Differential in vitro phosphorylation of clathrin light chains by the epidermal growth factor receptor-associated protein tyrosine kinase and a pp60c-src-related spleen tyrosine kinase.
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.
Mooibroek
M J
MJ
Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Alberta, Canada.
Cheng
H C
HC
Wang
J H
JH
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002966:Clathrin / Q000302:isolation & purification / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity
D002966:Clathrin; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)