FUNCTIONAL ANALYSIS OF CA/CALMODULIN-DEPENDENT PROTEIN KINASE II IN PANCREATIC BETS-CELLS WITH CONDITIONAL KNOCKOUT MICE.

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15590949
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Metabolomics
• Research Institution: KUMAMOTO UNIVERSITY
• Principal Investigator: MATSUMOTO Kazuya KUMAMOTO UNIVERSITY HOSPITAL, METABOLISM and ENDOCRINOLOGY, RESEARCH ASSISTANT, 医学部附属病院, 助手 (80346999)
• Co-Investigator(Kenkyū-buntansha): MIYAMURA Nobuhiro KUMAMOTO UNIVERSITY HOSPITAL, METABOLISM and ENDOCRINOLOGY, LECTURER, 医学部附属病院, 講師 (40274716) ; TOYONAGA Tetsushi KUMAMOTO UNIVERSITY, GRADUATE SCHOOL OF MEDICAL SCIENCE, METABOLIC MEDICINE, RESEARCH ASSISTANT, 大学院・医学薬学研究部, 助手 (60295128) ; ARAKI Eiichi KUMAMOTO UNIVERSITY, GRADUATE SCHOOL OF MEDICAL SCIENCE, METABOLIC MEDICINE, PROFESSOR, 大学院・医学薬学研究部, 教授 (10253733)
• Project Period (FY): 2003 – 2004
• Keywords: PANCREATIC BETA CELLS / CALCIUM / CALMODULIN / INSULIN SECRETION / KNOCKOUT MOUSE / PROMOTER ANALYSIS / INHIBITORY PROTEIN

Research Abstract

We have reported that Ca/calmodulin-dependent protein kinase II(CaM kinase II) was involved in insulin secretion. The aim of this study is to further analyze the roles of CaM kinase II in pancreatic beta cells. The 19 kb genomic clone of CaM kinase II delta subunit was isolated from C57/BL6 mouse genomic library and ascertained to contain the exon that encoded initial codon. Restriction enzyme sites were analyzed and three unique restriction enzyme sites were identified at upsteam(Nae I) or downstream(Sex AI, Pfl MI) of the exon. A lox P fragment was amplified, attached by Nae I recognition sequences in both ends and inserted into Nae I site of the clone. Furthermore, thymidine kinase cDNA attached by lox P and Sex AI sequence and diphtheria toxin B cDNA were planned to insert the clone to construct targeting vector. Also endogenous inhibitory peptide for CaM kinase II(CaM KIIN) cDNA was isolated from MIN6 cells. Adenoviral expression vector of CaM KIIN was preparing to analyze the role of CaM kinase II in beta cells. Moreover, promoter region of the CaM kinase II delta subunit gene was analyzed with luciferase reporter gene. The 2.1 kb upstream region from TATA-like sequence seemed to contain promoter activity. Possible transcriptional factor binding sites were identified in the region.