Abstract
Epidermal growth factor (EGF)-enhanced protein kinase activity in plasma membrane preparations of A-431 human epidermoid carcinoma cells was shown to involve the phosphorylation of tyrosine residues. Phosphotyrosine was detected in both endogenous membrane proteins and in histone when added as an exogenous protein substrate. The major phosphorylated amino acid in partial acid hydrolysates of 32P-labeled A-431 membranes was identified as phosphotyrosine on the basis of its identical behavior to authentic phosphotyrosine on paper electrophoresis and thin layer chromatography; its 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivative was indistinguishable from that of the authentic compound. Only traces, if any, of phosphoserine or phosphothreonine were detected. [32P]Phosphotyrosine was also detected in pronase digests of 32P-labeled membrane proteins. The EGF receptor . protein kinase complex, which was solubilized with Triton X-100 and purified by EGF affinity chromatography, was shown to phosphorylate tyrosine residues of the isolated membrane protein.
Highlights
From the Department of Biochemisty, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 membrane preparation was purified by Epidermal growth factor (EGF) affinity chromatography
Epidermal growth factor (EGF)-enhanced protkein- decyl sulfate-gel electrophoresis indicated the presence of one nase activity in plasma membrane preparations of A- major protein band of molecular weight 150O, OO and several
Phos-dalton protein band is the receptor for EGF and is a substrate photyrosine was detectedinbothendogenousmemof the phosphorylation reaction
Summary
Materials-EGF and membranes from A-431 cells were prepared by published procedures [5, 13]. A-431, which has been shown to contain an extraordinarily of standard phosphotyrosine were added 5pl of 10m~ dansyl chloride high concentration of membrane receptors for EGF [2, 3]. Standard Phosphorylation Procedure-A-431 membrane preparations and the purified EGF receptor. Pronase Digestion-The phosphorylated, precipitated membrane material (400 pgof protein) was suspended in 100 pl of 10 mM ammonium acetate containing 25 pg of pronase and adjusted with 0.1. The material was dissolved in 20 pl of electrophoresis buffer and aliquots (to which the standard phosphorylated amino acids were added) were subjected to electrophoresis. Kinase complex [8] was phosphorylated in the presence and absence of EGF and the resulting phosphorylated material was subjected to partial acidhydrolysis and electrophoresis.
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