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

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 RINm5f 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, Ca2+ 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 utilization 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 RINm5f 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 RINm5f cells contain a Type II CaM-PK which is activated during the secretion process.