FUNCTIONALLY IMPORTANT PKA PHOSPHORYLATION SITE IN A KIR3 CHANNEL SUBUNIT

KIR3 通道亚基中功能重要的 PKA 磷酸化位点

• 批准号: 8361551
• 负责人: Diomedes E. Logothetis
• 金额: $ 0.13万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361551
• 关键词: C-terminal; Cyclic AMP-Dependent Protein Kinases; Exhibits; Forskolin; Funding; Grant; In Vitro; Location; Mass Spectrum Analysis; Modification; Mutation; National Center for Research Resources; Paper; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphoproteins; Phosphorylation; Phosphorylation Site; Principal Investigator; Protein Kinase; Publishing; Regulation; Reporting; Research; Research Infrastructure; Resources; Role; Signal Transduction; Site; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staining method; Stains; United States National Institutes of Health; Work; Yang; cost; in vivo; inorganic phosphate; macromolecule; novel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Phosphorylation of the Kir3 channel by cAMP-dependent protein kinase (PKA) potentiates activity and strengthens channel-PIP2 interactions whereas phosphorylation by protein kinace C (PKC) leads to opposite effects. We utilized mass spectrometry to identify the phosphorylation sites within the Kir3.1 channel subunit upon treatment with protein kinases. We focused on the Kir3.1 C-terminal cytosolic domain that has been reported to be regulated by several modulators. In vitro phosphorylation by PKA exhibited a convincing signal upon treatment with a phosphoprotein stain. The phosphorylated C terminus was subjected to mass spectrometric analysis using MALDI-TOF/MS. Peptide peaks with a mass shift of 80u, which may relate to the addition of a phosphate group, were then subjected to tandem MS (MS2 and MS3) in order to determine the location of the modification. Using this approach, we identified S385 as an in vitro phosphorylation site. Mutation of this residue to an alanyl residue resulted in a reduced sensitivity of Kir3.1* currents to H89 and forskolin, suggesting an in vivo role for this novel site of the Kir3.1 channel subunit in its regulation by PKA. A paper describing this work has been published (Rusinova R, Shen YM, Dolios G, Padovan J, Yang H, Kirchberger M, Wang R, Logothetis DE. Mass spectrometric analysis reveals a functionally important PKA phosphorylation site in a Kir3 channel subunit. Pflugers Arch. 2009 Jun;458(2):303-14).

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