A New Monoclonal Antibody Which Selectively Recognizes the Active Form of Src Tyrosine Kinase

Kawakatsu, Hisaaki; Sakai, Takao; Takagaki, Yumiko; Shinoda, Yasuhiko; Saito, Masaki; Owada, M. Koji; Yano, Junichi

doi:10.1074/jbc.271.10.5680

Cited by 82 publications

(81 citation statements)

References 40 publications

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“…The monoclonal anti-Src antibody, clone 28, which selectively recognizes the active, carboxyl-terminal-dephosphorylated form of Src (29), was a gift from Drs. J. Yano and K. Owada (Kyota Pharmaceutical University, Kyoto, Japan).…”

Section: Methodsmentioning

confidence: 99%

Src Kinase Activity Is Regulated by the SHP-1 Protein-tyrosine Phosphatase

Somani¹,

Bignon²,

Mills³

et al. 1997

Journal of Biological Chemistry

158

123

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Activation of the cellular Src tyrosine kinase depends upon dephosphorylation of the carboxyl-terminal inhibitory tyrosine phosphorylation site. Herein we show that Src isolated from human platelets and Jurkat T cells is preferentially dephosphorylated at its inhibitory phosphotyrosine site by the SHP-1 tyrosine phosphatase. The data also revealed association of Src with SHP-1 in both platelets and lymphocytes and the capacity of Src to phosphorylate SHP-1 and interact with the SHP-1 NH 2 -terminal SH2 domain in vitro. Analysis of Src activity in thymocytes from SHP-1-deficient motheaten and viable motheaten mice revealed this kinase activity to be substantially lower than that detected in wild-type thymocytes, but to be enhanced by in vitro exposure to SHP-1. Similarly, immunoblotting analysis of thymocyte Src expression before and after selective depletion of active Src protein indicated that the proportion of active relative to inactive Src protein is markedly reduced in motheaten compared with wild-type cells. These observations, together with the finding of reduced Src activity in HEY cells expressing a dominant negative form of SHP-1, provide compelling evidence that SHP-1 functions include the positive regulation of Src activation.Both the kinase and transforming activities of the pp60 c-src (Src) protein-tyrosine kinase (PTK) 1 are repressed by phosphorylation of a tyrosine residue within the Src carboxyl terminus (1-5). The negative regulatory effect of this phosphotyrosine (Tyr-530 and Tyr-529 in human and murine Src, respectively) has been ascribed to its association with the Src SH2 domain and consequent SH2, as well as SH3 domain-mediated intramolecular interactions that repress activity of the kinase domain (6 -8). The catalytic activities of the other Src family members are similarly inhibited by phosphorylation of this conserved C-tail tyrosine (9), and activation of each of these PTKs thus depends on dephosphorylation at this site. However, while several lines of evidence implicate the Csk tyrosine kinase (10 -12) as well as Src-mediated autophosphorylation (13) in the phosphorylation of the COOH-terminal regulatory tyrosine, relatively little is known about the mechanisms whereby this inhibitory phosphotyrosine residue is dephosphorylated and Src activation achieved. In contrast to the Src-related Lck and possibly Fyn PTKs, the Src COOH-terminal phosphotyrosine does not appear subject to dephosphorylation by the transmembrane protein-tyrosine phosphatase (PTP), CD45 (14 -16). Moreover, while Src activity has been found to be increased in rodent cell lines overexpressing the receptor-like protein-tyrosine phosphatase, RPTP␣ (17, 18), a direct role for this PTP in dephosphorylating Tyr-529 in vivo has not been established. However, recent data from studies of the SHP-1 tyrosine phosphatase, a cytosolic, dual SH2 domain-containing protein expressed primarily in hemopoietic and epithelial cells (19 -22), have raised the possibility that this PTP plays a role in the dephosphorylation and activation ...

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Section: Methodsmentioning

confidence: 99%

Src Kinase Activity Is Regulated by the SHP-1 Protein-tyrosine Phosphatase

Somani¹,

Bignon²,

Mills³

et al. 1997

Journal of Biological Chemistry

158

123

View full text Add to dashboard Cite

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“…This antibody targets the C-terminal tail of Src, which is only detectable when Src is in its active conformation [16] Frozen sections were scored in a blinded fashion according to the intensity of staining of carcinoma cells on a scale of -, +/-, +, ++ or +++ by one observer (VJ) and confirmed by an independent reviewer (CW).…”

Section: Immunohistochemistrymentioning

confidence: 99%

Src inhibitors in early breast cancer: a methodology, feasibility and variability study

Jones¹,

Young

Renshaw

et al. 2008

Breast Cancer Res Treat

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“…Mouse monoclonal antibodies to nonphosphorylated Src-Tyr-530 (clone 28) were kindly provided by Dr. Hisaaki Kawakatsu [15] and monoclonal antibodies to p130Cas and paxillin were from BD Transduction Laboratories, to vinculin from Sigma, and to phosphotyrosine (clone 4G10) from Upstate Biotechnology.…”

Section: Antibodiesmentioning

confidence: 99%

Tac-β1 inhibits FAK activation and Src signaling

Berrier

Jones

LaFlamme

2008

Biochemical and Biophysical Research Communications

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The binding of integrins to extracellular matrix triggers signals that promote cell spreading. We previously demonstrated that expression of the integrin β1 cytoplasmic domain in the context of a chimeric transmembrane receptor with the Tac subunit of the interleukin-2 receptor (Tac-β1) inhibits cell spreading. To study the mechanism whereby Tac-β1-inhibits cell spreading, we examined the effect of Tac-β1 on early signaling events following integrin engagement namely FAK and Src signaling. We infected primary fibroblasts with adenoviruses expressing Tac or Tac-β1 and found that Tac-β1 prevented FAK activation by inhibiting the phosphorylation of FAK at Tyr-397. In contrast, Src activation was maintained, as phosphorylation of Src at Tyr-419 and Tyr-530 were not responsive to expression of Tac-β1. Importantly, adhesion-induced tyrosine phosphorylation of the Src substrates p130Cas and paxillin was inhibited, indicating that Src signaling was blocked by Tac-β1. These Src-dependent signaling events were found to require FAK signaling. Our results suggest that Tac-β1 inhibits cell spreading, at least in part, by preventing the phosphorylation of FAK at Tyr-397 and the assembly of signaling complexes necessary for phosphorylation of p130Cas and other downstream effectors.

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A New Monoclonal Antibody Which Selectively Recognizes the Active Form of Src Tyrosine Kinase

Cited by 82 publications

References 40 publications

Src Kinase Activity Is Regulated by the SHP-1 Protein-tyrosine Phosphatase

Src Kinase Activity Is Regulated by the SHP-1 Protein-tyrosine Phosphatase

Src inhibitors in early breast cancer: a methodology, feasibility and variability study

Tac-β1 inhibits FAK activation and Src signaling

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