Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Lee, Sungsoo; Lin, Xiaofeng; Nam, Nguyen Hai; Parang, Keykavous; Sun, Gongqin

doi:10.1073/pnas.2534493100

Cited by 83 publications

(104 citation statements)

References 41 publications

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“…For quantifying PTK activities, we measured the phosphorylation of polyE 4Y by using the acid precipitation assay (36). Standard assay reactions of 50 L contained 75 mM 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) (pH 8.0), 1 mg⅐mL Ϫ1 polyE4Y, 200 M [ 32 ]ATP (Ϸ1,000 dpm/pmol), 12 mM MgCl 2, 5% glycerol, and 0.005% Triton X-100.…”

Section: Methodsmentioning

confidence: 99%

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

Kemble

Sun

2009

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

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125

120

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Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8 -25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs. R eactive oxygen species (ROS), such as hydrogen peroxide and superoxide, can alter the function of proteins by oxidizing free sulfhydryl groups to sulfenic, sulfinic, or sulfonic acids (1, 2). Cellular responses to ROS are historically considered a damage-control mechanism to certain pathological situations that lead to oxidative stress. However, recent studies indicate that certain growth factors and cell adhesion also stimulate the production of ROS, which serve as secondary messengers to regulate downstream signaling pathways (3, 4). Numerous protein phosphorylation pathways respond to ROS (5-9), and identifying the proteins and the residues sensitive to oxidation will help elucidate the mechanism of cross-talk between redox and protein tyrosine phosphorylation.The level of protein tyrosine phosphorylation is the function of opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). All PTPs contain a catalytic Cys residue in the active site, and oxidation of this residue leads to the inactivation of the PTP activity (10). This response is recognized as a major mechanism by which ROS regulate the level of protein tyrosine phosphorylation. However, whether PTKs also directly respond to ROS is not established. PTK Src is a key regulator of cell survival, cytoskeleton reorganization, DNA synthesis, and cell division (11,12). A number of studies suggest that Src also plays an important role in cellular response to ROS, because Src specific inhibitors and dominantnegative Src mutants strongly attenuate cellular response to ROS (13-16). However, how ROS regulate Src activity has been controversial, likely reflecting the complexity of Src regulation. Src contains regulatory str...

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Section: Methodsmentioning

confidence: 99%

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

Kemble

Sun

2009

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

Self Cite

125

120

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“…The PS1/c-secretase system regulates Src-Csk association Csk kinase binds Src kinase, decreasing both phosphorylation of Src residue tyr418 and Src activity (Lee et al, 2003). Since the PS1/g-secretase system stimulates phosphorylation of Src tyr418, we asked whether this system affects the Src-Csk association.…”

Section: Ephrinb Ligands Are Cleaved By Mp and Ps1/c-secretasementioning

confidence: 99%

Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling

Georgakopoulos¹,

Litterst²,

Ghersi³

et al. 2006

EMBO J

145

178

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Bidirectional signaling triggered by interacting ephrinB receptors (EphB) and ephrinB ligands is crucial for development and function of the vascular and nervous systems. A signaling cascade triggered by this interaction involves activation of Src kinase and phosphorylation of ephrinB. The mechanism, however, by which EphB activates Src in the ephrinB-expressing cells is unknown. Here we show that EphB stimulates a metalloproteinase cleavage of ephrinB2, producing a carboxy-terminal fragment that is further processed by PS1/c-secretase to produce intracellular peptide ephrinB2/CTF2. This peptide binds Src and inhibits its association with inhibitory kinase Csk, allowing autophosphorylation of Src at residue tyr418. EphrinB2/CTF2-activated Src phosphorylates ephrinB2 and inhibits its processing by c-secretase. These data show that the PS1/c-secretase system controls Src activation and ephrinB phosphorylation by regulating production of Src activator ephrinB2/CTF2. Accordingly, csecretase inhibitors prevented the EphB-induced sprouting of endothelial cells and the recruitment of Grb4 to ephrinB. PS1 FAD and c-secretase dominant-negative mutants inhibited the EphB-induced cleavage of ephrinB2 and Src autophosphorylation, raising the possibility that FAD mutants interfere with the functions of Src and ephrinB2 in the CNS.

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“…Several side chains located in helix D outside the active site of Csk in a docking groove are important in maintaining efficient Src phosphorylation kinetics (19). In a previous study we showed that Csk does not bind with high affinity to Src based on equilibrium sedimentation and single turnover experiments (21).…”

mentioning

confidence: 99%

“…Mutagenesis studies have shown that Csk recognizes not only residues directly in the C terminus but also residues in the large lobe of the kinase domain of Src (17)(18)(19)(20). Several side chains located in helix D outside the active site of Csk in a docking groove are important in maintaining efficient Src phosphorylation kinetics (19).…”

mentioning

confidence: 99%

Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk

Lieser

Shaffer

Adams

2006

Journal of Biological Chemistry

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Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.

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Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

Cited by 83 publications

References 41 publications

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation

Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling

Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk

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