analysis of calcium signal-induced activation mechanisms of PKC using TIRFM

• Project/Area Number: 14570038
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: General physiology
• Research Institution: HAMAMATSU UNIVERSITY SCHOOL OF MEDICINE
• Principal Investigator: MOGAMI Hideo Hamamatsu University, School of Medicine, associate professor, 医学部, 助教授 (90311604)
• Co-Investigator(Kenkyū-buntansha): IHARA Hayato Hamamatsu University, School of Medicine, assistant professor, 医学部, 助手 (00223298)
• Project Period (FY): 2002 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
• Keywords: Ca^<2+> signal / protein kinase C / insulin / pancreatic β cell / Ca2+ signal / insulin secretion / Protein kinase C

Research Abstract

Protein kinase C (PKC) plays a pivotal role in a myriad of cellular functions. Ten isoforms of PKC have been identified so far are classified into three categories based on structural difference in the regulatory domain. Activation mechanisms of two distinct classes of PKC among them, conventinal PKC (cPKC ; PKCα) and novel PKC (nPKC ; PKCθ), by depolarization-evoked Ca^<2+> influx through voltage dependent Ca^<2+> channels were examined. We have demonstrated simultaneous translocation of both DsRed-tagged PKCα (PKC_α-DsRed) to the plasma membrane and green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) to the cytosol as a dual maker of PKC activity in response to depolarization-evoked Ca^<2+> in the PKCα-DsRed and MARCKS-GFP co-expresing cells. The result indicates that Ca^<2+> influx can generate diacylglycerol (DAG), since cPKC is activated by Ca^<2+> and DAG. We verified this by showing in three different ways : firstly, Ca^<2+> influx-induced translocation of GEP-tagged C1 domain of PKC_γ, secondly, Ca^<2+> influx-induced translocation of GFP tagged pleckstrin homology domain (GFP-PHD) and, thkdly, Ca^<2+> influx-induced translocation of PKCθ-GFP as a marker of DAG production and/or nPKC activity. Thus, Ca^<2+> influx alone via VDCC can generate DAG thereby activating cPKC and nPKC whose activation is independent of Ca^<2+>.

Research Products

• [Publications] H Mogami et al.: "Decoding of Short-Lived Ca^<2+> Signals into Long-Term Substrate Phospholylation through Activation of Two Distinct Classes of Protein Kinase C"journal of biological chemistry. 278. 9896-9904 (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Hideo Mogami, Hui Zhang, Yuko Suzuki, Naoaki Saito, Tetsumei Urano, Itaru Kojima, Ole H.Petersen: "Decoding of Short-Lived Ca^<2+> Influx Signals into Long-Term Substrate Phosphorylation through Activation of Two Distinct Classes of Protein Kinase C"J.Biol.Chem.. 278. 9896-9904 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Mogami H et al.: "Decoding of short-lived Ca^<2+> influx signals into long term substrate phosphorylation through activation of two distinct classes of protein kinase C"The Journal of Biological Chemistry. 278. 9896-9904 (2003)
• [Publications] Hideo Mogami et al.: "Decoding of short-lived CA2+ influx signals into long term substrate phosphorylation through activation of two distinct classes of protein kinase C"The Journal of Biological Chemistry. 278. 9896-9904 (2003)