pp60<sup><i>c‐src</i></sup> is a substrate for phosphorylation when cells are stimulated to enter cycle

Tamura, Teruko; Friis, Robert R.; Bauer, Heinz

doi:10.1016/0014-5793(84)81001-7

Cited by 34 publications

(17 citation statements)

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“…The scraped cytoskeletons were homogeneized with 12 strokes in a tight-fitting Dounce homogeneizer followed by centrifugation at 100000 x g for 1 h. The supernatant ( z 14 ml at 1 -1.2 mg/ml) was applied to a DEAE-cellulose column ( z 4 cm3) equilibrated with buffer D (20 mM Tris/HCl, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 10 mM 2-mercaptoethanol, 1 mM PhMeS02F). Preliminary experiments had shown that the peak of pp60"-"'" and pp60'-"'" activity eluted slightly below 0.1 M NaC1, as reported also by others [22]. Therefore, in order to achieve as concentrated preparations as possible, the fractions enriched in pp60"-"'" or in pp60"-"" were obtained by a one-step elution with 0.15 M NaCl in buffer D and collecting the proteins that eluted at the highest absorbance.…”

Section: Cells Viruses and Antibodiessupporting

confidence: 81%

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Serine/threonine‐specific protein kinase activity associated with viral pp60^src protein

David‐Pfeuty

Nouvian-Dooghe

1990

European Journal of Biochemistry

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Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60‐kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild‐type Rous sarcoma virus (SRD‐FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v‐src, the mAb327 [Lipsich, L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352–360] and to be phosphorylated in vitro at serine/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c‐src protein immunoprecipitated with mAb327 from extracts of NIH c‐src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor‐bearing rabbit (TBR) serum and reported in an earlier work [David‐Pfeuty, T. & Hovanessian, A. (1984) Eur. J. Biochem. 140, 325–342], together with those reported here, suggest that the TBR‐immunoprecipitated pp60v‐src coprecipitates with a cellular protein related to the 60‐kDa subunit of the Ca2+/calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v‐src, but not of pp60c‐src, are shown to contain a Ca2+/calmodulin‐dependent protein kinase activity that phosphorylates a 52‐kDa protein substrate.

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Section: Cells Viruses and Antibodiessupporting

confidence: 81%

“…The protein concentration of these fractions was generally around 0.5 mg/ml and their average NaCl concentration was 70 mM. An enrichment of approximately 20-fold in pp60"-"" and pp60'-"'" activities was achieved at that stage, as determined by measurement of TBRIgG phosphorylation [22].…”

Section: Cells Viruses and Antibodiesmentioning

confidence: 98%

Serine/threonine‐specific protein kinase activity associated with viral pp60^src protein

David‐Pfeuty

Nouvian-Dooghe

1990

European Journal of Biochemistry

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“…We (29) and others (26,62) have shown that pp60c src is phosphorylated rapidly and stoichiometrically at a normally unphosphorylated serine(s) when cells are treated with tumor promoters that bind and activate protein kinase C. The same sites, serine 12 (S12) in mammalian pp6Ocsrc and both S12 and serine 48 in chicken pp6fc-src, can be phosphorylated by purified protein kinase C in vitro (29). In this report, * Corresponding author. we demonstrate that all agonists of phosphatidylinositol turnover, which we have tested in fibroblasts, lead to the near stoichiometric phosphorylation at S12.…”

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confidence: 69%

Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

Gould¹,

Hunter²

1988

Mol. Cell. Biol.

234

112

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We have shown previously that pp69>-sc is a substrate for protein kinase C in vitro and in vivo and that the target of protein kinase C phosphorylation in mammalian pp6OcSrc is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in fibroblasts (platelet-derived growth factor, fibroblast growth factor, serum, vasopressin, sodium orthovanadate, and prostaglandin F2.) lead to the phosphorylation of pp60c-sr at serine 12. In addition to stimulating serine 12 phosphorylation in pp6Oc-"s, platelet-derived growth factor treatment of quiescent fibroblasts induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal 16 kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.pp6f-src is a membrane-associated phosphoprotein with intrinsic protein-tyrosine kinase activity (for a review, see reference 32). It can be activated to transform cells by C-terminal truncation or various point mutations (6,35,36,40,49,56). pp60c,src is expressed ubiquitously in animal cells, in which its transforming potential is normally suppressed and its specific tyrosine protein kinase activity is lower than that of its transforming counterparts (24,34,45). The highest levels of pp6csrc have been found in the brain (21), platelets (28), peripheral blood lymphocytes (28), and adrenal medullary chromaffin cells (46). Although this distribution has suggested that pp60c-src functions in development, maintenance of a differentiated state, or secretion, its role in normal cellular physiology and its mode of regulation are not understood.We are interested in the role of phosphorylation in the regulation of pp6c-src function. Under most conditions, serine 17 (S17) is the major site of serine phosphorylation in pp60c-src (48, 61). pp60csrc is also phosphorylated in cells to near stoichiometry on a tyrosine near its C terminus, tyrosine 527 (Y527) in chicken pp60csrc (16). Phosphorylation of Y527 constrains the protein kinase activity of the molecule (18,22), and deletion or mutation of this residue results in a protein with transforming ability and higher protein kinase activity (6,36,49,56). Another tyrosine residue within pp60c-src, tyrosine 416 (Y416), is the major site of autophosphorylation in vitro (47, 61). In vivo, Y416 is phosphorylated when Y527 has been mutated (as in Y527 to phenylalanine 527) or deleted (as in pp60vsrc) (6,36,49,56). If it is assumed that pp60c-src is active as a protein kinase under certain circumstances in normal cells, a mechanism(s) must exist for activating and subsequently inhibiting its activity. Such regulation could be achieved by the transient dephosphorylation of Y527, but to date, there have been no reports that this occurs in nontransformed cells.We (29) and others (26, 62) have shown that pp60c src is phosphorylated rapidly and stoichiometrically at a normally unphosphorylated serine(s) when cells are treated with tumor promoters that bind and ...

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“…Like its viral counterpart, the cellular src protein belongs to the tyrosine protein kinase family (review in Jove and Hanafusa, 1987). Biochemical studies have taken advantage of this property and have provided information on pp60~-s'~: the cellular src protein preferentially associates with plasma membranes, like pp60 .... , although, contrary to pp60 v-'~, such an interaction does not appear to occur via the non-ionic, detergent-insoluble cellular matrix (Loeb et al, 1987;Hamaguchi and Hanafusa, 1987); pp60 ~-'~ has been detected in intracellular membranes associated with the nuclear envelope, like pp60 ~-sr~ (Resh and Erikson, 1985) and with more specific structures such as chromaffin granules (Parsons and Greutz, 1986) and synaptic vesicles (Barnekow et al, 1990); platelets have also been shown to contain a relatively high level of pp60 ..... (Rendu et al, 1989;Ferrell et al, 1990); the cellular protein does not seem to form a soluble cytoplasmic complex with two cellular proteins, contrary to pp60 v-s' (Tamura et al, 1984).…”

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confidence: 99%

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

David‐Pfeuty

Nouvian-Dooghe

1990

The Journal of Cell Biology

129

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The mouse mAb, mAb 327, that recognizes specifically both pp60v-src and pp60c-src in a wide variety of cells, has been used to determine precisely the various locations of pp60c-src in NIH c-src overexpresser cells, using the technique of immunofluorescence microscopy. In interphase cells, the protein exhibits two main distributions: one that appears uniform and in association with the cell surface and the other that is patchy and juxtanuclear and coincides with the centrosomes. The juxtanuclear aggregation of pp60c-src-containing patches depends on microtubules and does not seem to occur within the Golgi apparatus and the rough ER. At the G2-to-M-phase transition, a drastic change in the localization patterns of pp60c-src takes place. We also report experiments in which the NIH c-src overexpresser cells were exposed to Con A for various times to induce a redistribution of the cell surface Con A receptors. We show that, at each stage of the Con A-mediated endocytotic process, the Con A-receptor complexes redistribute into structures to which pp60c-src appears also to be associated: at first, into patches that form at the cell surface level and then, into a cap that stands at the cell center in a juxtanuclear position and that coincides with the Golgi apparatus. During this capping process, pp60c-src-containing vesicles continue to accumulate in a centriolar spot, as in interphase, Con A-untreated cells, from which Con A is excluded. The significance of the intracellular locations of pp60c-src to the possible functions of the protein is discussed. Also, the distribution patterns of the cellular protein in the NIH c-src overexpresser cells are compared with those of pp60v-src in RSV-transformed cells. The differences observed are discussed in relation with the differences in transforming capacities of the two proteins. Finally, the possible physiological significance of the association between pp60c-src and the structures generated after the binding of Con A to its surface receptors is addressed.

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pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

Cited by 34 publications

References 34 publications

Serine/threonine‐specific protein kinase activity associated with viral pp60^src protein

Serine/threonine‐specific protein kinase activity associated with viral pp60^src protein

Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

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pp60c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

Cited by 34 publications

References 34 publications

Serine/threonine‐specific protein kinase activity associated with viral pp60src protein

Serine/threonine‐specific protein kinase activity associated with viral pp60src protein

Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity.

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

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pp60^c‐src is a substrate for phosphorylation when cells are stimulated to enter cycle

Serine/threonine‐specific protein kinase activity associated with viral pp60^src protein

Serine/threonine‐specific protein kinase activity associated with viral pp60^src protein