ID OF IN VITRO PHOSPHORYLATION SITES WITHIN ELONGATION FACTOR 2 KINASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。延伸因子2(EF2)是参与蛋白质合成的关键成分。一旦被特定的钙/钙调蛋白依赖的蛋白激酶EF2K(EF2K)磷酸化，EF2就完全失活。EF2的磷酸化的生理作用尚不清楚，但可能涉及对分裂细胞的细胞生长和未分裂细胞的局部蛋白质合成的调节。EF2K的周转似乎受其磷酸化状态的调节，可能是通过未知的激酶将DSGYPS基序(残基439-444)磷酸化而将酶靶向泛素化/蛋白小体途径。这些激酶可能与PKA协同作用来调节该酶。正在进行的研究的目标是确定能够磷酸化EF2K的蛋白激酶，无论是在DSGYPS基序中还是在其他地方。在目前的工作中，AMP激酶被证明可以磷酸化EF2K。体外与该激酶孵育后，对EF2K进行消化和质谱分析，以发现是否有磷酸化位点(S)。我们的数据表明，EF2K在394-400区域内的一个位置被AMP激酶磷酸化。确定磷酸化的确切位置的工作正在进行中。

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. Elongation factor 2 (EF2) is a key component involved in protein synthesis. Upon phosphorylation by the specific Ca2+/calmodulin-dependent protein kinase EF2 kinase (EF2K), EF2 is completely inactivated. The physiological role of phosphorylation of EF2 is unknown but may involve regulation of cell growth in dividing cells and of local protein synthesis in non-dividing cells. Turnover of EF2K appears to be regulated by its phosphorylation state, possibly by targeting of the enzyme to a ubiquitination/proteosome pathway by phosphorylation of the DSGYPS motif (residues 439-444) by unknown kinases. These kinases may act in concert with PKA to regulate the enzyme. The goal of ongoing studies is to identify protein kinases that are able to phosphorylate EF2K, either within the DSGYPS motif or elsewhere. In the current work, AMP kinase was shown to phosphorylate EF2K. After incubation with the kinase in vitro, EF2K was digested and analyzed by mass spectrometry to discover site(s) of phosphorylation, if any. Our data indicates that EF2K is phosphorylated by AMP kinase at one site within the region 394-400. Work to identify the exact site of phosphorylation is ongoing.