FUNCTIONALLY IMPORTANT PKA PHOSPHORYLATION SITE IN A KIR3 CHANNEL SUBUNIT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 cAMP依赖性蛋白激酶（PKA）对Kir 3通道的磷酸化增强活性并加强通道-PIP 2相互作用，而蛋白激酶C（PKC）的磷酸化导致相反的作用。我们利用质谱法来确定与蛋白激酶治疗后的Kir3.1通道亚基内的磷酸化位点。我们专注于Kir3.1的C-末端胞质结构域，已被报道由几种调节剂。在体外磷酸化PKA表现出令人信服的信号处理后，与磷蛋白染色。使用MALDI-TOF/MS对磷酸化的C末端进行质谱分析。然后对可能与磷酸基团的添加有关的具有80 u质量位移的肽峰进行串联MS（MS 2和MS 3）以确定修饰的位置。使用这种方法，我们确定S385作为体外磷酸化位点。该残基突变为丙氨酰残基导致Kir3.1* 电流对H89和毛喉素的敏感性降低，这表明Kir3.1通道亚基的该新位点在PKA调节中的体内作用。 [注]以下手稿已被接受发表在欧洲生理学杂志：RUSINOVA，R.;沈，Y. M.一、DOLIOS，G.;帕多万; YANG，H.; KIRCHBERGER，M.; Wang，R.; Logothetis，D. E.质谱分析揭示了Kir 3通道亚基中功能重要的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].