Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cells

Abstract

We have isolated a soluble 35-kDa protein from A-431 cells that in the presence of calcium can serve as a substrate for the epidermal growth factor (EGF)-receptor/kinase. The purification procedure exploits the reversible, Ca2+-dependent binding of the 35-kDa protein to the A-431 total particulate fraction. The 35-kDa protein was purified by 1) Ca2+-dependent adsorption to A-431 particulate fractions, 2) release by chelation of Ca2+ with ethyleneglycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 3) chromatography on Sephadex G-100, DEAE-cellulose, and CM-cellulose columns. When a plasma membrane preparation from A-431 cells is used as a source of the EGF-receptor/kinase, the phosphorylation of the 35-kDa protein occurs on tyrosine, is greatly enhanced in the presence of EGF, and occurs only when Ca2+ is added to the standard reaction mixture for phosphorylation. Autophosphorylation of the receptor does not require Ca2+. We have postulated that one of the roles of Ca2+ is to facilitate the interaction of the 35-kDa protein with cellular membranes. Ca2+ enhances, but apparently is not essential for, the direct phosphorylation of the 35-kDa protein by the Triton X-100-solubilized, purified EGF-receptor/kinase. Incubation of intact A-431 cells with EGF at 37 degrees C (but not 0 degrees C) enhances the ability of the particulate fraction prepared from these cells to bind and/or phosphorylate the 35-kDa protein. We suggest that this enhancement in the phosphorylation of the 35-kDa protein, a presumed physiological substrate, is associated with the clustering and internalization of the EGF receptor/kinase complex.