Amino Terminal Myristylation of the Protein Kinase p60
            <i>src</i>
            , a Retroviral Transforming Protein

Schultz, Alan M.; Henderson, Louis E.; Oroszlan, Stephen; Garber, Ellen A.; Hanafusa, Hidesaburô

doi:10.1126/science.3917576

Cited by 268 publications

(197 citation statements)

References 15 publications

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“…Here we have carried this point further by showing that the gag fusion and the accompanying myristylation are not necessary for oncogenic activation, since the truncated version of c-raf-1 in EC12 and the gag-c-raf fusion construct TCG, which both lack the myristylation site, have transforming activity. Independence from a membrane-anchoring site provided by the fatty-acid modification also reaffirms that the Raf kinase acts below the cell membrane (34,47) and distinguishes Raf protein kinase function from that of many tyrosine kinases encoded by oncogenes such as v-src (6,39,49) and v-abl (26,40) in which myristylation is critical for transformation. These results are also supported by our previous finding of a long terminal repeat (LTR)-activated transforming version of c-raf-1 which lacks these sequences and modifications (34).…”

Section: Resultsmentioning

confidence: 67%

Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Heidecker¹,

Huleihel²,

Cleveland³

et al. 1990

Mol. Cell. Biol.

222

184

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A series of wild-type and mutant rafgenes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine-and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.The raffamily of proto-oncogenes consists of three active members: A-raf-1, B-raf, and c-raf-1 (2,4,5,20,22,23). The three proteins show the greatest stretch of homology in the carboxy-terminal half, which, in the case of c-raf-1, has been shown to have serine-and threonine-specific protein kinase activity (33, 48). Physiologically, Raf protein kinases function as information shuttles that communicate between the cell surface and nucleus. From Ki-ras revertant and anti-ras antibody microinjection studies, it is known that c-raf-1 acts independently of and perhaps downstream from Ras in signal transduction (2,22,36,41,42,52). In addition, we recently showed that Raf is hyperphosphorylated and enzymatically activated in cells which are transformed by src, fms, or ras, and in cells which have been treated with platelet-derived growth factor or 12-0-tetradecanoylphorbol-13-acetate (TPA) (35). In response to platelet-derived growth factor and TPA, Raf protein is also translocated from the cytoplasm to the perinuclear space, as is evident from immune fluorescence and cell fractionation studies (44). Ultimately, rafis thought to exert its effect by modulation of transcription factor activity via phosphorylation (42). Evidence for this comes from experiments in which cells transfected with activated raf genes showed increased transcriptional activity from an APl-dependent promoter in transient transfection assays, suggesting that Raf is involved in the regulation of transcription factors of the jun-AP1 gene family (56). Raf function is essential for normal rates of cell proliferation, as demonstrated by the lethal effect of mutations in the Drosophila melanogaster D-raf-1 locus (37).c-raf-1, the most-studied member of the family, encodes a * Corresponding auth...

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Section: Resultsmentioning

confidence: 67%

Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Heidecker¹,

Huleihel²,

Cleveland³

et al. 1990

Mol. Cell. Biol.

222

184

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“…This protein has covalently bound fatty acid (myristic acid) at its N terminus (24,46,49), which appears to be essential for its association with the plasma membrane and for cell transformation (17). Endogenous p60c"src in CEF was reported to fractionate with plasma membranes, measured by an immune complex kinase reaction (15 cells was fractionated by equilibrium centrifugation on a sucrose gradient (Fig.…”

Section: Methodsmentioning

confidence: 99%

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

Iba¹,

Cross²,

Garber³

et al. 1985

Mol. Cell. Biol.

183

167

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We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60csrc overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60jcsrc had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60csrc was similar to transforming p6Os in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p6o-src accounts for its inability to transform cells.The viral src gene of Rous sarcoma virus (RSV) encodes p60v-sr( (2) which has protein kinase activity (9,23,34) specific for tyrosine residues (10, 29), and this enzyme activity correlates with transforming activity (47). The cellular src (c-src) gene, the cellular counterpart of the v-src gene (56), encodes p60C-sr( (7), a closely related tyrosine kinase expressed at a low level in normal cells (32,38). Previously, Hanafusa and co-workers found that RSV variants in which the v-src gene is entirely replaced with the chicken c-src gene are unable to transform cells, although they induce overproduction of p60csrc (27,30). However, variants in which v-src is partially replaced with c-src are active in transformation. Transforming viruses were generated from the nontransforming c-src-containing viruses at a high frequency (30). Therefore, p60csrc has critical structural differences from p60v-src, accounting for its lack of transforming activity. The inability of overproduced chicken p60csr( to transform mammalian cell lines was demonstrated by Parker et al. (40) and Shalloway et al. (50).In this paper we examine subcellular localization and tyrosine kinase activity of various p6Os, to try to find a basis for the inability of p60c-r( to transform cells. MATERIALS AND METHODSVirus strains and infection. The virus strains used here were replication-competent RSV variants described by Hanafusa and co-workers (27, 30). Their p60 structures are shown in Fig. 1. Secondary cultures of chicken embryo fibroblasts (CEF) were infected with viruses, kept under soft agar medium (0.4% agar), and transferred once to achieve full infection before biochemical analys...

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“…Membrane association of Src is in part due to a postranslational modi®cation at its amino terminus ± Willingham et al, 1979;Courtneidge et al, 1980;Focal adhesions;actin factor RTKs** and GPCRs; cell Resh and Erikson, 1985;Henderson and Rohrschneider, 1987;cytoskeleton;microtubule adhesion and spreading; cell Kaplan et al, 1992;Twamley-Stein et al, 1993;Kaplan et al, 1994; bundles at points of cell-cell migration; cell-cell contact Okamura and Resh, 1994;Fincham et al, 1996;Li et al, 1996;contact;adherens junctions;Luttrell et al, 1996;Schaller et al, 1999;Fincham et al, 2000;caveolae Owens et al, 2000 Cytoplasmic and perinuclear Protein trafficking; endosomal Willingham et al, 1979;Tanaka and Kurth, 1984 the covalent attachment of a 14-carbon fatty acid moiety, myristate (Buss and Sefton, 1985;Schultz et al, 1985;Kaplan et al, 1988). However, myristoylation is not su cient for membrane anchorage (Kaplan et al, 1990b).…”

Section: Src Localizationmentioning

confidence: 99%

Selected glimpses into the activation and function of Src kinase

2000

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Since the discovery of the v-src and c-src genes and their products, much progress has been made in the elucidation of the structure, regulation, localization, and function of the Src protein. Src is a non-receptor protein tyrosine kinase that transduces signals that are involved in the control of a variety of cellular processes such as proliferation, di erentiation, motility, and adhesion. Src is normally maintained in an inactive state, but can be activated transiently during cellular events such as mitosis, or constitutively by abnormal events such as mutation (i.e. v-Src and some human cancers). Activation of Src occurs as a result of disruption of the negative regulatory processes that normally suppress Src activity, and understanding the various mechanisms behind Src activation has been a target of intense study. Src associates with cellular membranes, in particular the plasma membrane, and endosomal membranes. Studies indicate that the di erent subcellular localizations of Src could be important for the regulation of speci®c cellular processes such as mitogenesis, cytoskeletal organization, and/or membrane tra cking. This review will discuss the history behind the discovery and initial characterization of Src and the regulatory mechanisms of Src activation, in particular, regulation by modi®cation of the carboxyterminal regulatory tyrosine by phosphatases and kinases. Its focus will then turn to the di erent subcellular localizations of Src and the possible roles of nuclear and perinuclear targets of Src. Finally, a brief section will review some of our present knowledge regarding Src involvement in human cancers. Oncogene (2000) 19, 5620 ± 5635.

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Amino Terminal Myristylation of the Protein Kinase p60 src , a Retroviral Transforming Protein

Cited by 268 publications

References 15 publications

Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

Selected glimpses into the activation and function of Src kinase

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