 kDa. Ser residues are primarily phosphorylated although a low level of phosphorylation is observed on Thr residues also. Ca(2+) and Mn(2+) inhibit PKx activity in the micromolar range. Staurosporine is found to inhibit the PKx activity to a significant level at sub-nanomolar concentration. Lyso-phosphatidylcholine and certain detergents at very low concentrations (<0.05%) stimulate enzyme activity to some extent. The immuno-crossreactivity study with antibody against different PKC isotypes suggests that the protein kinase under study is not related to any known PKC family. Even the antibody against PKN (a related protein kinase reported in rat testis found to be activated by arachidonic acid) does not cross-react with this protein kinase. Hence we believe that the protein kinase (PKx) reported here is different even from the PKN of rat testis. The phosphorylation of endogenous proteins by the protein kinase may be involved in cell regulation including fertility regulation and signal transduction.
Roy
K
K
Department of Chemistry, Bose Institute, 93/1 A.P.C. Road, Calcutta, India.
Mandal
A K
AK
Sikdar
R
R
Majumdar
S
S
Ono
Y
Y
Sen
P C
PC
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D003636:DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology*; D006041:Goats; D008297:Male; D011494:Protein Kinases / Q000302:isolation & purification*; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
D005231:Fatty Acids, Unsaturated; D016718:Arachidonic Acid; D003636:DEAE-Cellulose; D011494:Protein Kinases