No abstract

The CI subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S-ethyl-cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic-AMP-dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2 + . These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A 'multiphosphorylation loop' uniquely present in this but not in the homologous p subunit contains all the phosphoserine residues so far identified in the a subunit. Separation of the subunits by HPLC has shown that the CI subunit contains 2.7 f: 0.4 mol phosphate/mol protein 131. In the test tube, this subunit can be phosphorylated additionally by a variety of protein kinases. Best studied are phosphorylations catalyzed by the cyclic-AMP-dependent protein kinase as well as by phosphorylase kinase itself, termed selfphosphorylation. The surrounding sequence with only one serine in the c1 subunit, phosphorylated by the cyclic-AMPdependent protein kinase, is known [4] ; the sites phosphorylated by self-phosphorylation, by cyclic-GMP-dependent protein kinase, by Ca2+-calmodulin-dependent protein kinase as well as by casein protein kinase are unidentified as yet By protein and cDNA sequencing, we have determined recently the complete primary structure of the CI subunit comprising 1237 amino acids [9]. This knowledge is the basis by which to localize phosphorylation sites in the primary structure of this high-molecular-mass protein.If self-phosphorylation is a mechanism occurring in the intact cell, we would expect to find identical sites labelled [5 -81.