Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation.

Cooper, Jonathan A.; MacAuley, A

doi:10.1073/pnas.85.12.4232

Cited by 171 publications

(149 citation statements)

References 40 publications

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“…Moreover, 8-azido-ATP failed to induce crosslinking, establishing that the Q-phosphate-azido substitution was critical (data not shown). Since Csk can phosphorylate Src and Src can autophosphorylate intermolecularly [8,9], it should be possible to observe a variety of crosslinked adducts, consistent with the heterogeneous pattern observed in Fig. 4.…”

Section: Protein Crosslinking By Compoundsupporting

confidence: 72%

Development of photo‐crosslinking reagents for protein kinase–substrate interactions

et al. 2002

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The identification of relevant protein kinase^protein substrate partners remains a serious challenge on a genome-wide scale. The design and synthesis of a photo-activatable nucleotide reagent to crosslink protein kinases with their substrates is described in which an azido group is appended to the Q Q-phosphoryl and purine moieties of ATP. In the absence of UV, compounds of this class were shown to act as competitive inhibitors versus ATP and non-competitive inhibitors versus peptide substrate for the protein tyrosine kinase Csk, suggesting that they can form a ternary complex with kinase and protein substrate. In vitro experiments with protein kinases indicate the bifunctional reagent can induce covalent protein^protein crosslinking that is dependent on UV irradiation. That significant kinase^substrate crosslinking occurs is suggested by the fact that this crosslinking is competitively inhibited by ATP. The crosslinked adducts can be readily cleaved by phosphodiesterase which supports the model for crosslinking and provides a simple method to deconvolute the linked protein partners. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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Section: Protein Crosslinking By Compoundsupporting

confidence: 72%

Development of photo‐crosslinking reagents for protein kinase–substrate interactions

et al. 2002

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“…Our results agree with data presented by others who showed that the Src tyrosine kinase retains activity when phosphorylated on Tyr-416 and Tyr-527 (41). 3 Previous work in our laboratory as well as kinetic data by other groups suggest that the activating phosphorylation of Tyr-394 in Src family members is an intermolecular event rather than intramolecular reaction (22,(42)(43)(44). 4 Intermolecular phosphorylation of Tyr-394 may be carried out by Lck in vivo, but it is quite possible that other Src family members, or non-Src tyrosine kinases, may also act to phosphorylate Tyr-394 and activate Lck.…”

Section: Discussionmentioning

confidence: 97%

The Activated Form of the Lck Tyrosine Protein Kinase in Cells Exposed to Hydrogen Peroxide Is Phosphorylated at Both Tyr-394 and Tyr-505

Hardwick

Sefton

1997

Journal of Biological Chemistry

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Members of the Src family of non-receptor tyrosine protein kinases are known to be inhibited by the intramolecular association between a phosphorylated carboxyl-terminal tyrosine residue and the SH2 domain. We have previously shown that exposure of cells to H 2 O 2 strongly activates Lck, a lymphocyte-specific Src family kinase, by inducing phosphorylation on Tyr-394, an absolutely conserved residue within the activation loop of the catalytic domain. Here we show that Lck that has been activated by H 2 O 2 is simultaneously phosphorylated at both the carboxyl-terminal tyrosine (Tyr-505) and Tyr-394. Thus, dephosphorylation of Tyr-505 is not a prerequisite for either phosphorylation of Lck at Tyr-394 or catalytic activation of the kinase. These results indicate that activation of Lck by phosphorylation of Tyr-394 is dominant over any inhibition induced by phosphorylation of Tyr-505. We propose that these results may be extended to all Src family members.p56 lck , a member of the Src family of non-receptor tyrosine protein kinases (1, 2), is expressed predominantly in T cells. Lck function is critical both for T-cell development in the thymus (3, 4) and activation of mature T cells in the periphery by antigen (5, 6). Lck stably associates with the inner surface of the plasma membrane as a result of myristoylation of Gly-2 and palmitoylation of Ser-3 and Ser-5 (7-10). There it binds to the T-cell receptor-associated glycoproteins CD4 and CD8 as well as other proteins through its unique amino terminus (11-15). The activity of Lck is regulated by phosphorylation of two conserved tyrosine residues. Tyr-505 (equivalent to Tyr-527 in c-Src) is located near the carboxyl terminus of Lck and, when phosphorylated, associates intramolecularly with the SH2 domain in the amino-terminal half of the protein. This helps stabilize Lck in a conformation that, biologically, is relatively inactive (16 -20). In the absence of phosphorylation at Tyr-505, intramolecular binding of the carboxyl terminus to the SH2 domain does not occur, and Lck exhibits increased activity in vivo. In contrast, phosphorylation of Tyr-394 (equivalent to Tyr-416 in c-Src) stimulates the catalytic activity of Lck (21-23). Phosphorylation of Tyr-394 allows the formation of hydrogen bonds between the phosphate of Tyr(P) 1 -394 and amino acid residues in the catalytic cleft. These interactions allow the enzyme to assume a conformation resembling that of activated cyclic AMP-dependent protein kinase A (19, 20, 24 -26).We have previously demonstrated that hydrogen peroxide, a potent activator of Lck, acts by inducing phosphorylation of Lck on 37). It is likely that the effects of exposing cells to H 2 O 2 are mediated by global inhibition of tyrosine phosphatases (27-31). The increase in phosphorylation of Lck at Tyr-394 that we observe in the presence of H 2 O 2 may therefore result largely from reduced dephosphorylation of this site. Our previous work did not address the question of whether or not activation of Lck by H 2 O 2 -induced phosphorylation of Tyr-394...

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“…Normally, inactive c-Src exists as a tight complex in which the SH2 domain interacts with phosphotyrosine at position 527 localized in the C-terminal region of the protein. Src activation results in the displacement of C termini from the SH2 domain proteins, promoting interactions with SH2 and/or SH3 ligands that prime c-Src for activation and thereby allowing phosphorylation of the kinase on Tyr 416 (26,27).…”

Section: Discussionmentioning

confidence: 99%

mda -9/Syntenin promotes metastasis in human melanoma cells by activating c-Src

Boukerche

Prévot

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

118

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The scaffold PDZ-domain containing protein mda-9/syntenin functions as a positive regulator of cancer cell progression in human melanoma and other tumors. mda-9/Syntenin regulates cell motility and invasion by altering defined biochemical and signaling pathways, including focal adhesion kinase (FAK), p38 mitogenactivated protein kinase (MAPK) and NF-B, but precisely how mda-9/syntenin organizes these multiprotein signaling complexes is not well understood. Using a clinically relevant human melanoma model, we demonstrate that mda-9/syntenin physically interacts with c-Src and this communication correlates with an increase in FAK/c-Src complex formation and c-Src activation. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin or addition of c-Src siRNA, suppresses melanoma cell migration, anchorage-independent growth, and spontaneous tumor cell dissemination in vivo in a human melanoma animal metastasis model. These data are compatible with a model wherein interaction of MDA-9/syntenin with c-Src promotes the formation of an active FAK/c-Src signaling complex, leading to enhanced tumor cell invasion and metastatic spread. These provocative findings highlight mda-9/ syntenin and its interacting partners as promising therapeutic targets for intervention of metastasis. Understanding and preventing this process and its dire consequences remain formidable obstacles in achieving successful treatment outcomes in advanced cancer patients. In the context of advanced melanoma, the response rate of patients with metastatic disease to single agent chemotherapy or combination therapies is frequently less than 10% (1). Given this bleak picture, it is vital to develop new, efficacious, and rationally designed treatment strategies to, ideally, prevent or effectively treat metastasis. In this context, defining relevant genes that are seminal regulators of metastasis provide an entry point for developing improved therapies.Melanoma differentiation associated gene-9 (mda-9) was cloned in our laboratory (2, 3) as a unique gene displaying biphasic expression during terminal differentiation of human melanoma cells treated with a combination of fibroblast IFN (IFN-␤) and the antileukemic compound mezerein (MEZ). These changes are consistent with early enhancement followed by decreased expression during the course of reversion of the cancer phenotype in melanoma cells (2, 3). mda-9, also called syntenin, expression displays an inverse relationship with tumor progression, with lowlevel expression in melanocytes and radial growth phase (RGP) primary melanoma versus elevated expression in vertical growth phase (VGP) primary melanoma and metastatic melanomas (4, 5). The level of mda-9/syntenin is also elevated in multiple additional cancers, including breast and gastric carcinomas, suggesting an expanded involvement in tumor progression (6). Recently, we confirmed the importance of mda-9/syntenin in metastasis in vivo, providing direct support for mda-9/syntenin as an essential gene controlling melanoma progres...

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Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation.

Abstract: The product of the protooncogene c-src is a protein-tyrosine kinase, p0C-src, that is normally inhibited by phosphorylation at a tyrosine residue close to the C terminus

Cited by 171 publications

References 40 publications

Development of photo‐crosslinking reagents for protein kinase–substrate interactions

Development of photo‐crosslinking reagents for protein kinase–substrate interactions

The Activated Form of the Lck Tyrosine Protein Kinase in Cells Exposed to Hydrogen Peroxide Is Phosphorylated at Both Tyr-394 and Tyr-505

mda -9/Syntenin promotes metastasis in human melanoma cells by activating c-Src

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