Mooibroek M J, Cheng H C, Wang J H
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Arch Biochem Biophys. 1992 Feb 1;292(2):448-55. doi: 10.1016/0003-9861(92)90015-o.
The epidermal growth factor (EGF) receptor-associated protein tyrosine kinase activity has been suggested to play important roles in the EGF-enhanced, clathrin-coated pit-mediated receptor internalization (W. S. Chen, C. S. Lazar, M. Peonie, R. Y. Tsien, G. N. Gill, and M. G. Rosenfeld, 1987, Nature 328, 820-823) but the kinase substrate important for this process has not been identified. This study demonstrates that the EGF receptor, partially purified from A431 epidermoid carcinoma cells, catalyzes the phosphorylation of one of the two clathrin light chains, clathrin light chain a (LCa). The phosphorylation activity is stimulated by EGF and immunoprecipitated by an EGF receptor monoclonal antibody. The phosphorylation occurs exclusively on tyrosine residues. Amino acid composition of the major tryptic phosphopeptide of the EGF receptor-phosphorylated LCa corresponds closely to that of residues 1 to 97 of LCa. A stoichiometry of 0.2 mol phosphate/mol LCa was attained after 60 min at 30 degrees C and a Km value of 1.7 microM was determined for the reaction. LCa of either neuronal or non-neuronal origin could serve as a substrate. In addition to the EGF receptor tyrosine kinase, a particulate src-related protein tyrosine kinase purified from bovine spleen (C. M. E. Litwin, H.-C. Cheng, and J. H. Wang, 1991, J. Biol. Chem. 226, 2557-2566) was shown in this study to also phosphorylate the light chains. However, in contrast to the EGF receptor phosphorylation, both clathrin light chains a and b were phosphorylated by the spleen kinase, suggesting that the two tyrosine kinases have differential site specificities. Given the specificity of LCa phosphorylation by the EGF receptor, we propose that LCa phosphorylation on a tyrosine residue(s) may be important in EGF-induced receptor internalization.
表皮生长因子（EGF）受体相关蛋白酪氨酸激酶活性被认为在EGF增强的、网格蛋白包被小窝介导的受体内化过程中起重要作用（W.S.陈、C.S.拉扎尔、M.皮奥尼、R.Y.钱恩、G.N.吉尔和M.G.罗森菲尔德，1987年，《自然》328卷，820 - 823页），但这一过程中重要的激酶底物尚未被鉴定。本研究表明，从A431表皮样癌细胞中部分纯化得到的EGF受体催化网格蛋白轻链之一，即网格蛋白轻链a（LCa）的磷酸化。磷酸化活性受EGF刺激，并被一种EGF受体单克隆抗体免疫沉淀。磷酸化仅发生在酪氨酸残基上。EGF受体磷酸化的LCa的主要胰蛋白酶磷酸肽的氨基酸组成与LCa的1至97位残基的氨基酸组成密切对应。在30℃下反应60分钟后，达到0.2摩尔磷酸盐/摩尔LCa的化学计量比，反应的米氏常数（Km）值为1.7微摩尔。神经元或非神经元来源的LCa都可以作为底物。除了EGF受体酪氨酸激酶外，本研究还表明，从牛脾脏中纯化得到的一种颗粒状src相关蛋白酪氨酸激酶（C.M.E.利特温、H.-C.程和J.H.王，1991年，《生物化学杂志》226卷，2557 - 2566页）也能使轻链磷酸化。然而，与EGF受体磷酸化不同的是，脾脏激酶使网格蛋白轻链a和b都发生了磷酸化，这表明这两种酪氨酸激酶具有不同的位点特异性。鉴于EGF受体对LCa磷酸化的特异性，我们提出酪氨酸残基上的LCa磷酸化可能在EGF诱导的受体内化中起重要作用。
