Activation of calcium and calmodulin dependent protein kinase II during stimulation of insulin secretion

Abstract Pancreatic islets contain an alloxan sensitive, calcium and calmodulin dependent protein kinase (CaM-PK) which may play an important part in the cellular control of insulin secretion. We have studied this activity in islets and the insulin secreting tumor cell line RIN m 5f with particular interest in the changes in kinase activity that accompany stimulation of secretion. Initial experiments showed that the CaM-PK activity enriched in microsomal preparations from RIN cells was similar to the islet cell kinase in terms of apparent endogenous substrates, Ca 2+ and calmodulin dependence, and inactivation by alloxan. For studies of protein substrate specificity, tumor cell CaM-PK was isolated from other kinase activities and substantially purified by affinity chromatography with calmodulin-agarose. The major protein substrates of CaM-PK (54 kD and 57 kD) co-purified with the kinase activity, representing autophosphorylation of subunits of the enzyme. Exogenous substrates phosphorylated by these preparations included microtubule-associated protein 2, synapsin, and glycogen synthase; this pattern of substrate utllization identified the kinase as the Type II multifunctional kinase which has been extensively characterized in brain. A polyclonal antibody to rat brain CaM-PK II was employed to immunoprecipitate the kinase from RIN m 5f cells incubated with secretagogues to measure the effect of stimulation of secretion on autophosphorylation of CaM-PK (which reflects kinase activation). d -Glyceraldehyde (22 mM) and depolarizing concentrations of potassium increased autophosphorylation and insulin secretion in a parallel fashion. Potassium stimulated autophosphorylation was dose dependent and saturable, and was increased to near maximal levels at times as short as 1 min. These studies demonstrate that pancreatic islets and RIN m 5f cells contain a Type II CaM-PK which is activated during the secretion process.