A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.

Abstract

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp6Ov-rc. In this mutant, lysine-295 is replaced with methionine. SF2 pp6ov-src was found to have a half-life similar to that of wild-type pp6Ov-c and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp6Ov-src were morphologically untransformed and do not form tumors. The SF2 pp6Ov-S isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp6Ov-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp6OVS-was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp6Ov-sr itself. By contrast, SF2 pp6Ov-srC could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp6Ov-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp6O-src elicits an allosteric change required for phosphorylation of serine in the protein.The tumorigenicity and in vitro transforming ability of Rous sarcoma virus (RSV) reside in pp60v-src (4), the protein encoded by the viral oncogene v-src (3, 33, 48). pp60v-src has tyrosine-specific protein kinase activity (10,11,21,26,27,36) and is itself phosphorylated, both on serine-17 (and, to a lesser degree, on at least one other serine) in the aminoterminal half of the protein (9, 16) and on tyrosine-416 (32, 41). Recent evidence has also demonstrated the existence of phosphotyrosines in the amino-terminal half of the protein that may play a role in regulation of enzymatic activity (7,12,34).Evidence from studies on mutants of v-src suggests that pp60v-src may have as-yet-undiscovered functions independent of the kinase activity which are necessary for full expression of transformation (5, 13, 45). To examine this possibility more closely, it would be desirable to identify residues involved in kinase activity and then mutate them to eliminate this activity while preserving other possible functions of pp6Ov-src. Both indirect (2) and direct (23) evidence now exists that lysine-295 is intimately involved with the ATP binding of pp60v-src. We wanted to obtain a mutation at this site that would eliminate tyrosine kinase activity without altering overall structure or other active functions. In this paper, we report on the construction and expression of such a mutant, which we call RSV-SF2, and the effect of this mutation on the structure and activity of pp6Ov-src and on the expression of some parameters of transformation. Site-specific mutagenesis. Procedures for site-specific mutagenesis on M13 and selection of mutated DNA have been described earlier (43). The 19-base oligonucleotide used for converting lysine-295 of v-src to me...

RACK1: a novel substrate for the Src protein-tyrosine kinase

“…We used three Src mutants: (1) Y527F c-Src (constitutively-active) (Cartwright et al, 1987); (2) K297M Src (kinase-inactive) (Snyder et al, 1985); and (3) dl155 Src (contains a three amino acid deletion in the phosphotyrosine binding pocket of the SH2 domain) (Moyers et al, 1993). Previously, we showed that the dl155 Src mutant has reduced binding to RACK1 (Chang et al, 2001).…”

“…pGEM K297M src was a gift from Tony Hunter (The Salk Institute for Biological Studies, La Jolla, CA, USA; Snyder et al, 1985). Src inserts from these plasmids or Y527F c-src (Cartwright et al, 1987) were subcloned into pcDNA3 (Introgen, La Jolla, CA, USA) to create pcDNA3 c-src, dl155 c-src, K297M c-src and Y527F c-src, and transiently expressed in CHO cells (Chang et al, 1998(Chang et al, , 2001).…”

Rapid activation of sodium–proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades

“…DN-SHPT1D, a mutant phosphotyrosine phosphatase type 1D which incorporates a carboxy terminal deletion of 60 amino acids required for its tyrosine phosphorylation, was from Dr. Ben Neel (Beth Israel Hospital) [40]. Wild-type Src, dominant negative Src (K298MSrc) [41] and constitutively activated Src (Src Y527F) which lacks a key inhibitory phosphorylation site [42] were from Dr. L. Luttrell (Duke University).…”

“…Genistein, a broad-spectrum tyrosine kinase inhibitor, markedly attenuated the ability of 5-HT to activate ERK and NHE, whereas the structurally similar but inactive compound, daidzein, had no effect, supporting the involvement of tyrosine kinase(s) in both pathways (Table 1). PTP1D is a cytosolic phosphotyrosine phosphatase which has been shown to be a critical positive regulator of tyrosine kinase signals (DNA synthesis and ERK activation) initiated by both growth factor and G proteincoupled receptors [38][39][40][41][42][43][44][45][46][47][48][49]. Transfection of a cDNA construct encoding a mutant inactive PTP1D (∆PTP1D) molecule effectively blocked 5-HT-stimulated ERK, but had no effect on NHE activity.…”

Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

“…1). NYmet295 (10) and 5Hmet295 (21), derivatives of RSV and NY5H, respectively, are tyrosine kinase-defective mutants with point mutations in the ATP binding site (38). NYT10-1 is a substitution of Arg-Ser-Asp for residues 412 to 416 of p60vsrc (6).…”