ENDOGENOUS SUBSTRATE PROTEINS FOR cAMP, Ca²⁺-CALMODULIN AND Ca²⁺-PHOSPHOLIPID-DEPENDENT PROTEIN KINASES, AND FOR Ca²⁺-DEPENDENT

CALMODULIN-STIMULATED PROTEIN PHOSPHATASE IN RAT PANCREATIC ISLETS

Abstract

Whole homogenates of rat islets of Langerhans were incubated with [γ-³²P] ATP to investigate endogenous substrate proteins for three major protein kinases, cAMP-dependent (CAMP-PK-A), Ca²⁺-calmodulin-dependent (CaM kinase), and Ca²⁺-phospholipid-dependent protein kinases (PK-C) . Phosphorylated proteins were separated by SDS-polyacrylamide gel electrophoresis and assesed by autoradiography. Endogenous substrates for Ca²⁺-dependent calmodulin-stimulated protein phosphatase were also studied. Substrates of cAMP-PK-A were 15 proteins of 15 kDa et al. CaM kinase substrates were 13 proteins of 16 kDa et al. PK-C substrates were seven proteins of 16 kDa et al. Three proteins, 21, 30 and 31 kDa, were also common substrates for cAMP-PK-A, CaM kinase and PK-C. In addition, common substrates for cAMP-PK-A and CaM kinase were four 17, 20, 32 and 60 kDa proteins, and those for CaM kinase and PK-C were four 16, 22, 42 and 70 kDa proteins. The Ca²⁺-dependent calmodulin-stimulated protein phosphatase substrates were proteins that included most of the CAMP-PK-A substrates, and two 38 and 47 kDa proteins. Multiple interactions between the cAMP and Ca²⁺ second messenger systems, and the phosphorylation and dephosphoylation of islet proteins may be important to the release of insulin.