Ca2+/calmodulin-dependent protein kinase II (CaMK11) is expressed in insulin-secreting beta cells. However, the effects of CaMK11 on insulin synthesis are unknown. Although Ser133 phosphorylation of cyclic AMP-responsive element-binding protein (CREB) typically increases CREB transcriptional activity, CaMK11 phosphorylates CREB at Ser142 and at Ser133 to exert a dominant inhibitory effect. Our objective was to characterize the role of CaMK11 in insulin gene expression. In MIN6 cells, insulin gene promoter activity was significantly down-regulated by wild-type (WT) CaMK11 delta 2, but was significantly upregulated after small interfering RNA (siRNA) knockdown of CaMK11 delta expression. These results were independent of glucose concentrations and membrane depolarization. Insulin mRNA levels were also decreased by WT CaMK11 delta 2 and increased by CaMK11 delta siRNA. Downregulation of insulin gene promoter activity by WT CaMK11 delta 2 was partly mediated via cyclic AMP-responsive element 2 (CRE2). CaMK11 delta 2 significantly increased CREB phosphorylation at Ser142 and significantly decreased binding to CREB binding protein (CBP), whereas kinase dead CaMK11 delta 2 did not. Our results indicate that CaMK11 delta 2 downregulates insulin gene expression by Ser142 phosphorylation of CREB and reducing binding of CREB to CBP. (C) 2012 Elsevier Inc. All rights reserved.