Biologically active mutants with deletions in the v-mos oncogene assayed with retroviral vectors.

1988

Mol. Cell. Biol.

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The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.

Section: Methodsmentioning

confidence: 99%

Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

Sauer¹,

1988

Mol. Cell. Biol.

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“…These mutants contain either an aspartate codon or an arginine codon in place of lysine codon 121 of the v-mos gene. The effect of these mutations on the biological activity of the v-mos gene was assayed using a Mo-MLV-derived vector to provide for expression of the v-mos gene in NIH 3T3 cells (23,24). This vector, pDD102, is derived from a DNA clone of Mo-MLV (26) and contains a unique Xho I site for the insertion of foreign genes in place ofthe envelope (env) gene of Mo-MLV.…”

Section: Resultsmentioning

confidence: 99%

“…Using the anti-C-terminal serum C3, we have been unable to identify a protein kinase activity associated with immunoprecipitates of p37mOS (unpublished data). However, this discrepancy may be a result of the different antisera used, since the C-terminal 23 amino acids of p37mos are required for biological activity (24) and antiserum binding to the C terminus of p37mos may inhibit the associated kinase activity found with the anti-N-terminal serum.…”

Section: Discussionmentioning

confidence: 99%

Lysine residue 121 in the proposed ATP-binding site of the v-mos protein is required for transformation.

Hannink¹,

Proc. Natl. Acad. Sci. U.S.A.

1985

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The transforming gene product encoded byMoloney murine sarcoma virus clone 124, p37", contains a lysine residue (lysine-121) that is conserved among all members of the protein kinase family. This lysine has been shown to be part of a conserved ATP-binding site in both the catalytic subunit of the cAMP-dependent protein kinase and p60O'. We wished to determine whether this lysine is required for the transforming activity of p3711". Two site-specific mutations were therefore constructed, which result in the substitution of an aspartic acid or arginine codon in place of the codon for lysine-121. and has been localized to the cytoplasm by indirect immunofluorescence, using antiserum specific for the predicted C terminus of p37mOS (13).Comparison of the predicted amino acid sequence of p37mOS with those of the catalytic subunit of the cAMPdependent protein kinase and of p60"-" has revealed the existence of limited regions of sequence homology (14). A region of particular interest is that surrounding lysine-121 of p37mOS; in both the catalytic subunit of the cAMP-dependent protein kinase (15), and in p60v`src (16), the homologous lysine specifically binds the ATP analog, p-fluorosulfonylbenzoyladenosine (FSBA) with concomitant loss of kinase activity. This suggests the existence of a conserved ATP-binding site among members of the protein kinase family, containing a Gly-Xaa-Gly-Xaa-Xaa-Gly sequence that is also found in other nucleotide-binding proteins (16).To test the importance of lysine-121 of p37mOS in transformation, we have constructed two mutants of the v-mos gene. These mutations result in the substitution of a codon for aspartate or for arginine in place of the codon for lysine-121. Both of these mutations abolish the ability of the v-mos gene to transform NIH 3T3 cells. In transient expression assays in COS-l cells, p37mos and p37m s(Rl2i) are expressed at equal levels and have comparable half-lives. However, no p37mos(Dl21) was detected in vivo. These results indicate that lysine-121 of p37mOS is required for its biological activity and by analogy suggest that p37mos, like the cAMP-dependent protein kinase and p60vsrc, possesses an ATP-binding site. MATERIALS AND METHODSOligonucleotide Site-Directed Mutagenesis. The following synthetic oligonucleotides were used to construct the sitedirected mutants: (i) a 16-mer, TACTTGATCGATGGCC, used for the aspartate mutation, and (il) a 20-mer, CCATC-CGGCAAGTAAACAAG, used for the arginine mutation. Gapped circular heteroduplex molecules, constructed from restriction fragments ofplasmid subclones ofthe v-mos gene, were used as substrates for the mutagenic oligonucleotides. The plasmid subclones were derivatives ofpDDO, which was constructed from a cDNA clone of . The heteroduplex molecules were formed by using NaOH to denature the DNA and formamide to control the reannealing of the strands (17). Mutagenic oligonucleotides were hybridized to the gapped heteroduplex molecules at room temperature for 30 min in H20, using a 50-to 100-fold molar excess of oligonuc...

“…Previously described mos-encoded mutant proteins devoid of in vitro autophosphorylating activity were unable to transform NIH 3T3 cells (17,19 showed a corresponding decrease in transforming activity (approximately 13-fold less efficient than wild-type v-mos) (2,17). These results suggested a correlation between in vitro autophosphorylation of p37ntS and transformation by mos.…”

mentioning

confidence: 99%

Transforming mutant v-mos protein kinases that are deficient in in vitro autophosphorylation.

Freeman¹,

1989

Mol. Cell. Biol.

Self Cite

We investigated the importance of specific serine residues for autophosphorylation and transformation by serine-threonine protein kinase p37mOS. When either serine 326 or 358 was replaced with alanine, the resulting mutant protein retained the ability to transform NIH 3T3 cells but failed to autophosphorylate in vitro. These studies represent the first functional uncoupling of these two activities for p37.OS* The transforming protein encoded by the v-mos oncogene, p37mos, belongs to the family of serine-threonine protein kinases (14). Although the transforming retrovirus which encodes the mos gene was isolated in 1966 (13), the extremely low level of p37mos in transformed cells (estimated to be approximately 0.0005% of the total cellular protein [14]) has hindered studies on the biochemistry of mos transformation. p37mos is phosphorylated both in vivo and in vitro predominately on serine residues (11, 14); however, the phosphorylation sites have not been identified. Although many protein kinases are regulated by phosphorylation, a role for phosphorylation in the activation of p37mos remains to be found.A p37mos-associated in vitro kinase activity was first identified in immune complexes by using an antibody against residues 37 to 55 (11). Results from previous studies with a variety of mos mutations have suggested a general correlation between in vitro autophosphorylation of p37mos and transformation by the mos gene (17,19). We undertook this study to investigate the importance of selected serine residues for the transforming and kinase activities of p37"'. Using site-directed mutagenesis, we replaced 6 of the 37 serine codons in mos with alanine codons (see Fig. 1). Serine codons 49, 88, 210, 263, 326, and 358 were selected for mutagenesis for the following reasons. (i) Serine 263 is positioned between two short sequence motifs conserved among protein kinases, Asp-Phe-Gly and Ala-Pro-Glu (5). This region contains a tyrosine autophosphorylation site in many tyrosine protein kinases (8), as well as a site of threonine autophosphorylation in the catalytic subunit of the cyclic AMP-dependent protein kinase (10, 16). (ii) Serine 358 is the last senine in the primary sequence of p37mos and is located 16 residues from the C terminus. Anti-peptide sera which bind the C terminus of p37hOs have been shown to inhibit in vitro kinase activity (12). (iii) Serines 88 and 358 are the only serine residues not conserved in p35mos-HT, the transforming gene product of Moloney murine sarcoma virus variant strain HT-1 (15). At the time these mutations were constructed, p35mos-HT-1 had been observed to autophosphorylate in vitro to a much lower extent than p37"OS (18).(iv) Results from in vivo kinase assays with the Ala-358 mutant protein (discussed below) raised the possibility that serine 358 was a site of autophosphorylation. Since p37mOs is likely to contain multiple sites of in vitro autophosphorylation (11), we looked for other serines in p37hOs with neigh-* Corresponding author.boring amino acids similar to those of serine 3...