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

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Abstract

The epidermal growth factor (EGF) receptor-associated protein tyrosine kinase activity has been suggested to play important roles in the EGF-enhanced, clathrincoated 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 °C and a Km value of 1.7 μm was determined for the reaction. LCa of either neuronal or nonneuronal 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.

References (35)

  • M.N. Khan et al.

    J. Biol. Chem

    (1989)
  • K.A. Lund et al.

    J. Biol. Chem

    (1990)
  • H.S. Wiley et al.

    J. Biol. Chem

    (1991)
  • A.M. Honegger et al.

    Cell

    (1987)
  • F.M. Brodsky et al.

    J. Mol. Biol

    (1983)
  • M.J. Mooibroek et al.

    J. Biol. Chem

    (1987)
  • C. DeLuca-Flaherty et al.

    Cell

    (1990)
  • D. Bar-Zvi et al.

    J. Biol. Chem

    (1986)
  • D. Bar-Zvi et al.

    J. Biol. Chem

    (1988)
  • C.M.E. Litwin et al.

    J. Biol. Chem

    (1991)
  • T. Akiyama et al.

    Biochem. Biophys. Res. Commun

    (1985)
  • R.A. Fava et al.

    J. Biol. Chem

    (1984)
  • J.A. Cooper et al.
  • W.S. Chen et al.

    Cell

    (1989)
  • J.R. Glenney et al.

    Cell

    (1988)
  • A. Sorkin et al.

    J. Biol. Chem

    (1991)
  • K. Helin et al.

    J. Biol. Chem

    (1991)
  • Cited by (6)

    This work was supported by grants from the Medical Research Council of Canada.

    2

    Recipient of an Alberta Heritage Foundation for Medical Research studentship (1984–1988). Present address: Department of Biochemistry, University of Ottawa, Ottawa K1H 8M5, Canada.

    3

    Recipient of an Alberta Heritage Foundation for Medical Research Postdoctoral fellowship (1988–1989) and a Medical Research Council of Canada postdoctoral fellowship (1989–1992).

    4

    Alberta Heritage Foundation for Medical Research scientist.

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