Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

Abstract

A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S. A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

“…It is a relatively abundant protein, and upon transformation of the host cells by RSV, this protein becomes up to 10-fold more phosphorylated on tyrosine than in normal cells. Recent studies by several groups have revealed that the 36K protein is localized in the plasma membrane or the detergent insoluble matrix of the cell or both (8,14,19,32,41). In the transformed cell, only 5% of the total 36K protein is phosphorylated on tyrosine and the localization of this phosphorylated form appears to be no different from the major nonphosphorylated form, within the detection limits of the experiments.…”

The p36 substrate of tyrosine-specific protein kinases co-localizes with non-erythrocyte alpha-spectrin antigen, p230, in surface lamina of cultured fibroblasts.

“…It has become evident from cell fractionation, morphologic and immunochemical studies that p36, or at least a fraction of it, is closely associated with plasma membranes (Cooper and Hunter, 1982;Amini and Kaji, 1983;Courtneidge et al, 1983;Nigg et al, 1983), with detergent-resistant cytoskeletal structures (Cheng and Chen, 1981;Cooper and Hunter, 1982) or, according to one study, with ribonucleoprotein particles (Arrigo et al, 1983). However, distinctly specific interactions, such as that of vinculin, another important tyrosine kinase substrate, and focal adhesion sites (Sefton et al, 1981), have not been found between p36 and other membrane or cytoskeletal components.…”

Epidermal growth factor (urogastrone)-mediated phosphorylation of a 35-kDa substrate in human placental membranes: relationship to the beta subunit of the guanine nucleotide regulatory complex.

“…Nonetheless, once isolated, the 35-kDa substrate from A431 cells could serve as substrate for the EGF(Uro) receptor kinase in a reconstituted system. The 35-kDa substrate from A431-cell membranes appeared to differ from other potential EGF(Uro) receptor substrate(s) of comparable molecular mass (34-39 kDa) that have been detected in 32P-labeled cells after Rous sarcoma virus infection (19)(20)(21)(22)(23)(24)(25)(26)(27) and that have been isolated from intestinal epithelium (28). The relationships of the 35-kDa A431 substrate (19) and the 36-kDa intestinal substrate (28) to the G-protein f subunit remain to be determined.…”