Site‐specific phosphorylation of the purified receptor for calcium‐channel blockers by cAMP‐ and cGMP‐dependent protein kinases, protein kinase C, calmodulin‐dependent protein kinase II and casein kinase II

Abstract

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase I1 and casein kinase 11 phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/ 28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase I1 phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase I1 phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase I1 or casein kinase I1 and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or fivc peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase I1 and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase I1 and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.The calcium current of a variety of cells is modulated by hormones which increase or decrease the current through activation of GTP-binding proteins and/or phosphorylation of the channel protein (see [I] for a recent introduction). cAMP kinase, cGMP kinase and protein kinase C have been identified as in vivo modulators of calcium currents in neuronal and nonneuronal cells [2 -131. Detailed studies in cardiac myocytes [2-41 and neurons [5, 61 show that cAMP kinase increases the calcium current by raising the probability of channel opening and by stabilization of the channel life time. It was even suggested [S] that CAMP-dependent phosphorylation of the channel is a prerequisite for its voltagedependent opening. The importance of the phosphorylation state for the function of calcium channel is further strengthened by the observation that calcium-dependent dephosphorylation of the channel promotes its inactivation [5]. Similar effects to those observed with cAMP b a s e are induced by cGMP kinase in specific neurons of Helix aspera [9]. In contrast, opposing effects of protein kinase C have been observed [lo-131. Protein kinase C increases the number of calcium channels [lo, 1 I] and/or the density of dihydropyridine-bind- ing sites [12] in some cells, whereas in other cells a decrease in calcium current has been reported [13]. These results suggest that, similar to many enzymes involved in energy m...

The Structure of the Skeletal Muscle Calcium Channel

“…The number of the kinase-specific phosphopeptide(s) is shown. See also Jahn et al (1988). Cattcrall, 1985;Flockerzi et al, 1986a,b) and other kinases in vitro O'Callahan et al, 1988;Jahn et al, 1988).…”

“…See also Jahn et al (1988). Cattcrall, 1985;Flockerzi et al, 1986a,b) and other kinases in vitro O'Callahan et al, 1988;Jahn et al, 1988). The α,-subunit is a good substrate for cAMP-kinase and casein kinase II, whereas the 55-kDa subunit is preferentially phosphorylated by protein kinase C and cGMP-dependcnt protein kinase.…”

“…The 165-and 55-kDa subunits of the purified CaCB-receptor are phosphorylated readily by cAMP-dependent protein kinase (Curtis and The purified receptor was phosphorylatcd by each kinase as described by Jahn et al, (1988).…”

“…The α,-subunit is a good substrate for cAMP-kinase and casein kinase II, whereas the 55-kDa subunit is preferentially phosphorylated by protein kinase C and cGMP-dependcnt protein kinase. Two-dimensional peptide maps yield 11 phosphopeptides from the 165-kDa subunit and 11 from the 55-kDa subunit using these kinases (Jahn et al, 1988). With the exception of protein kinase C, each kinase apparently phosphorylates one or two peptides specifically in each subunit (Tabic 1).…”

Cyclic GMP‐Dependent Protein Kinase and Cellular Signaling in the Nervous System

“…Phosphorylation of the a subunit by PKA is activating, and PKG can also phosphorylate the same site with greatly reduced kinetics (Hell et al, 1993(Hell et al, , 1994. The /3 subunit is phosphorylated 10-50 times faster by PKG than PKA (Jahn et al, 1988). There is strong evidence that PKG and cGMP regulate the activity of this channel in intact cells.…”