Characterization of p47gag-crk, a novel oncogene product with sequence similarity to a putative modulatory domain of protein-tyrosine kinases and phospholipase C.
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Biomedical subjects
B J Mayer
Publications and source records attributed to B J Mayer.
Analysis of p60v-src mutants carrying lesions involved in temperature sensitivity.
Analysis of the src genes of three temperature-sensitive (ts) mutants of Rous sarcoma virus (tsNY68, tsNY72-4, and PA104) showed that each has two C-terminal mutations in the kinase domain required for temperature sensitivity, as assayed by morphological alteration and anchorage-independent growth. In all three mutants, one of the mutations is a valine-to-methionine change at position 461. To assess the contribution of each mutation to the biochemical properties of the src protein, we analyzed the kinase activity and the interaction with cellular proteins p50 and p90 of recombinant src gene products in which only one mutation was combined with wild-type src sequences. Chimeric src protein containing only the Met-461 mutation was indistinguishable from the wild type by all criteria examined, while the effect of the second C-terminal mutation alone varied with the defectiveness of the parental ts mutant. The second mutation alone, while not sufficient to cause ts transformation, altered p60src complex formation with cellular proteins p50 and p90 and altered the in vitro thermolability of src kinase activity. The results indicate that these biochemical properties of p60src are more sensitive to mutation than others, such as in vivo kinase activity, which require more profound structural alterations.
Animals↗
Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters.
The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology.
Amino Acid Sequence↗
Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants.
The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.
Amino Acid Sequence↗
Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant.
We molecularly cloned the src coding region of tsNY68, a mutant of Rous sarcoma virus temperature sensitive (ts) for transformation, and constructed a series of ts wild-type recombinant src genes. DNA containing the hybrid genes was transfected into chicken cells together with viral vector DNA and helper viral DNA, and infectious transforming viruses were recovered. Characterization of these recombinant viruses indicated that at least two mutations are present in the 3' half of the mutant src gene, both of which are required for ts. Nucleotide sequence analysis revealed three differences in the deduced amino acid sequence compared with the parental virus. Two of these changes, a deletion of amino acids 352 to 354 and an amino acid substitution at position 461, are responsible for the ts phenotype.
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Induction of tumors and generation of recovered sarcoma viruses by, and mapping of deletions in, two molecularly cloned src deletion mutants.
td108 , a transformation-defective (td) deletion mutant of the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A (SR-A), was molecularly cloned. Two isolates of td viruses, td108 -3b and td108 -4a, obtained by transfection of the molecularly cloned td108 DNAs into chicken embryo fibroblasts, were tested for their ability to induce tumors and generate recovered avian sarcoma viruses ( rASVs ) in chickens. Both td viruses were able to induce tumors with a latency and frequency similar to those observed previously with biologically purified td mutants of SR-A. rASVs were isolated from most of the tumors examined. The genomic RNAs of those newly obtained rASVs were analyzed by RNase T1 oligonucleotide fingerprinting. The results showed that they had regained the deleted src sequences and contained the same set of marker src oligonucleotides as those of rASVs analyzed previously. The src oligonucleotides of rASVs are distinguishable from those present in SR-A. We conclude that those rASVs must have been generated by recombination between the molecularly cloned td mutants and the c-src sequence. The deletions in the td mutants were mapped by restriction enzyme analysis and nucleotide sequencing. td108 -3b was found to contain an internal src deletion of 1,416 nucleotides and to retain 57 and 105 nucleotides of the 5' and 3' src coding sequences, respectively. td108 -4a contained a src deletion of 1,174 nucleotides and retained 180 and 225 nucleotides of the 5' and 3' src sequences, respectively. Comparison of sequences in the 5' src and its upstream region of td108 -3b with those of SR-A, rASV1441 (a td108 -derived rASV analyzed previously), and c-src suggested that the 5' recombination between td108 and c-src occurred from 7 to 20 nucleotides upstream from the beginning of the src coding sequence.
Animals↗
Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun.
The stress-activated protein kinases (SAPKs), which are distantly related to the MAP kinases, are the dominant c-Jun amino-terminal protein kinases activated in response to a variety of cellular stresses, including treatment with tumour-necrosis factor-alpha and interleukin-beta (refs 1, 2). SAPK phosphorylation of c-Jun probably activates the c-Jun transactivation function. SAPKs are part of a signal transduction cascade related to, but distinct from, the MAPK pathway. We have now identified a novel protein kinase, called SAPK/ERK kinase-1 (SEK1), which is structurally related to the MAP kinase kinases (MEKs). SEK1 is a potent activator of the SAPKs in vitro and in vivo. An inactive SEK1 mutant blocks SAPK activation by extracellular stimuli without interfering with the MAPK pathway. Although alternative mechanisms of SAPK activation may exist, as an immediate upstream activator of the SAPKs, SEK1 further defines a signalling cascade that couples cellular stress agonists to the c-Jun transcription factor.
Amino Acid Sequence↗