Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene.

We used myristylated and nonmyristylated c-src-based variants and phosphotyrosine-specific antibodies to reevaluate the role of tyrosine phosphorylation in cellular transformation by pp60src. Prior methods used to detect tyrosine-phosphorylated proteins failed to discriminate predicted differences in tyrosine phosphorylation which are clearly observed with phosphotyrosine-specific antibodies and Western blotting (immunoblotting). Here we report the observation of a 120,000-Mr protein whose phosphorylation on tyrosine correlates with the induction of morphological transformation. p120 was not observed in cells overexpressing the regulated, nononcogenic pp60c-src, whereas phosphorylation of p120 was greatly enhanced in cells expressing activated, oncogenic pp60527F. Furthermore, phosphorylation of p120 was not induced by expression of the activated but nonmyristylated src variant pp602A/527F, which is transformation defective. p120 partitioned preferentially with cellular membranes, consistent with the observation that transforming src proteins are membrane associated. Although a number of additional putative substrates were identified and partially characterized with respect to intracellular localization, tyrosine phosphorylation of these proteins was not tightly linked to transformation.

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Activation of c-Src in cells bearing v-Crk and its suppression by Csk

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4706-4713.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4706-4713

Author(s):

H Sabe ◽

M Okada ◽

H Nakagawa ◽

H Hanafusa

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Src Kinase ◽

Cellular Protein ◽

Morphological Transformation ◽

Protein Tyrosine ◽

In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

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Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

Molecular Biology of the Cell ◽

10.1091/mbc.6.8.953 ◽

1995 ◽

Vol 6 (8) ◽

pp. 953-966 ◽

Cited By ~ 7

Author(s):

M F Verderame ◽

J L Guan ◽

K M Woods Ignatoski

Keyword(s):

Host Range ◽

Tyrosine Phosphorylation ◽

Kinase Activity ◽

Biochemical Properties ◽

Morphological Transformation ◽

Anchorage Independent Growth ◽

Parental Allele ◽

Morphological Phenotype ◽

In Cells

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.

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Activation of c-Src in cells bearing v-Crk and its suppression by Csk.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4706 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4706-4713 ◽

Cited By ~ 64

Author(s):

H Sabe ◽

M Okada ◽

H Nakagawa ◽

H Hanafusa

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Src Kinase ◽

Cellular Protein ◽

Morphological Transformation ◽

Protein Tyrosine ◽

In Cells

The protein product of the CT10 virus, p47gag-crk (v-Crk), which contains Src homology region 2 (SH2) and 3 (SH3) domains but lacks a kinase domain, is believed to cause an increase in cellular protein tyrosine phosphorylation. A candidate tyrosine kinase, Csk (C-terminal Src kinase), has been implicated in c-Src Tyr-527 phosphorylation, which negatively regulates the protein tyrosine kinase of pp60c-src (c-Src). To investigate how c-Src kinase activity is regulated in vivo, we first looked at whether v-Crk can activate c-Src kinase. We found that cooverexpression of v-Crk and c-Src caused elevation of c-Src kinase activity, resulting in an increase of tyrosine phosphorylation of cellular proteins and morphological transformation of rat 3Y1 fibroblasts. v-Crk and c-Src complexes were not detected, although v-Crk bound to a variety of tyrosine-phosphorylated proteins in cells overexpressing v-Crk and c-Src. Overexpression of Csk in these transformed cells caused reversion to normal phenotypes and also reduced the level of c-Src kinase activity. However, Csk did not cause reversion of cells transformed by v-Src or c-Src527F, in which Tyr-527 was changed to Phe. These results strongly suggest that Csk acts on Tyr-527 of c-Src and suppresses c-Src kinase activity in vivo. Because Csk can suppress transformation by cooverexpression of v-Crk and c-Src, we suggest that v-Crk causes activation of c-Src in vivo by altering the phosphorylation state of Tyr-527.

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Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.905-913.1987 ◽

1987 ◽

Vol 7 (2) ◽

pp. 905-913

Author(s):

W Yonemoto ◽

A J Filson ◽

A E Queral-Lustig ◽

J Y Wang ◽

J S Brugge

Keyword(s):

Tyrosine Phosphorylation ◽

Tumor Antigen ◽

Tyrosine Kinases ◽

Cellular Transformation ◽

Cellular Protein ◽

Cellular Proteins ◽

Transformed Cells ◽

Major Protein ◽

Rous Sarcoma ◽

Phosphotyrosine Phosphatases

Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.

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Role of SH2 Domain in JAK2-V617F Mediated Transformation.

Blood ◽

10.1182/blood.v108.11.3608.3608 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3608-3608 ◽

Cited By ~ 1

Author(s):

Sivahari P. Gorantla ◽

Tobias Dechow ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Point Mutation ◽

Jak2 V617f ◽

Cellular Transformation ◽

Sh2 Domain ◽

Cytokine Receptor ◽

Wild Type ◽

Therapeutic Implications ◽

Proliferation Assays ◽

In Cells

Abstract A point mutation in JAK2 (V617F) has been described recently in patients with myeloproliferative diseases like polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (IMF). This V617F point mutation in JAK2 has been shown to activate several downstream pathways including STAT5 and ERK. This mutation also renders haematopoietic progenitors cytokine-independent. The role of the V617F mutation in oncogenesis is not fully understood. In this study we aim to dissect the role of the SH2 domain in JAK2-V617F mediated transformation. Stable Ba/F3 cell lines expressing JAK2-wild type (wt), JAK2-V617F, JAK2-R439K (SH2 domain mutation) and JAK2-V617F/R439K mutants were generated. Cell proliferation assays showed that JAK2-V617F transforms Ba/F3 cells and renders them IL3 independent, while wild type JAK2 and JAK2-R439K could not. Surprisingly, JAK2-V617F/R439K was not able to induce a transformed phenotype in Ba/F3 cells. Imunoblotting revealed strong activation of JAK2, STAT5 and ERK in cells expressing JAK2-V617F, whereas no such activation could be found in JAK2-wt, JAK2-R439K and in JAK2-V617F/R439K expressing cells. Thus the SH2 domain in JAK2-V617F seems to play a crucial role in the transformation of Ba/F3 cells containing a heterodimeric (IL-3) cytokine receptor. It has been demonstrated that JAK2-V617F induces cellular transformation more efficiently in cells expressing a homodimeric cytokine receptor such as the erythropoetin receptor. We therefore established Ba/F3 cells overexpressing EpoR together with JAK2-wt, JAK2-V617F, JAK2-R439K and JAK2-V617F/R439K. In contrast to parental Ba/F3 cells, EpoR expressing Ba/F3 cells could be transformed by both JAK2-V617F as well as JAK2-V617F/R439K. Both the single and double mutant Ba/F3 cells showed strong activation of STAT5 and ERK. This suggests that an intact SH2 domain is not required for homodimeric cytokine receptor expressing cells. These results show that transformation by JAK2-V617F requires an intact SH2 domain only in cells expressing a heterodimeric cytokine receptor. In contrast, cells containing a homodimeric cytokine receptor are able to induce transformation in the presence of JAK2-V617F with an additional SH2 mutation. Further progress in understanding the role of the SH2 domain in JAK2-V617F mediated transformation may help in delineating downstream signalling with therapeutic implications.

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Proteomic identification of the differentially expressed and phosphorylated proteins in 20-hydroxyecdysone (20E) signal transduction pathway in salivary gland of Drosophila melanogaster

10.32469/10355/41913 ◽

2010 ◽

Author(s):

◽

Yaning Sun

Keyword(s):

Signal Transduction ◽

Drosophila Melanogaster ◽

Salivary Gland ◽

Intracellular Localization ◽

Signal Transduction Pathway ◽

Phosphorylated Proteins ◽

Rnai Technology ◽

Pkc Isoforms ◽

First Time

Protein kinase C (PKC) plays important role in 20-hydroxyecdysone (20E) signal transduction, however, little is known about the exact role of PKC in this process. In my research, PKC-regulated phosphorylation in 20E signal transduction is investigated in the salivary gland of Drosophila melanogaster. Our experiments demonstrate that PKCregulated phosphorylation is responsible for the intracellular localization of ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle protein (USP), which is possibly due to the forming of receptor complex with chaperons. We also confirmed PKC-regulated phosphorylation is required in 20E induced protein expression and identified 14 proteins induced by 20E but inhibited by PKC inhibitor. Using 2D Western blot and phospho-(Ser) PKC substrate, we were able to identify four phosphorylated PKC substrates in 20E signal transduction process, which may function in 20E-induced gene transcription/translation process or in ecdysteroid transporting. In addition, PKC isoforms in the salivary gland were also investigated by RNA interference (RNAi). For the first time, we showed the successful application of RNAi technology by soaking the salivary glands of D. melanogaster with dsRNAs.

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Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.905 ◽

1987 ◽

Vol 7 (2) ◽

pp. 905-913 ◽

Cited By ~ 22

Author(s):

W Yonemoto ◽

A J Filson ◽

A E Queral-Lustig ◽

J Y Wang ◽

J S Brugge

Keyword(s):

Tyrosine Phosphorylation ◽

Tumor Antigen ◽

Tyrosine Kinases ◽

Cellular Transformation ◽

Cellular Protein ◽

Cellular Proteins ◽

Transformed Cells ◽

Major Protein ◽

Rous Sarcoma ◽

Phosphotyrosine Phosphatases

Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.

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Integrin signaling is critical for pathological angiogenesis

Journal of Experimental Medicine ◽

10.1084/jem.20060807 ◽

2006 ◽

Vol 203 (11) ◽

pp. 2495-2507 ◽

Cited By ~ 131

Author(s):

Ganapati H. Mahabeleshwar ◽

Weiyi Feng ◽

David R. Phillips ◽

Tatiana V. Byzova

Keyword(s):

Endothelial Cells ◽

Tyrosine Phosphorylation ◽

Ex Vivo ◽

Regulatory Function ◽

Vegf Receptor ◽

Wild Type ◽

Pathological Angiogenesis ◽

Β3 Integrin ◽

In Cells

The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant β3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant β3 knock-in mice. In an ex vivo angiogenesis assay, mutant β3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant β3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and β3 integrin in wild-type but not in mutant β3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant β3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin–VEGF axis in pathological angiogenesis.

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