Phosphorylation of a Src kinase at the autophosphorylation site in the absence of Src kinase activity.

Exposure of cells to oxidants increases the phosphorylation of the Src family tyrosine protein kinase Lck at Tyr-394, a conserved residue in the activation loop of the catalytic domain. Kinase-deficient Lck expressed in fibroblasts that do not express any endogenous Lck has been shown to be phosphorylated at Tyr-394 following H(2)O(2) treatment to an extent indistinguishable from that seen with wild type Lck. This finding indicates that a kinase other than Lck itself is capable of phosphorylating Tyr-394. Because fibroblasts express other Src family members, it remained to be determined whether the phosphorylation of Tyr-394 was carried out by another Src family kinase or by an unrelated tyrosine protein kinase. We examined here whether Tyr-394 in kinase-deficient Lck was phosphorylated following exposure of cells devoid of endogenous Src family kinase activity to H(2)O(2). Strikingly, treatment of such cells with H(2)O(2) led to the phosphorylation of Tyr-394 to an extent identical to that seen with wild type Lck, demonstrating that Src family kinases are not required for H(2)O(2)-induced phosphorylation of Lck. Furthermore, this efficient phosphorylation of Lck at Tyr-394 in non-lymphoid cells suggests the existence of an ubiquitous activator of Src family kinases.

Members of the Src family of non-receptor tyrosine protein kinases have important roles in controlling growth, proliferation, and differentiation (1). For example, the Src family kinases c-Src, Yes, and Fyn are required for integrin-mediated signaling in response to cell adhesion to the extracellular matrix (2). Additionally, Lck, a lymphoid cell-specific member of the Src family (3), is essential for both the development of T cells in the thymus and the response of mature T cells to signals arising from the T-cell antigen receptor (4,5).
Src family kinases are 52-62-kDa cytoplasmic proteins con-sisting of an acylated N-terminal unique domain that mediates association with the plasma membrane, an SH3 1 domain, an SH2 domain, a catalytic domain, and a short C-terminal regulatory tail (6). The SH3 domain interacts with poly-proline type II helices (7), whereas the SH2 domain binds sites of tyrosine phosphorylation (8). Both the SH3 and SH2 domains play a role in the intramolecular regulation of Src family kinase activity (9,10). The kinase activity of Src family members can be both inhibited and activated by phosphorylation. Phosphorylation of a conserved tyrosine near the C terminus (Tyr-505 in Lck, Tyr-527 in c-Src) by the ubiquitous tyrosine protein kinase Csk (11,12) induces formation of a biologically inactive conformation by allowing intramolecular binding of the SH2 domain to the phosphorylated C terminus (9,13,14). This inactive "closed" conformation is further stabilized by binding of the SH3 domain to the linker between the SH2 domain and the catalytic domain (9,10). Dephosphorylation of this site by the tyrosine phosphatase CD45, in the case of Lck, or R-PTP-␣, in the case of c-Src, activates the kinase (15)(16)(17)(18).
In contrast, phosphorylation of a conserved tyrosine in the activation loop (Tyr-394 in Lck, Tyr-416 in c-Src) enhances kinase activity (19 -21). In Lck, the phosphorylation of Tyr-394 is activating because it stabilizes the catalytic active site by forming hydrogen bonds with Arg-387 and Arg-363 and hydrophobic interactions with Ile-361 and Ile-389 (22). Phosphorylation of this site can be carried out by the kinase itself, and it is the major site of "autophosphorylation" in vitro (23,24). Studies of c-Src activity in vitro and in yeast indicate that phosphorylation of the activation loop in c-Src is an intermolecular autophosphorylation event (25,26). The same appears to be the case with Lck and Lyn (21,27). Dephosphorylation of Tyr-394 in Lck is mediated by the tyrosine phosphatases PEP and SHP-1 (28). 2 Exposure of cells to oxidants such as hydrogen peroxide and pervanadate induces the rapid tyrosine phosphorylation of multiple cellular proteins (29) and mimics stimulation by growth factors such as epidermal growth factor or plateletderived growth factor (30,31) or antigen receptor cross-linking (32). These agents have this effect because both inhibit the activity of protein tyrosine phosphatases through the oxidation of an essential catalytic cysteine (33)(34)(35)(36). The induced phosphorylation on tyrosine results, at least in part, from cessation of dephosphorylation in the oxidant-treated cells.
Hydrogen peroxide stimulates the activity of Src family kinases. Endogenously produced H 2 O 2 stimulates the activity of the Src family kinases c-Fgr and Lyn in adherent neutrophils (37). Exposure of Jurkat T cells or Lck-expressing rat 208F fibroblasts to H 2 O 2 induces the phosphorylation of Tyr-394 and enhances Lck kinase activity (21). Interestingly, this oxidantinduced phosphorylation of Tyr-394 in Lck occurs at an undiminished rate in fibroblasts expressing only kinase-deficient forms of Lck. Therefore, the kinase activity of Lck is not required for the oxidant-induced phosphorylation of Tyr-394, and * This work was supported in part by Grants CA14195 and CA42350 from NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. another tyrosine protein kinase may function as an activator of Lck (21). The kinase that phosphorylates Tyr-394 in H 2 O 2treated fibroblasts could be either another Src kinase or an as yet unidentified kinase. To distinguish between these possibilities, we took advantage of the cell line SYF, which is derived from mutant mouse embryos lacking the Src family members c-Src, Yes, and Fyn (2). Additionally, the SYF cell line does not express Lyn (2). We expressed kinase-deficient Lck in SYF cells and analyzed the phosphorylation of Tyr-394 following cellular exposure to H 2 O 2 .

EXPERIMENTAL PROCEDURES
Cell Culture-293, a human embryonic kidney cell line, was grown in DMEM (Cellgro, Mediatech) supplemented with 10% calf serum (Hyclone). Jurkat, a human leukemic T-cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (Intergen) and 2 mM glutamine. SYF (2), a murine embryonic fibroblast cell line derived from mutant embryos deficient for c-Src, Yes, and Fyn (a kind gift from Drs. R. Klinghoffer and P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, WA), was maintained in DMEM supplemented with 10% fetal calf serum.
DNA Constructs and Retroviral Infections-Wild type Lck (WT) and kinase-deficient Lck (R273) cDNAs have been previously described (21,38). The lck cDNAs were subcloned into the retroviral vector MSCVhph (39) from the retroviral vector LXSN (40) to utilize hygromycin B phosphotransferase as a selectable marker. Recombinant retroviruses were produced by cotransfecting the MSCV-Lck constructs along with the viral helper plasmid SV-⌿ Ϫ E-MLV (41) into 293 cells using a calcium phosphate transfection system (Life Technologies, Inc.). 48 h post-transfection, supernatant containing recombinant virus particles was collected, filtered through an 0.45-m filter to remove cells and debris, and used to infect SYF cells. 48 h post-infection, cells expressing WT Lck or R273 Lck were selected with 400 g/ml hygromycin B (Calbiochem) in DMEM supplemented with 10% fetal calf serum.
Immunoblotting-Immunoprecipitated Lck was resolved by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore). Western blotting was carried out with rabbit anti-Lck antibodies as described previously (42) and enhanced chemiluminescence.
In Vivo Labeling and Two-dimensional Tryptic Peptide Mapping-SYF cells were plated at a density of 1.5 ϫ 10 6 cells/5-cm plate, 16 h prior to labeling. Immediately prior to labeling, cells were washed twice in phosphate-free DMEM. Cells were biosynthetically labeled in 2 ml of phosphate-free DMEM supplemented with 10% dialyzed fetal calf serum containing 32 P i (H 3 32 PO 4 , ICN, 0.5 mCi/ml) for 2 h at 37°C. For H 2 O 2 treatment, H 2 O 2 was added to a final concentration of 5 mM, and cells were incubated for 15 min at 37°C. Cells were washed with Tris-buffered saline and lysed in RIPA buffer, and 32 P-labeled Lck was isolated by immunoprecipitation as described. 32 P-labeled Lck was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and excised from the membrane following identification by autoradiography. The excised membrane containing the labeled Lck was then digested with TPCK-trypsin as described previously (43). Two-dimensional tryptic peptide mapping was carried out on cellulose thin layer chromatography plates (EM Science) by electrophoresis at pH 8.9 in the first dimension followed by ascending chromatography in phosphochromatography buffer in the second dimension as described previously (44). The labeled peptides were visualized with a PhosphorImager (Molecular Dynamics).

Stable Expression of WT Lck and R273 Lck in SYF Cells-To
express wild type Lck and the kinase-deficient R273 Lck in SYF cells, we infected the cells with recombinant MSCVhph retroviruses. Following antibiotic selection, pools of selected cells were tested for both Lck expression and kinase activity. Anti-Lck immunoprecipitates were analyzed by Western blotting using anti-Lck antibodies or were subjected to an in vitro kinase reaction. Uninfected SYF cells did not express Lck (Fig.  1A, lane 1). SYF cells infected with WT-or R273 Lck-encoding virus expressed readily detectable levels of Lck (Fig. 1A, lanes  2 and 3). Wild type Lck from the SYF cells exhibited robust kinase activity (Fig. 1B, lane 2). In contrast, the R273 Lck did not exhibit any detectable kinase activity (Fig. 1B, lane 3). Because we were able to detect phosphorylated bands with 0.5% of the intensity of the wild type Lck signal, we estimate that the activity of the R273 Lck is at least 200-fold lower than that of the wild type kinase. H 2 O 2 treatment of SYF cells expressing R273 Lck had no detectable effect on the activity of the mutant kinase (data not shown).
Kinase-deficient Lck Is Phosphorylated at Tyrosine 394 in SYF Cells following H 2 O 2 Treatment-It has been shown previously that exposure of both Jurkat T cells and 208F fibroblasts expressing wild type Lck to H 2 O 2 induces the phosphorylation of Lck at Tyr-394 (21). To determine whether this is also the case in SYF cells, we biosynthetically labeled cells expressing wild type Lck with 32 P i and immunoprecipitated Lck both before and after exposure to 5 mM H 2 O 2 . The phosphorylation of the protein was examined by two-dimensional tryptic peptide analysis (Fig. 2). Wild type Lck was predominately phosphorylated on Tyr-505 in untreated cells ( Fig. 2A). A trace of phosphorylation at Tyr-394 (2% of the level of Tyr-505) was also observed in untreated cells. Following exposure of the labeled cells to H 2 O 2 , wild type Lck became phosphorylated at Tyr-394 to an extent equal to that of Tyr-505 (Fig. 2B).

Phosphorylation of Lck at Tyr-394 6056
The identity of the tryptic peptide containing Tyr-394 was confirmed by its co-migration with the predominant phosphorylated peptide from Lck labeled in vitro by incubation with [␥-32 P]ATP, when mixtures of peptides from two samples were examined by two-dimensional tryptic peptide analysis (Fig. 2, E and F, and data not shown). The trace phosphorylation at Tyr-394 in wild type Lck observed in untreated cells is presumably due to Lck autophosphorylation because kinase-deficient Lck was not phosphorylated at Tyr-394 in untreated cells (Fig.  2, compare A with C) To determine whether kinase-deficient Lck could be phosphorylated at Tyr-394 in cells lacking all Src family kinases, we treated SYF cells expressing R273 Lck with H 2 O 2 and analyzed Lck phosphorylation as detailed above. R273 Lck was phosphorylated at Tyr-505 with no detectable Tyr-394 phosphorylation in untreated SYF cells (Fig. 2C). Strikingly, H 2 O 2 treatment of SYF cells expressing kinase-deficient Lck led to the phosphorylation of Tyr-394 to an extent equal to that of Tyr-505 (Fig.  2D). Pervanadate treatment of SYF cells expressing R273 Lck gave identical results to that seen with H 2 O 2 , indicating that this phenomenon is not specific to H 2 O 2 (data not shown). It is clear that the lack of Lck kinase activity has no effect on H 2 O 2 -induced phosphorylation of tyrosine 394, even in cells completely lacking Src family kinases. DISCUSSION Many serine/threonine protein kinases are activated by phosphorylation in the activation loop. This activating phos-phorylation is usually carried out by a specific upstream activator. For example, the cyclin-dependent kinases (Cdks) are stimulated by the Cdk-activating kinase, CAK, in this manner (45). Additionally, protein kinase B/c-Akt, cAMP-dependent protein kinase, p70 S6 kinase, and several protein kinase C isoforms are all activated by the 3-phosphoinositide-dependent kinase, PDK1 (46 -50). Furthermore, mitogen-activated protein (MAP) kinases are activated by phosphorylation by MAP kinase kinases, such as MEK1 (51)(52)(53)(54).
Src family kinases are also activated by phosphorylation of a conserved tyrosine in the activation loop. This phosphorylation traditionally has been thought to occur as an intermolecular event carried out by the kinase itself (25,26). Our data, however, suggest that phosphorylation of the activation loop tyrosine need not be performed by Src family kinases. Using SYF cells, which are devoid of all known Src family kinase activity, we have demonstrated that H 2 O 2 and pervanadate induce phosphorylation of kinase-deficient Lck at Tyr-394 to an extent equal to that of wild type Lck. Therefore, our results argue strongly that the oxidant-induced phosphorylation of Lck at Tyr-394 is not catalyzed exclusively by Src family members and may be carried out by another tyrosine protein kinase(s) that is functionally analogous to activators of serine/threonine protein kinases.
In the absence of a completely sequenced mammalian genome, the formal possibility that the tyrosine protein kinase responsible for the phosphorylation of Lck at Tyr-394 is a Src family kinase that has escaped detection to date cannot be excluded. However, the defective integrin-mediated signaling phenotype of the SYF cells (2) argues against the expression of a heretofore unidentified Src family member that can functionally complement for the c-Src, Yes, and Fyn deficiency in these cells. Therefore, the simplest interpretation of our results is that a non-Src family kinase is responsible for the oxidantinduced phosphorylation of Lck. We have not yet determined whether the kinase(s) responsible for phosphorylation of Lck at Tyr-394 is itself stimulated by hydrogen peroxide or whether its activity is constitutive and the increased phosphorylation of Tyr-394 is due simply to inhibition of a tyrosine phosphatase. Because Lck is normally expressed only in lymphoid cells, the fact that kinase-deficient Lck is phosphorylated in non-lymphoid cells following exposure to oxidants suggests that a general activator of Src family kinases may exist. Characterization of this kinase(s) in SYF cells may provide insight into this alternative mechanism of Src family kinase activation. We suspect that phosphorylation of the activation loop tyrosine in Src kinases will be shown to be carried out by Src family kinases in some circumstances and by one or more non-Src kinases in others. Phosphorylation of Lck at Tyr-394 6057