FUNCTIONALLY IMPORTANT PKA PHOSPHORYLATION SITE IN A KIR3 CHANNEL SUBUNIT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 CAMP依赖的蛋白激酶(PKA)使Kir3通道的活性增强，增强通道与PIP2的相互作用，而蛋白激酶C(PKC)的磷酸化则导致相反的作用。我们利用质谱仪鉴定了蛋白激酶处理后Kir3.1通道亚单位内的磷酸化位点。我们重点研究了Kir3.1C-末端的胞液结构域，据报道该结构域受几个调节器的调节。在体外，PKA的磷酸化在用磷蛋白染色处理时显示出令人信服的信号。磷酸化的C末端用MALDI-TOF/MS进行了质谱分析，质量位移为80U的多肽峰可能与磷酸基团的添加有关，然后经过串联MS(MS2和MS3)以确定修饰的位置。利用这种方法，我们确定了S385是一个体外磷酸化位点。该残基突变为丙氨酰残基，导致Kir3.1*电流对H89和forskolin的敏感性降低，这表明Kir3.1通道亚单位的这个新位点在PKA对其调节中起到了体内作用。 [注]以下手稿已被接受发表在《欧洲生理学杂志》上：Rusinova，R.；沈，Y.-M.A.；DOLIOS，G.；Padovan，J.C.；Yang，H.；Kirchberger，M.；Wang，R.；Logothetis，D.E.质谱分析显示Kir3通道亚基中具有重要功能的PKA磷酸化位点[2008]。

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].