The role of protein phosphorylation at tyrosine in transformation and mitogenesis

Martin, G S; Radke, Kathryn; Carter, C.; Moss, Paul S.; Dehazya, Philip; Gilmore, Thomas D.

doi:10.1002/jcp.1041210417

Cited by 8 publications

(2 citation statements)

References 41 publications

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“…However, in previous studies only the tyrosine phosphorylation of a 42-kDa protein was found to be consistently increased by many distinct mitogens, including the mitogenic protease trypsin and tumor promotors (Martin et al, 1985;Hunter and Cooper, 198513). In addition, p36, or calpactin, a peripheral membrane protein and a substrate of many transforming protein tyrosine kinases (Cooper and Hunter, 19831, was previously found to become phosphorylated on tyrosine residues in cells exposed to EGF and PDGF (Cooper and Hunter, 1983).…”

Section: Discussionmentioning

confidence: 95%

Comparative study of the tyrosine phosphorylation of proteins in Swiss 3T3 fibroblasts stimulated by a variety of mitogenic agents

Pasquale

Maher

Singer

1988

Journal Cellular Physiology

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We have carried out a comparative study of the protein tyrosine phosphorylation induced by a wide range of mitogenic stimuli on a single cell type, Swiss 3T3 mouse fibroblasts. For this purpose we have used high-affinity antibodies directed to phosphotyrosine residues on proteins (Wang: Mol. Cell. Biol. 5:3640-3643, 1985) in immunoblotting and immunofluorescence microscopy experiments. Immunoblotting experiments showed that all of the mitogens tested, including epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, fetal calf serum, trypsin, and 12-O-tetradecanoylphorbol-13-acetate, increased the phosphorylation on tyrosine of a number of proteins. Most of the increase in tyrosine phosphorylation induced by each factor involved a small set of proteins with apparent molecular weights (Mr) above 50,000. Following stimulation with epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor, increased phosphotyrosine modification of proteins with molecular weights corresponding to those of the respective receptors was observed. A protein band of apparent Mr 160,000 contained substantially increased levels of phosphotyrosine following insulin treatment, but tyrosine phosphorylation of the insulin receptor was apparently below the level of detectability. The phosphotyrosine content of proteins with apparent Mr of 220,000, 120,000, and 70,000 was increased by all the agents tested. Phosphorylation on tyrosine of most of the proteins increased within a few minutes of the mitogenic stimulation, reached a peak, and returned more slowly to basal levels. Immunofluorescence labeling with the antibodies specific for phosphotyrosine showed a substantial increase in the amount of phosphotyrosine containing proteins only in the presence of platelet-derived growth factor and fetal calf serum. This finding suggests that most of the proteins phosphorylated on tyrosine in Swiss 3T3 fibroblasts are not concentrated in specific subcellular structures, but rather are diffusely distributed throughout the cell and are therefore not detectable by immunofluorescence microscopy.

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Section: Discussionmentioning

confidence: 95%

Comparative study of the tyrosine phosphorylation of proteins in Swiss 3T3 fibroblasts stimulated by a variety of mitogenic agents

Pasquale

Maher

Singer

1988

Journal Cellular Physiology

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“…They have important functions in the regulation of enzyme activities (19), in the initiation of protein biosynthesis (25) and in the process of cell proliferation (13,22). Besides the well studied oneogene protein kinases, a number of virus associated protein kinases has been described, not only for enveloped viruses (2,4,5,11,13), but, also for adenoviruses (1,33) and pieornaviruses (10,29).…”

Section: Introductionmentioning

confidence: 99%

Properties of poliovirus associated protein kinase

Lackmann

Ueckermann

Engelmann

et al. 1987

Archives of Virology

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Highly purified virulent poliovirus preparations harbour an endogenous protein kinase. Enzyme activity increases significantly upon purification of infectious virus particles from infected HeLa cells. Enzyme activity is stimulated by divalent cations. The substrate specificity and the degree of stimulation of the kinase are dependent on the nature of the divalent cations included in the assay. The preferred substrates for this kinase are the viral capsid proteins. Exogenously added proteins such as alpha-casein, phosvitin and protamine are also phosphorylated by the kinase. Moreover, these proteins enhance the phosphorylation of viral proteins. In the presence of Mg++ VP 2 and VP 0 are highly phosphorylated, while in the presence of Zn++ only VP 2 and VP 4, but not VP 0 or exogenous proteins are phosphorylated. Poliovirus associated protein kinase exhibits optimal activity at pH 7.9 in the presence of 10 mM Mg++. The Km for ATP is shown to be 40 microM. By testing different nucleotides as phosphate donors a specificity of the phosphorylation reaction for ATP is demonstrated. Phosphoamino acid analysis of hydrolysates of the substrates phosphorylated in the presence of Mg++ by thin layer electrochromatography and HPLC yielded phosphoserine and phosphothreonine from viral capsid proteins while hydrolysates of protamine yield only phosphoserine. Destabilization of the viral capsid, e.g. by preincubation at 42 degrees C for 20 minutes results in a stimulation of kinase activity. Moreover, phosphorylation of the poliovirus capsid proteins itself results in destabilization of the viral capsid. These findings suggest that phosphorylation of the viral coat proteins triggers or enhances the uncoating of poliovirus leading to the release of viral RNA.

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