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

Abstract

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...

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

“…At least for Jurkat T cells it was described that Tyr 394 /Tyr 505 doubly phosphorylated Lck was catalytically active, demonstrating that phosphorylation of the positive regulatory site Tyr 394 is dominant over the C-terminal inhibitory Tyr 505 phosphorylation (38). In line with this finding, the significant FRET increase of our Y394F-mutated biosensor in comparison to the parental construct shows the strong mechanical link between the activation loop and the overall conformation of Lck in living cells.…”

Autophosphorylation of Src and Yes blocks their inactivation by Csk phosphorylation

“…Once autophosphorylated on Y a , Src becomes hyperactive regardless of the phosphorylation status of Y r , although Y r can still be phosphorylated to form the doubly phosphorylated form. In support of this model, Lck that is phosphorylated on both Y a and Y r and hyperactive was recently isolated from Jurkat human leukemic T cells (Hardwick and Sefton, 1997). Csk phosphorylation can only inactivate Src that is not autophosphorylated.…”

Differential Requirement for p56lck in HIV-tat Versus TNF-Induced Cellular Responses: Effects on NF-κB, Activator Protein-1, c-Jun N-Terminal Kinase, and Apoptosis

“…Thus it is possible that p56 lck -induced ROI generation mediates the activation of NF-B, AP-1, JNK, MEK, and apoptosis. H 2 O 2 has been shown to activate p56 lck enzyme (38), but whether p56 lck can mediate ROI generation, has not been reported.…”

“…We have shown that ROI are also required for HIV-tat-induced cellular responses (18). Moreover p56 lck has been shown to be a redox-sensitive protein kinase (38). Whether p56 lck is required for the ligand-induced generation of ROI is not known.…”