A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

Abstract

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-rc lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p6ov-srC and related oncogene products.Cell transformation by Rous sarcoma virus (RSV) is mediated by the v-src gene product p60vsrc which is a tyrosine-specific kinase (3, 21, 34) located primarily at the cytoplasmic face of the plasma membrane of transformed cells (31). Many cellular proteins have been found to contain increased levels of phosphotyrosine in RSV-transformed cells and are thus considered as potential substrates of p60v-src. These proteins include p50 (21, 41) and a 120-kilodalton (kDa) protein (32, 33) which are detected by their association with p60vsrc. Other potential substrates are calmodulin (13), vinculin (1, 48), ezrin (15), talin (9, 43), and the fibronectin (FN) receptor (20), which are identified by immunoprecipitation with specific antibodies. Some other substrates, like a 36-kDa protein (calpactin I) (44) and glycolytic enzymes (6), were found by two-dimensional gel analysis. However, most of these proteins have low stoichiometries (1 to 6%) of phosphorylation (5,6,48). Moreover, many of these proteins are phosphorylated on tyrosine in nontransformed cells infected with RSV mutants that encode nonmyristoylated p60vsrc.Recently, a sensitive method to detect phosphotyrosine (ptyr)-containing proteins has been developed by using antiptyr antibodies (4,12,40,42,46,50). With this technique, a more comprehensive picture of overall changes has been obtained for cellular proteins phosphorylated in RSV transformation, and this has led to identification of some new ptyr-containing protein species whose phosphorylation correlates with transformation (16,18,26,36,37,45,52).We have also analyzed the subcellular localization of ptyr-containing proteins in cells transformed with RSV by cell fractionation followed by immunoblotting with an antiptyr antibody (18). Most ptyr-containing proteins were present in the plasma membrane matrix structures, where p60v-src is localized (2, 18). Furthermore...

Biochemical and cytological changes associated with expression of deregulated pp60src in Xenopus oocytes.

“…Myristoylation of the deregulated pp60( is required to elicit all of the effects that we see in injected oocytes. Our findings support the hypothesis that phosphorylation of a substrate of pp60c`at or near the plasma membrane is involved with morphological changes associated with transformation (34,59). Our findings additionally suggest that the availability of potential substrates for pp60'S is regulated during the cell cycle.…”

v-Src suppresses SHPS-1 expression via the Ras-MAP kinase pathway to promote the oncogenic growth of cells

“…It was somewhat unexpected, however, that our antibodies detected only a trace of SHPS-1 protein in SR3Y1 compared to that of 3Y1 (Figure 1c). To exclude the inhibitory e ect of tyrosine phosphorylation of SHPS-1 on antibody binding, tyrosine phosphorylated glycoproteins puri®ed from SR3Y1 by WGA agarose (Hamaguchi et al, 1990) was dephosphorylated by potato acid phosphatase (PAP) and, subsequently, immunoblotted with anti-SHPS-1 antibodies. As shown in Figure 1d, PAP treatment did not enhance SHPS-1 detection by the antibody raised against type-c GST-SHPS-1 in SR3Y1, although tyrosine phosphorylated glycoprotein of 130 kDa (Hamaguchi et al, 1990) was dephosphorylated.…”

“…By use of mutant src genes, several cellular proteins have been described as transformation-relevant substrates of v-Src kinase. These include; b-catenin (Hamaguchi et al, 1993a); glycoproteins of 95 kDa and 130 kDa (Hamaguchi et al, 1990;Kozuma et al, 1990); connexin-43 (Crow et al, 1992;Kanemitsu et al, 1997); 120-kDa protein (Linder and Burr, 1988). Thus, mutant src genes have been utilized as an e cient tool to characterize substrate proteins.…”

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

“…Tyrosine phosphorylation of proteins with apparent Mr of 130-135 kD (27,40), 120 kD (53), and 95 kD (40) has recently been found to correlate with v-src-induced transformation. It was thus of interest to see whether the inhibition of transformation of the 2R cells by DFMO was associated with changes in tyrosine phosphorylation of such proteins.…”