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Insulinoma Cells Contain an Isoform of Ca²⁺/Calmodulin-Dependent Protein Kinase II Associated with Insulin Secretion Vesicles¹

Department of Internal Medicine (M.M., S.W., P.M., M.O., P.A.H., H.S., A.P.), BG Kliniken Bergmannsheil, University of Bochum, Medical School, D-44789 Bochum, Germany; Division of Clinical Biochemistry (J.L.), University of Geneva, Medical School, CH-1211 Geneva, Switzerland

Address all correspondence and requests for reprints to: Prof. Dr. Andreas Pfeiffer, Medizinische Klinik und Poliklinik, Berufsgenossenschafliche Kliniken Bergmannsheil, Universitätsklinik, Bürkle de la Camp-Platz 1, D-44789 Bochum, Germany. E-mail: Andreas.Pfeiffer{at}rz.ruhr-uni-bochum.de

The Ca²⁺/calmodulin dependent protein kinase II (CaM kinase II) is thought to play an important part in glucose-stimulated insulin secretion. To determine which of the known subtypes (, ß, , ) occur in insulin-secreting cells, we amplified all types of CaM kinase II by RT-PCR and found the ß₃-, -, ₂- and ₆-subtypes in RINm5F insulinoma cells. None of the other 8 -subtypes was present. Antibodies generated against the bacterially expressed association domain of the ₂-subtype recognized the recombinant and -subtypes. In INS-1 and RINm5F cells, as well as freshly isolated rat islets, only a 55-kDa protein corresponding in size to the ₂-subtype expressed in NIH3T3 fibroblasts was detected. The ₂-subtype therefore appears to represent the predominant subtype of CaM kinase II present in insulin secreting cells. The enzyme was primarily associated with cytoskeletal structures, and very little was present in the soluble compartment or detergent soluble fraction in INS-1- or RINm5F-cells. An analysis of its subcellular distribution was performed by sucrose and Nycodenz density gradient fractionation of INS-1 cells and detection of CaM kinase II by immune blots. The enzyme codistributed with insulin used as a marker for secretory granules but not with the lighter synaptic-like microvesicles detected with an antibody against synaptophysin, plasma membranes (syntaxin 1), lysosomes (arylsulfatase), or mitochondria (cytochrome c oxidase).

CaM kinase II ₂ thus is identified as the subtype associated with insulin secretory granules and is likely to be involved in insulin secretion.

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