Thomas Y. Shih

Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry

Using electron microscopic immunocytochemistry, we have investigated the intracellular location of the src protein (p21) in cells transformed by the Harvey strain of Murine Sarcoma Virus (Ha-MSV). Antibodies to p21 were derived from tumor-bearing rats inoculated with Ha-NRK cells. The distribution of p21 in intracellular sites in MDCK dog cells transformed by Ha-MSV was examined and quantified using a recently developed immunocytochemical technique. More than 95% of p21 was localized to the inner surface of the plasma membrane in these Ha-MSV-transformed cells; p21 was not exposed on the outside surface of the plasma membrane. A similar location was observed by immunofluorescence in other Ha-MSV-transformed cell lines, including cells derived from rat, mouse and mink. This finding, and the previous demonstration that p60src of avian sarcoma virus is concentrated on the inner surface of the plasma membrane, suggests that the plasma membrane is a major site of action for transforming proteins.

Identification of a sarcoma virus-coded phosphoprotein in nonproducer cells transformed by Kirsten or Harvey murine sarcoma virus

A similar protein of 21,000 MW (p21) coded for by Harvey or Kirsten murine sarcoma virus has been identified in nonproducer cells transformed by these two viruses. Antisera prepared from rats bearing tumors induced by syngeneic transplantation of NRK cells transformed by Harvey murine sarcoma virus (Ha-MuSV) specifically precipitated the Ha-MuSV p21 from a nonproducer Balb/c mouse cell and a nonproducer dog cell transformed by Ha-MuSV. The same antisera also precipitated a similar protein, Ki-MuSV p21, from a nonproducer mink cell transformed by Kirsten murine sarcoma virus (Ki-MuSV). Both the p21 of Ha-MuSV and of Ki-MuSV are phosphoproteins. Previous studies have reported a virus-specific p21 polypeptide from translation of Ha-MuSV RNA in cell-free protein synthesis systems (W. P. Parks and E. M. Scolnick, 1977, J. Virol. 22, 711-719; T. Y. Shih, D. R. Williams, M. O. Weeks, J. M. Maryak, W. C. Vass, and E. M. Scolnick, 1978, J. Virol 27, 45-55). This p21 protein was specifically precipitated by the same anti-tumor sera. Similarly, a p21 polypeptide translated from Ki-MuSV RNA was also specifically precipitated by the antitumor sera. Therefore, it is concluded that the p21 of Ha-MuSV and Ki-MuSV are homologous proteins coded for bv homologous sequences found in the recombinant genomes of Ha-MuSV and Ki-MuSV.

Identification of a normal vertebrate cell protein related to the p21 src of harvey murine sarcoma virus

Abstract Harvey murine sarcoma virus (Ha-MSV) is a recombinant virus between genetic sequences of a helper-independent mouse type C virus and rat nucleic acid sequences. The rat sequences of Ha-MSV encode a 21,000-dalton protein (p21) which is responsible for the maintenance of the virus-induced transformation. Employing rat antisera containing antibodies directed against Ha-MSV p21, we have detected lower amounts of a 21,000-dalton cellular homolog in uninfected cells derived from a variety of vertebrate species including rat, mouse, mink, hamster, rabbit, turkey, bat, cat, dog, horse, monkey, and human. Although we could detect with the same antisera a phosphorylated form of the viral p21, we were unable to immunoprecipitate a phosphorylated form of the endogenous p21. Partial proteolytic peptide mapping by Staphylococcus aureus V8 protease demonstrated that the Ha-MSV p21 is structurally very similar to the endogenous cellular homolog which is itself chemically highly conserved in a variety of vertebrate cells. The present data suggest that a normal cellular gene encoding an antigenically and chemically related protein of the Ha-MSV p21 is present in many vertebrate species.

Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.

Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.

The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

Loss and restoration of glucagon receptors and responsiveness in a transformed kidney cell line

A kidney cell line (MDCK) retains an adenylate cyclase system sensitive to glucagon, vasopressin, isoproterenol and prostaglandin E1. The stimulatory effect of glucagon on cAMP production was selectively lost in a cloned line derived from MDCK cells transformed by Harvey murine sarcoma virus. Sensitivity to glucagon was largely restored by treatment of the transformed cells with prostaglandin E1 or butyrate. Loss and reappearance of glucagon receptors seemed to be responsible for the observation. The parental MDCK line produced prostaglandins and in the transformed line, this function was abolished. These observations suggest that synthesis of glucagon receptors is controlled by endogenously produced prostaglandin in MDCK cells and that loss of glucagon receptors and their responsiveness in the transformed cells occurs as a consequence of the inability of these cells to synthesize this prostaglandin.

Markedly elevated levels of an endogenous sarc protein in a hemopoietic precursor cell line.

The src gene product of Harvey murine sarcoma virus is a 21,000-dalton guanine nucleotide-binding protein. We have recently shown that a wide variety of vertebrate cell strains and cell lines express much lower levels of an endogenous p21 immunologically related to the Harvey murine sarcoma virus-coded p21. In this report, we have examined the levels of endogenous p21 in a unique hemopoietic precursor cell line, 416B, which was originally described as a continuous cell line of a hemopoietic stem cell, CFU-S. The currently available 416B cells express markedly elevated levels of endogenous p21. The level of endogenous p21 in the 416B cells is 5- to 10-fold higher than the level of p21 in Harvey murine sarcoma virus-infected cells and more than 100 times higher than the level of endogenous p21 that we have observed in a variety of other fresh or cultured cells. The results indicate that marked regulation of the levels of an endogenous sarc gene product can occur, and speculation about a possible role for endogenous p21 in normal hemopoietic stem cells is discussed.

Revertants of Ha-MuSV-transformed MDCK cells express reduced levels of p21 and possess a more normal phenotype

Four subclones of the originally cloned Harvey murine sarcoma virus-transformed Madin Darby canine kidney (MDCK) cells have been isolated. These subclones fall into two general classes. Two subclones have a fibroblastic morphology, have lost the growth requirement for prostaglandin E1 (PGE1), do not respond to glucagon or vasopressin, and, in general, appear transformed. Two other subclones have epithelioid morphologies, are growth-stimulated by PGE1, respond to vasopressin with an increase in intracellular cAMP. We propose that these cells represent revertants to a more non-transformed phenotype. Unlike normal cells, however, these revertants grow under anchorage-independent conditions, express detectable but reduced amounts of the transforming gene product, p21, and grow in nude mice. The appearance of such revertants may be one cause of the observed heterogeneity of tumor cells.

Biological activity of a K-ras mutant that contains the 12R/59T/116Y mutations

The 12R/59T/116Y mutations have been shown to confer a dominant negative activity on H-ras oncogene (H-ras 116Y). To determine whether this event is unique for H-ras, we introduced the same mutations into K-ras oncogene. This mutant, K-ras 116Y, suppressed transformed phenotypes induced by overexpression of H-ras proto-oncogene. NIH3T3 cells expressing K-ras 116Y were resistant to transformation by v-fes oncogene. Analysis of chimaeras between H- and K-ras 116Y showed that the C-terminal variable region determines the level of suppressor activity. These results suggest that these mutations are applicable to other GDP/GTP binding proteins.

Structure and Function of ras p21: Studies BY Site-Directed Mutagenesis

Are studies by analogy fruitful to understanding protein structure and function? With protein sequences accumulating at an astonishing rate through gene cloning and DNA sequencing, a great deal of protein structure and function can be learned by analogy with members of the superfamily whose structures have been determined and whose molecular mechanisms of action are known. The best case in point, perhaps is in unraveling the elusive function of ras p21. The molecular model of p21 has been constructed by analogy with the crystal structure of the E. coli elongation factor, EF-Tu (McCormick et al., 1985; Jurnak, 1985). This p21 model is remarkably consistent with the actual three dimensional structure of p21 later determined by X-ray crystallography (De Vos et al., 1988). The recent identification of the GAP protein which stimulates GTPase activity of p21 (Trahey and McCormick, 1987), is a conceptual offspring of similar biochemical mechanism well understood for the function of EF-Tu in protein synthesis (Kaziro, 1978). Furthermore, analogy with the well characterized G-proteins, which regulate transmembrane cell signalling in the adenylate cyclase systems and light transduction in retina, forms the foundation for the current belief that ras p21 mediates transmission of growth signals to their intracellular effectors that control cell proliferation and differentiation (Bourne and Sullivan, 1986).