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: 7954149
• 负责人: Diomedes E. Logothetis
• 金额: $ 0.12万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954149
• 关键词: C-terminal; Computer Retrieval of Information on Scientific Projects Database; Cyclic AMP-Dependent Protein Kinases; European; Exhibits; Forskolin; Funding; Grant; In Vitro; Institution; Journals; Location; Manuscripts; Mass Spectrum Analysis; Modification; Mutation; Peptides; Phosphoproteins; Phosphorylation; Phosphorylation Site; Physiology; Protein Kinase; Publications; Regulation; Reporting; Research; Research Personnel; Resources; Role; Signal Transduction; Site; Source; Staining method; Stains; United States National Institutes of Health; Yang; 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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. [Note] The following manuscript has been accepted for publication in the European Journal of Physiology: RUSINOVA, R.; SHEN, Y.-M. A.; DOLIOS, G.; PADOVAN, J. C.; YANG, H.; KIRCHBERGER, M.; WANG, R.; LOGOTHETIS, D. E. Mass spectrometric analysis reveals a functionally important PKA phosphorylation site in a Kir3 channel subunit [2008].

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 通过CAMP依赖性蛋白激酶（PKA）对KIR3通道的磷酸化增强了活性并增强了通道PIP2相互作用，而蛋白质Kinace C（PKC）磷酸化会导致相反的影响。我们利用质谱法确定用蛋白激酶治疗Kir3.1通道亚基内的磷酸化位点。我们专注于Kir3.1 C末端胞质结构域，该结构域据报道受几个调节剂的调节。 PKA的体外磷酸化在用磷蛋白染色治疗后表现出令人信服的信号。使用MALDI-TOF/MS对磷酸化的C末端进行质谱分析。然后，将肽峰为80U，可能与磷酸组的添加有关，然后对串联MS（MS2和MS3）进行串联，以确定修饰的位置。使用这种方法，我们将S385确定为体外磷酸化位点。该残基对丙酰基残基的突变导致Kir3.1*对H89和福斯科林的敏感性降低，这表明在PKA调控其调节中，Kir3.1通道亚基的这个新颖位点的体内作用。 [注意]以下手稿已被接受在《欧洲生理学杂志》中发表：Rusinova，R。； Y.-M。Shen一个。; Dolios，G。； Padovan，J.C。; Yang，H。； Kirchberger，M。； Wang，R。; Logothetis，D。E.质谱分析揭示了在KIR3通道亚基[2008]中功能上重要的PKA磷酸化位点。