| Abstract: | Protein I from intestinal epithelium is biochemically and immunologically related to the fibroblast 36-kDa substrate of the Rous sarcoma virus-encoded tyrosine protein kinase (Gerke and Weber (1984) EMBO J. 3, 227-233). Protein I is a Ca2+-binding protein containing two copies each of a 36- and 10-kDa subunit. Denaturation/renaturation experiments show that the 36-kDa subunit is a monomer, whereas the 10-kDa subunit forms a dimer. Mixing of the subunits leads to reconstituted protein I. Physicochemical properties of protein I and its isolated subunits reveal a Ca2+-dependent conformational change in the 36-kDa subunit which involves the exposure of 1 or more tyrosine residues to a more aqueous environment. This change points to a Ca2+ binding constant of about 10(4) M-1 in the presence of 2 mM Mg2+ and induces the ability of protein I and the 36-kDa subunit to bind in vitro to F-actin and nonerythroid spectrin. The same high Ca2+ requirement has been reported for the in vitro tyrosine phosphorylation of a 35-kDa protein from A-431 carcinoma cells by the epidermal growth factor receptor kinase (Fava and Cohen (1984) J. Biol. Chem. 259, 2636-2645). Here we show that this 35-kDa substrate is biochemically and immunologically related to the 36-kDa subunit of protein I, which in turn corresponds to the substrate of the Rous sarcoma virus kinase. The protein of A-431 cells exists not only as a monomer but also as a dimer. The latter fraction contains a 10-kDa polypeptide immunologically related to the corresponding subunit of protein I. Given past results on the A-431 system, we speculate that the monomer rather than the dimer is the preferred in vitro substrate for the epidermal growth factor receptor kinase. Thus, the 10-kDa subunit, which induces dimerization of the phosphorylatable large subunit, may act as an inhibitor. |
