Rossomando A J, Wu J, Michel H, Shabanowitz J, Hunt D F, Weber M J, Sturgill T W
Department of Microbiology, University of Virginia, Charlottesville, VA 22908.
Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5779-83. doi: 10.1073/pnas.89.13.5779.
Tyrosine phosphorylation of 42-kDa mitogen-activated protein kinase (p42mapk) occurs during expression of the recombinant protein in Escherichia coli, as well as during in vitro phosphorylation of the protein purified from this source. Structural analyses were performed to identify the site(s) of tyrosine phosphorylation of recombinant p42mapk, both during expression of the protein in E. coli and during in vitro incubations with ATP/Mg2+/Mn2+. Mass spectrometry and phosphopeptide mapping showed that tyrosine phosphorylation of recombinant p42mapk occurs on Tyr-185, the site of regulatory tyrosine phosphorylation that occurs in mitogen-stimulated mammalian cells.
在大肠杆菌中表达重组蛋白的过程中，以及在体外对从该来源纯化的蛋白进行磷酸化的过程中，42 kDa丝裂原活化蛋白激酶（p42mapk）都会发生酪氨酸磷酸化。进行了结构分析，以确定重组p42mapk在大肠杆菌中表达蛋白期间以及在与ATP/Mg2+/Mn2+进行体外孵育期间酪氨酸磷酸化的位点。质谱分析和磷酸肽图谱显示，重组p42mapk的酪氨酸磷酸化发生在Tyr-185上，这是在有丝分裂原刺激的哺乳动物细胞中发生调节性酪氨酸磷酸化的位点。
