INSULINOMA CELLS CONTAIN AN ISOFORM OF CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-II-DELTA ASSOCIATED WITH INSULIN-SECRETION VESICLES/

The Ca2+/calmodulin dependent protein kinase II (CaM kinase II) is tho ught to play an important part in glucose-stimulated insulin secretion . To determine which of the known subtypes (alpha, beta, gamma, delta) occur in insulin-secreting cells, we amplified all types of CaM kinas e II by RT-PCR and found the beta(3)-, gamma-, delta(2)- and delta(6)- subtypes in RINm5F insulinoma cells. None of the other 8 delta-subtype s was present. Antibodies generated against the bacterially expressed association domain of the delta(2)-subtype recognized the recombinant gamma and delta-subtypes. In INS-1 and RINm5F cells, as well as freshl y isolated rat islets, only a 55-kDa protein corresponding in size to the delta(2)-subtype expressed in NIH3T3 fibroblasts was detected. The delta(2)-subtype therefore appears to represent the predominant subty pe of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little wa s present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution wa s performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II delta by immune blots. The enzyme codistributed with insulin used as a marker for secretory granu les but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lys osomes (arylsulfatase), or mitochondria (cytochrome c oxidase). CaM ki nase II delta(2) thus is identified as the subtype associated with ins ulin secretory granules and is likely to be involved in insulin secret ion.