Characterization of Ca(2+)-dependent endogenous phosphorylation of 160,000- and 150,000-Dalton proteins of sarcoplasmic reticulum.
The 160 and 150 kDa proteins of sarcoplasmic reticulum (SR) are phosphorylated endogenously. The phosphorylation of both proteins has a marked requirement for Ca2+. Half-maximal and maximal phosphorylation was obtained at about 1 nM- and 1 microM-Ca2+ respectively, and a Hill coefficient of about 0.5 was calculated. The phosphorylation is also dependent on NaF as an inhibitor of the SR phosphoprotein phosphatase. The phosphorylation of these proteins is very rapid, and maximal phosphorylation is achieved in less than 15 s. The phosphorylation of the 160 kDa and 150 kDa polypeptides is completely inhibited by 5 mM-MgCl2 and by 75 microM-LaCl3, by very low concentrations of different detergents, and by preincubation of the SR for 2 min at 60 degrees C. The inhibition by Mg2+ is due to stimulation of ATP hydrolysis, thereby decreasing ATP concentration. Different phosphorylated peptides were obtained by digestion with protease V8 of the 160 kDa and 150 kDa protein bands, suggesting that the 160 kDa and 150 kDa proteins are distinct. The two phosphorylated proteins are present in different fractions and preparations of SR, with or without [3H]PN200-110 binding capacity. These and other results suggest that the phosphorylated SR proteins are distinct from the alpha 1 and alpha 2 subunits of the voltage-gated Ca2+ channel of the T-system membranes. Different inhibitors and activators of protein kinase C and calmodulin-dependent protein kinase have no effect on the endogenous phosphorylation of both polypeptides, suggesting that the phosphorylation is regulated solely by Ca2+. A possible regulatory function for this phosphorylation system is described in the accompanying paper [Gechtman. Orr & Shoshan-Barmatz (1991) Biochem. J. 276.97-102].
Orr
I
I
Department of Biology, Ben-Gurion University of the Negev, Beer Sheva, Israel.
Gechtman
Z
Z
Shoshan-Barmatz
V
V
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D010750:Phosphoproteins; D011506:Proteins; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D008274:Magnesium; D002118:Calcium