Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation

Kmiecik, Thomas E.; Shalloway, David

doi:10.1016/0092-8674(87)90756-2

Cited by 637 publications

(506 citation statements)

References 31 publications

Supporting

Mentioning

487

Contrasting

Unclassified

Order By: Relevance

“…CEF that overexpress p60csrc do not show a significant alteration in cell morphology. Substitution of Phe-527 for Tyr-527 converts p60csrc into a kinase-active transforming protein p6OC-527F (Cartwright et al, 1987;Kmiecik and Shalloway, 1987;Piwnica-Worms et al, 1987;Reynolds et al, 1987). Cells transformed by p6o,-527F exhibit a typical transformed morphology.…”

Section: Introductionmentioning

confidence: 99%

Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

Wang

Erikson

1992

MBoC

View full text Add to dashboard Cite

We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.

show abstract

Section: Introductionmentioning

confidence: 99%

Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

Wang

Erikson

1992

MBoC

View full text Add to dashboard Cite

show abstract

“…Comparison of the sequence of v-src from a number of RSV strains, with the cellular c-src gene, has shown that the transforming proteins contain a number of common amino acid substitutions as well as deletions of the terminal nineteen amino acids (summarised in Hunter, 1987). Demonstration that a single point mutation is capable of activating the oncogenic properties of pp60C-src has been obtained from in vitro mutagenesis studies and analysis of c-src transformation-competent mutants (Kmiecik & Shalloway, 1987; Piwnica-Worms et al, 1987;Cartwright et al, 1987;Levy et al, 1986). There is considerable evidence that TYR 527 is a negative regulator of kinase activity of pp6Oc-src and its deletion in v-src contributes to the elevated kinase activity of the viral protein.…”

mentioning

confidence: 99%

c-src structure in human cancers with elevated pp60c-src activity

Wang

Fromowitz²,

Koslow³

et al. 1991

Br J Cancer

View full text Add to dashboard Cite

Summary We used RNAase protection and restriction fragment length polymorphism assays to detect activating mutations of c-src in a spectrum of human tumours. No mutations were detected at codons 98, 381, 444, and 530. We conclude that mutational activation is not the mechanism of enhancement of pp60C.srcspecific kinase activity found in a number of human cancer types.The src proto-oncogene (c-src) is the cellular homologue of the transforming gene of the Rous sarcoma virus (RSV). Both genes encode Mr 60,000 phosphoproteins (pp6O) which are membrane bound and have tyrosine-specific protein kinase activity (Collett & Erikson, 1978; Hunter & Sefton, 1980;Levinson et al., 1980). Comparison of the sequence of v-src from a number of RSV strains, with the cellular c-src gene, has shown that the transforming proteins contain a number of common amino acid substitutions as well as deletions of the terminal nineteen amino acids (summarised in Hunter, 1987). Demonstration that a single point mutation is capable of activating the oncogenic properties of pp60C-src has been obtained from in vitro mutagenesis studies and analysis of c-src transformation-competent mutants (Kmiecik & Shalloway, 1987; Piwnica-Worms et al., 1987;Cartwright et al., 1987;Levy et al., 1986). There is considerable evidence that TYR 527 is a negative regulator of kinase activity of pp6Oc-src and its deletion in v-src contributes to the elevated kinase activity of the viral protein. Substitution of TYR 527 with amino acid residues which cannot be phosphorylated also enhances the specific kinase activity of the molecule and activates its transforming properties. Additional single point mutations which activate the transforming ability of chicken pp60csrc include amino acid substitutions in the kinase domain (THR 338, GLU 378, ILE 441) and mutations in the amino terminus modulation domain, particularly at ARG 95. All of the activating mutations of pp60csrc result in an increase in the specific kinase activity of the molecule (Hunter, 1987). pp60c-src tyrosyl kinase activity has been shown to be elevated in a number of human cancers including colon cancer, neuroblastoma, breast cancer and sarcomas (Jacobs & Rubsamen, 1983;Bolen et al., 1985;Barnekow et al., 1987;Bolen et al., 1987;Cartwright et al., 1989 (Gibbs et al., 1985) encoding amino acids 452-532 (Figure 1, probe D). To test the ability of the RNAase protection assay to detect single base mismatches at codon 530, we constructed a T to A mutation at the second base of that codon using oligonucleotide-directed in vitro mutagenesis (Kunkel, 1985) and cloned the mutated fragment into a SP65-based vector. Using in vitro transcription we generated sense strand RNA which was hybridised to a radioisotopically-labelled anti-sense RNA probe. The hybrid was then subjected to the RNAase cleavage assay (described in Figure 2). After hybridisation to the mutated RNA and digestion with RNAase, the 317 nucleotide probe was cleaved into the predicted fragments of 282 and 35 nucleotides, indicating that the co...

show abstract

“…Tyr-416 is the major site of phosphorylation in vitro (27,35). Enzymatically active forms of p60c-src are also phosphorylated at Tyr-416 in the cell (3,4,15,18,20,28,30). Tyr-527 is the major site phosphorylated in repressed forms of p60c-src in fibroblasts (7,19).…”

mentioning

confidence: 99%

“…Transformation by the polyomavirus middle T antigen leads to dephosphorylation of Tyr-527 and stimulation of p60csrc kinase activity (2,4,11). Alteration of c-src codon 527 to specify Phe, a residue which is not phosphorylated, activates the transforming potential of c-src (3,18,28,30). Dephosphorylation of Tyr-527 in vitro results in a 10-to 20-fold stimulation of the specific activity of p60c-src in an in vitro kinase reaction (8).…”

mentioning

confidence: 99%

“…The presence of the Ick sequence was confirmed by dideoxynucleotide sequencing (31). To provide an activated version of c-src as a positive control for transformation, the Tyr-527 codon of pMX was changed to code for Phe by using oligonucleotide-directed mutagenesis (3,18,28,30). The modified c-src genes were excised from the plasmids by using BamHI and BglII and cloned into the BamHI site of pLJ, a murine retroviral expression vector that carries a neomycin phosphotransferase minigene kindly provided by R. Mulligan (28).…”

mentioning

confidence: 99%

See 1 more Smart Citation