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Publications and source records attributed to B M Willumsen.
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A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.
A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could not be altered, even conservatively, without a loss of function (codons 32 and 35); one which retained detectable biologic activity with conservative changes but which lost function with more drastic substitutions (codons 36 and 40); and one which retained function even with a nonconservative substitution (codon 39).
Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.
We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.
Members of the ras multigene family have been found in virtually all eukaryotes, from yeast to mammals. ras is required for normal cell growth in the yeast Saccharomyces cerevisiae and in at least some mammalian cells. These genes induce tumorigenic transformation of established NIH 3T3 cells by increased expression of a normal ras gene, certain point mutations or amino acid deletion. In tumours, point mutation appears to be the most common mechanism of activation. The ras proteins are found at the plasma membrane, bind guanine nucleotides GDP and GTP and possess a GTPase activity. At least some ras proteins that have been activated by single amino acid substitutions possess a GTPase activity that is lower than that of the normal version. These results are consistent with the hypothesis that ras protein stimulates its putative target(s) when GTP is bound to it, as is true for the G regulatory proteins or elongation factor Tu. In Saccharomyces cerevisiae, ras has been shown to stimulate adenylate cyclase. However, there does not appear to be a direct interaction between ras and adenylate cyclase in mammalian cells.
The mammalian p21 ras proteins contain a 20-amino acid region that is highly divergent, in contrast to the strong sequence conservation that is common to other regions of these proteins. This major variable region is located near the C terminus just upstream from a conserved cysteine residue that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor the protein in the membrane.
Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein was processed normally, as was the p21 encoded by a transformation-competent deletion mutant from which amino acids 166-175 had been deleted. The Ser-186 mutant was defective for transformation. The p21s encoded by the Ser-186 mutant and by the previously described transformation-defective mutants did not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue is required for all ras proteins.
The sequences encoding the 21-kilodalton transforming protein (p21 ras) of Harvey murine sarcoma virus have previously been localized genetically to a 1.3-kilobase segment of the viral DNA (E. H. Chang, R. W. Ellis, E. M. Scolnick, and D. R. Lowy, Science 210:1249-1251, 1980). Within this segment, DNA sequence analysis has found a single open reading frame large enough to encode the viral p21 (R. Dhar, R. W. Ellis, T. Y. Shih, S. Oroszlan, B. Shapiro, J. Maizel, D. Lowy, and E. M. Scolnick, Science 217:934-937, 1982). There are three potential in-frame ATG initiation codons at the 5' end of this open reading frame. By constructing a mutant of Harvey murine sarcoma virus DNA from which the first two ATG codons of this open reading frame have been deleted, we now show by transfection of the mutant viral DNA into NIH 3T3 cells that only the third ATG codon is necessary and sufficient for synthesis of the viral p21 and for cellular transformation.
Two types of 'spontaneous' malignant alteration in vitro of ST/a mouse lung fibroblasts (ST-L) have been observed. In contrast to cells which retained their fibroblastic appearance (R- ST-L cells), cells showing morphological signs of transformation (R+ ST-L cells) developed strong isoimmunizing properties. Both types of cells expressed MuLV antigens which were found to be responsible for serum as well as cell-mediated immune reactions in vitro. The higher concentration of gp70 in R+ ST-L cells as compared to R- cells and possibly also the morphological differences in surface structure between the two cell types may account for the differences in immunogenicity. Preimmunization with R+ ST-L cells protected ST/a mice against secondary challenge with two ascites tumors (STABAL leukemia and Ehrlich). R- ST-L cells did not have a similar protective effect. However, the two ascites tumors only showed weak or no cross-reactions in vitro with sera and lymphoid cells from ST/a mice sensitized to R+ ST-L cells, and the in vitro reaction between the latter cells and sera or lymphoid cells from mice immunized against the two ascites tumors was moderate. This discrepancy between in vivo and in vitro observations is discussed.
ST-L1 is a cell line established from lung explants from a normal ST/a mouse. The ST-L1 cells have undergone spontaneous malignant alteration in vitro. The cells were rejected after inoculation into syngeneic immunocompetent hosts, and a syngeneic humoral immuneresponse against the ST-L1 cells has been detected. The specificity of this humoral response was investigated. The syngeneic response to ST-L1 was characterized by indirect immunofluorescence tests and by immunoprecipitation of radiolabelled cells and of a C-type virus produced by the cell lines. The specific anti-ST-L1 reactivities were found to be directed against the envelope glycoprotein of an endogenous C-type virus expressed by the antigenic cell line.
Low-infectious, nontransforming type C virus was isolated from an in vitro spontaneously transformed ST/a mouse cell line, ST-L1. The virus released by ST-L1 cells was NB-tropic and XC(-). It gave rise to very small peroxidase antibody plaques (PAP) in cultures which initially were nonproducing. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the structural proteins of the ST-L1 virus showed an envelope glycoprotein with an apparent mass of 65 kilodaltons (kdal). The mouse cells SC-1, BALB/3T3, and NIH/3T3 could be productively infected with cell-free supernatants from the ST-L1 cell line; however, virus was detected in supernatant fluids only after two to four subcultures of the infected cells. The virus thus produced was XC(+) and a large plaque former. The virus released from infected SC-1 cells was N-tropic, whereas the viruses from infected NIH/3T3 and BALB/3T3 cells were NB-tropic. The structural proteins of the N- and NB-tropic viruses could be distinguished on SDS polyacrylamide gels, the major dissimilarity being a difference in the mobility of the p30. All these viruses had an envelope glycoprotein with an apparent mass of 70 kdal. The infectivity of the viruses, measured as PAP per nanogram of p30, was 30- to 60-fold lower for the virus released from the ST-L1 cell line than that of the viruses after passage in SC-1, NIH/3T3, and BALB/3T3 cells. None of the viruses could infect rabbit or mink cells. Inoculation of the viruses into newborn mice showed that the ST-L1 virus was non-leukemogenic, whereas the NB-tropic virus selected from this after passage in BALB/3T3 or NIH/3T3 cells was highly leukemogenic. Viruses isolated from leukemic animals were indistinguishable with respect to host range and protein mobilities in SDS gels from the ones with which the mice were inoculated. Although the SC-1-selected virus was highly infectious in vitro, it was only weakly, if at all, leukemogenic.
Mutants in the spo T gene have been isolated as stringent second site revertants of the relC mutation. These show varying degrees of the characteristics associated with the spoT1 gene, viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of 3H-guanosine into GTP and ppGpr pools in spoT+ and spoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower in spoT- than in spoT+ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower in spoT than in spoT+ cells. In one of the "intermediate" spoT mutants the rate of entry of 3H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is: GDP leads to GTP leads to pppGpp leads to ppGpp leads to Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in various spoT+ and spoT- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis.
The ras genes encode GTP/GDP-binding proteins that participate in mediating mitogenic signals from membrane tyrosine kinases to downstream targets. The activity of p21ras is determined by the concentration of GTP-p21ras, which is tightly regulated by a complex array of positive and negative control mechanisms. GAP and NF1 can negatively regulate p21ras activity by stimulating hydrolysis of GTP bound to p21ras. Other cellular factors can positively regulate p21ras by stimulating GDP/GTP exchange.
The Harvey murine sarcoma virus (Ha-MuSV) transforming gene, v-rasH, encodes a 21,000 molecular weight protein (p21) that is closely related to the p21 proteins encoded by the cellular transforming genes of the ras gene family. The primary translation product (prop21), which is found in the cytosol, undergoes posttranslational modification and the mature protein subsequently becomes associated with the inner surface of the plasma membrane and binds lipid tightly. The p21 proteins have the capacity to bind guanine nucleotides non-covalently in vitro. To assess the biological relevance of these biochemical features of the protein, we have now studied a series of deletion mutants located at or near the C-terminus of the viral p21 protein. Our tissue culture studies indicate that amino acids located at or near the C-terminus are required for cellular transformation, membrane association and lipid binding.