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Originally published In Press as doi:10.1074/jbc.M503498200 on April 25, 2005

J. Biol. Chem., Vol. 280, Issue 25, 23918-23925, June 24, 2005
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Inactivation of Src Family Tyrosine Kinases by Reactive Oxygen Species in Vivo*

Hua Tang{ddagger}§, Qin Hao{ddagger}, Stacey A. Rutherford{ddagger}, Brad Low{ddagger}, and Z. Joe Zhao¶

From the {ddagger}Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas 75708 and the Division of Hematology/Oncology, Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232

Received for publication, March 30, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species, including H2 O2, and are constantly produced in the human body and are involved in the development of cardiovascular diseases. Emerging evidence suggests that reactive oxygen species, besides their deleterious effects at high concentrations, may be protective. However, the mechanism underlying the protective effects of reactive oxygen species is not clear. Here, we reported a novel finding that H2O2 at low to moderate concentrations (50-250 µM) markedly inactivated Src family tyrosine kinases temporally and spatially in vivo but not in vitro. We further showed that Src family kinases localized to focal adhesions and the plasma membrane were rapidly and permanently inactivated by H2O2, which resulted from a profound reduction in phosphorylation of the conserved tyrosine residue at the activation loop. Interestingly, the cytoplasmic Src family kinases were activated gradually by H2O2, which partially compensated for the loss of total activities of Src family kinases but not their functions. Finally, H2O2 rendered endothelial cells resistant to growth factors and cytokines and protected the cells from inflammatory activation. Because Src family kinases play key roles in cell signaling, the rapid inactivation of Src family kinases by H2 O2 may represent a novel mechanism for the protective effects of reactive oxygen species.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Src family tyrosine kinases (SFKs)1 play initiating and key roles in the control of cell proliferation, differentiation, survival, adhesion, cytoskeletal rearrangement, and specialized cell signals by a diverse set of cell surface receptors such as growth factor receptors, G protein-coupled receptors, antigen receptors, cytokine receptors, and integrins (1). SFKs comprise nine family members, Src, Fyn, Yes, Lck, Hck, Blk, Fgr, Lyn, and Yrk, and share a conserved domain structure, including a myristoylated N-terminal unique sequence that mediates association with the inner face of the plasma membrane, followed by Src homology (SH)3, SH2, and kinase domains and a short C-terminal tail with regulatory function (2). The ability of SFKs to initiate and mediate signaling from cell surface receptors is dependent on their catalytic activities, locations, and binding partners (3, 4).

The enzymatic activities of SFKs are positively and negatively regulated by tyrosine phosphorylation. Phosphorylation of a conserved tyrosine (Tyr-418, using human Src numbering throughout) in the activation loop enhances kinase activity (5-7). Phosphorylation of Src Tyr-418, the major site of auto-phosphorylation in vitro, has been traditionally thought to occur as an intermolecular event carried out by the kinase itself (8, 9). However, a set of evidence suggests that phosphorylation of the activation loop tyrosine can be achieved by a kinase other than SFK (7, 10). On the other hand, phosphorylation of a conserved tyrosine (Tyr-529 in human Src) in the C-terminal tail by tyrosine kinase Csk or its family member Chk inhibits the activity of Src (11, 12). Phosphorylation of Tyr-529 causes an intramolecular binding of the SH2 domain to the phosphorylated C terminus, followed by binding of the SH3 domain to the linker between the SH2 and the catalytic domains, thus resulting in formation of an inactive conformation of Src (13, 14). Dephosphorylation of this site is mediated by receptor tyrosine phosphatases (RPTP{alpha} and CD45) (15), which facilitates SFKs activation.

Reactive oxygen species (ROS), such as H2O2, superoxide (), and hydroxyl radical (OH·), are constantly produced in the human body and are involved in the pathogenesis of cardiovascular diseases, cancer, and Alzheimer disease (16-18). ROS can be generated in activated phagocytes as one of the defense mechanisms and in non-phagocytes stimulated with cytokines, growth factors, and agonists for G protein-coupled receptors (19-21). Moreover, tissue injuries such as ischemia/reperfusion and surgical angioplasty, hypercholesterolemia, normal body metabolism, and exercise all promote ROS production (22-24). ROS have major impacts on vascular cells, including endothelial cells (ECs) that are at the center of most common clinical pathologies. The conventional thought has generally regarded ROS as being harmful to the vasculature, leading to such pathological processes as atherosclerosis, coronary ischemia, angiogenesis, retinopathy, restenosis, and hypertension (16, 24, 25). However, controlled clinical trials have failed to show a consistent benefit of antioxidants on these cardiovascular diseases (26-28). Although a number of factors may contribute to this lack of efficacy of antioxidants, one intriguing possibility is that ROS may play both a physiological and pathophysiological role in vascular homeostasis, i.e. ROS may be both protective and deleterious. It has been shown that endothelial preconditioning by ROS reduces EC inflammatory responses to tumor necrosis factor-{alpha} (TNF-{alpha}) (29). Preconditioning by ROS also protects reperfusion injury of cardiomyocytes (24). However, the mechanism underlying the protective effect of ROS has not been elucidated yet.

In the present study, we report a novel finding that H2O2 at a low to moderate concentration (50-250 µM) markedly inactivates SFKs temporally and spatially in vivo, but not in vitro, and renders ECs resistant to growth factors and cytokines, thereby protecting the cells from inflammatory activation. Furthermore, we have shown that a reduced phosphorylation of the tyrosyl residue in the activation loop (Tyr-418 in human Src) is responsible for the inactivation of SFKs by H2O2 in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—H2O2 was from Sigma and Fisher. Recombinant human full-length Src and Fyn, Src substrate peptide (KVEKIGEGTYGVVYK), and P81 phosphocellulose paper were from Upstate Cell Signaling Solutions. Reagents for chemiluminescence detection were from Cell Signaling.

Antibodies—Antibodies against phospho-Src (Tyr-418) and phospho-Src (Tyr-529) were from BIOSOURCE. Antibodies against phospho-p130Cas (Tyr-410), phospho-paxillin (Tyr-118), phospho-ERK (Thr-202/Tyr-204), phospho-Src (Tyr-416), and phospho-Src (Tyr-527) were from Cell Signaling. A phospho-specific antibody against Fyn (Tyr-528) or Src (Tyr-530) was from BD Pharmingen. Antibodies against SFK (SRC-2) and vascular cell adhesion molecule-1 (VCAM-1) were from Santa Cruz Biotechnology. Monoclonal antibodies against Src (GD11) and phosphotyrosine (4G10) were from Upstate Cell Signaling Solutions. CyTM3-conjugated donkey anti-rabbit antibody was from Jackson ImmunoResearch.

Cell Culture—Human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) were from Cambrex Bio Science; they were cultured in EGM-2 medium and used for experiments within 10 passages. 293 human embryonic kidney cells and Chinese hamster ovary-K1 were from American Type Culture Collection and cultured, respectively, in Dulbecco's modified Eagle's medium or Kaighn's modification of Ham's F12 medium supplemented with 10% fetal bovine serum. Murine embryonic E6 fibroblasts (30) were kindly provided by Dr. Jan Sap (New York University, New York) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

Immunoprecipitation and Immunoblotting—Immunoblotting and immunoprecipitation were performed essentially as described previously (31). Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed on ice in Nonidet P-40 lysis buffer (25 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of leupeptin and aprotinin). The extract was clarified by centrifugation and incubated sequentially with primary antibodies and protein A- or G-agarose. The immunoprecipitates were collected and washed three times with the lysis buffer. For immunoblotting, whole cell lysates or immunoprecipitates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was probed with various primary antibodies as indicated and detected using the ECL system with horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's protocol.

In Vitro SFK Assay—The activities of SFKs were measured essentially according to the manufacturer's protocol. To determine the effect of H2O2 on SFKs in vitro, purified recombinant human full-length Src (6 units) or Fyn (25 ng) was incubated with various concentrations of H2O2 (0-1000 µM) for 20 min at 30 °C. Catalase was added at final concentration of 100 ng/µl for 10 min at room temperature to remove the excess H2O2. Kinase reactions were initiated by the addition of 100 µM ATP, 25 mM MgCl2, 5 mM MnCl2, 20 µM Na3VO4, and 10 µCi of [{gamma}-32P]ATP in the presence of 150 µM SFK-specific substrate peptide (KVEKIGEGTYGVVKK) in a total volume of 40 µl. After incubation at 30 °C for 12 min, the kinase reaction was stopped by adding 20 µl of 40% trichloroacetic acid, and the reaction mixture was applied onto P81 phosphocellulose filter paper. Papers were washed four times with 0.75% phosphoric acid and washed once with acetone, dried, and then counted in a scintillation counter. To determine the effect of H2O2 on SFKs in vivo, cells were treated with H2O2 (250 µM) for various time periods and SFKs were immunoprecipitated with an antibody (SRC-2) that recognizes the C-terminal sequence of SFK. The immune complexes were washed with Nonidet P-40 lysis buffer and incubated in 40 µl of kinase reaction buffer containing 100 µM ATP, 25 mM MgCl2, 5 mM MnCl2, 20 µM Na3VO4, 10 µCi [{gamma}-32P]ATP, and 150 µM SFK-specific substrate peptide. After incubation at 30 °C for 12 min, 20 µl of reaction mixture was spotted onto P81 phosphocellulose paper, washed with 0.75% phosphoric acid, and counted in a scintillation counter.

Purification of SHP-2 Tyrosine Phosphatase—Full-length SHP-2 was purified to homogeneity as described by Zhao et al. (32).

In Vitro PTP Assay—Cells were washed twice with ice-cold PBS and then lysed on ice in PTP lysis buffer (25 mM sodium acetate, pH 5.5, 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of leupeptin and aprotinin. The phosphatase activity was measured essentially as described previously (33). Briefly, the synthetic peptide Raytide (Oncogene) was labeled at its tyrosine residue using [{gamma}-32P]ATP and Src tyrosine kinase. Cell lysates were mixed with 32P-labeled Tyr-Raytide in 50 µl of phosphatase reaction buffer (25 mM Hepes, pH 7.4, and 5 mM EDTA) and incubated at 37 °C for 15 min. The reaction was terminated by the addition of acidic charcoal mixture (0.9 M HCl, 90 mM sodium pyrophosphate, 2 mM NaH2PO4, and 4% (w/v) Norit A). After centrifugation in a microcentrifuge, the amount of radioactivity present in the supernatant was determined by scintillation counting. The phosphatase activity was evaluated by the extent of Tyr-Raytide dephosphorylation in vitro. To determine the effect of H2O2 on SHP-2 in vitro, purified full-length SHP-2 (0.5 µg/each) was incubated with various concentrations of H2O2 (0-1000 µM) for 30 min at 25 °C. Catalase was added at final concentration of 100 ng/µl for 10 min at room temperature to remove the excess H2O2. The activity of SHP-2 was measured using the 32P-labeled Tyr-Raytide or substrate p-nitrophenyl phosphate (10 mM) as described previously (32).

Immunofluorescence Microscopy—Immunofluorescence microscopy was performed essentially as described recently (34). Cells grown on glass coverslips in a 6-well plate were treated with H2O2 for the indicated time periods at 37 °C and then washed with PBS and fixed in 3.7% formaldehyde solution in PBS for 10 min at 25 °C. The fixed cells were extracted in ice-cold acetone at -20 °C for 5 min, washed, and preincubated in PBS containing 1% bovine serum albumin for 30 min at 25 °C. Cells were then incubated with antibodies (1:150 dilution) against Tyr-418-phosphorylated Src for 1 h at 25 °C in PBS containing 1% bovine serum albumin, washed three times with PBS, and then incubated with CyTM3-conjugated donkey anti-rabbit secondary antibody (1:100 dilution) for 1 h at 25 °C in PBS containing 1% bovine serum albumin. Control stainings were performed without either primary or secondary antibodies. After washing with PBS, coverslips were mounted on slides with the cell side down with CytosealTM and examined and photographed using a PerkinElmer Ultra VIEW LCI confocal imaging system configured with a Nikon TE2000-S fluorescence microscope fitted with PlanApo 60 and x100 oil objectives. Adobe Photoshop 6.0 software was used for image processing.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
H2O2 Suppresses the Tyrosine Phosphorylation of Focal Adhesion Proteins in Vivo—H2O2, the most stable form of ROS, can easily diffuse across the membrane and has been widely used to study the role of ROS in cells (16). To determine H2O2 signaling, primary HAECs and murine embryonic E6 fibroblasts (30) were serum-starved for 2 h and treated with a subcytolytic concentration of H2O2 (300 µM) for the indicated time periods. Lysates were immunoblotted with a phosphotyrosine antibody. As shown in Fig. 1, A and B, H2O2 markedly suppressed the tyrosine phosphorylation of two major protein bands (~130 and 70 kDa) in a time-dependent manner in HAECs and E6 fibroblasts. Furthermore, the inhibitory effect of H2O2 on tyrosine phosphorylation of the 130-kDa protein band, but not the 70-kDa protein, was reversible. In addition, we found that H2O2 enhanced the tyrosine phosphorylation of a 190-kDa protein in HAECs. To determine whether the 130- and the 70-kDa protein bands represent, at least, the focal adhesion proteins p130Crk-associated substrate (p130Cas) and paxillin (68 kDa), which have similar molecular sizes and are known to be heavily tyrosine-phosphorylated (35), we determined the effects of H2O2 on the tyrosine phosphorylation of p130Cas and paxillin using phospho-specific antibodies in E6 fibroblasts. As shown in Fig. 1C, the phosphorylation of p130Cas on Tyr-410 was profoundly suppressed by H2O2 at 5 min but was fully recovered at 30 min. However, the H2O2-induced reduction in paxillin phosphorylation on Tyr-118 could not be reversed at 30 min. Because p130Cas and paxillin are native substrates of SFKs (35), these data suggest that H2O2 may inactivate SFKs in vivo.



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  		FIG. 1.
H2O2 suppresses the tyrosine phosphorylation of focal adhesion proteins in vivo. HAECs (A) or E6 fibroblasts (B) were serum-starved for 2 h and treated with H2O2 (300 µM) for the indicated time periods. Cell lysates were immunoblotted with a phosphotyrosine antibody (pTyr). C, lysates from E6 fibroblasts treated with H2O2 (250 µM) were subjected to immunoblotting (IB) with phospho-specific antibodies against Tyr-410-phosphorylated p130Cas or Tyr-118-phosphorylated paxillin. Results shown are representative immunoblots of two independent experiments.

 
H2O2 Inactivates SFKs in Vivo—SFKs are activated when the conserved tyrosine (Tyr-418 in human Src) in the activation loop is phosphorylated (5-7). Thus, the activated form of SFKs can be detected with a phospho-specific antibody against the conserved tyrosine. Fig. 2 shows a time-dependent effect of H2O2 (250 µM) on phosphorylation of the conserved tyrosine in various types of cells using an antibody against Tyr-418-phosphorylated Src (BIOSOURCE). In agreement with previous reports (36, 37), several bands of ~60 kDa were detected with the phospho-Src (Tyr-418) antibody (Fig. 2), indicating that the phospho-specific antibody also recognizes other SFK members when phosphorylated at the equivalent sites. We found that the phosphorylation of Src Tyr-418 and the equivalent sites of other members was suppressed more than 80% by H2O2 within 10 min and gradually returned to a near basal level by 60 min in primary HUVECs, HAECs, and E6 fibroblasts (Fig. 2, A-C). A similar time-dependent effect of H2O2 was observed in 293 human embryonic kidney cells (Fig. 2D) and Chinese hamster ovary-K1 cells (data not shown). These findings indicate that SFKs can be inactivated by H2O2 in vivo. We next determined the dose-dependent inactivation of SFKs by H2O2 using the phospho-Src (Tyr-418) antibody. As shown in Fig. 3A, the phosphorylation of Src Tyr-418 was suppressed 51% by 5 min of treatment of HUVECs with as low as 50 µM H2O2, 54% by 100 µM H2O2, and ~85% by 250-500 µM H2O2. Similar dose-dependent inactivation of SFKs by H2O2 was observed in HAECs (Fig. 3B). In addition, H2O2 over 100 µM was required to inactivate SFKs in E6 fibroblasts (Fig. 3C) and 293 cells (data not shown), and the maximal inactivation of SFKs can be achieved by 250 µM H2O2 in both cells. Thus, SFKs can be inactivated maximally by H2O2 at 250 µM in all the cells we examined. It seems that the primary human ECs are more sensitive to H2O2 than fibroblasts and 293 cells on the inactivation of SFKs. Similar results were obtained by using an antibody against Tyr-416-phosphorylated Src (Cell Signaling) (data not shown).



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  		FIG. 2.
Time-dependent inactivation of SFKs by H2O2 in vivo. HAECS (A), HUVECs (B), E6 fibroblasts (C), or 293 cells (D) were serum-starved and treated with H2O2 (250 µM) for the indicated time periods. Lysates were subjected to immunoblotting (IB) with Src (pY418) antibody that recognizes Tyr-418-phosphorylated Src and other SFK members when phosphorylated at the equivalent sites. The phosphorylation of Tyr-418 is also shown as percentage of untreated control cells determined by densitometric analyses. The same blot was stripped and reprobed with SRC-2 antibody against the C-terminal sequence of SFKs to show the equal loading. Results shown are representative immunoblots of three independent experiments.

 
The effect of H2O2 on the catalytic activities of SFKs was then determined in ECs. SFKs were immunoprecipitated with an antibody (SRC-2) that recognizes the C-terminal sequence of SFKs, and kinase activities of the immune complexes toward a SFK-specific peptide substrate were measured. As shown in Fig. 4A, the activities of SFKs were profoundly inhibited by H2O2 in a time-dependent manner in ECs. At 1 min, the activities of SFKs were inhibited 90 or 80% by H2O2 in HUVECs and HAECs, respectively. The pattern of the inhibition in kinase activity correlated well with the H2O2-induced suppression in the phosphorylation of Tyr-418 shown in Fig. 2. Because SFKs interact with substrates to transduce signal, inactivation of SFKs by H2O2 should suppress the tyrosine phosphorylation of SFK-bound substrates. As shown in Fig. 4B, the tyrosine phosphorylation of all the SFK-bound substrates was markedly inhibited by H2O2 (250 µM) at 5 min in HUVECs. Excluding the protein bands indicated with arrows, the tyrosine phosphorylation of several protein bands, including the 130-kDa protein, was recovered to basal levels by 1 h after H2O2 treatment. Taken together, these findings from different approaches demonstrate that H2O2 strongly inactivates SFKs in vivo.



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  		FIG. 3.
Dose-dependent inactivation of SFKs by H2O2 in vivo. HUVECs (A), HAECs (B), or E6 fibroblasts (C) were serum-starved and treated with various concentrations of H2O2 for 5 min. Lysates were subjected to immunoblotting (IB) with Src (pY418) antibody that recognizes Tyr-418-phosphorylated Src and other SFK members when phosphorylated at the equivalent sites. The phosphorylation of Tyr-418 is also shown as percentage of untreated control cells determined by densitometric analysis. The same blot was stripped and reprobed with SRC-2 antibody against the C-terminal sequence of SFKs to show the equal loading. Results shown are representative immunoblots of three independent experiments.

 



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  		FIG. 4.
H2O2 inhibits the catalytic activities of SFKs and the tyrosine phosphorylation of SFK-bound substrates in ECs. A, HAECs or HUVECs were treated with H2O2 (250 µM) for the indicated time periods. SFKs were immunoprecipitated the with SRC-2 antibody that recognizes the C-terminal sequence of SFKs, and kinase activities of the immune complexes toward an SFK-specific peptide substrate were measured. indicates HAECs; {circ} HUVECs. Error bars indicate S.D. B, HUVECs were treated with H2O2 (250 µM) for the indicated time periods. Lysates were immunoprecipitated (IP) with SRC-2 antibody and subjected to immunoblotting (IB) with a phosphotyrosine antibody (pTyr). Arrows indicate protein bands in which the tyrosine phosphorylation was not recovered to basal levels by 1 h after H2O2 treatment. Results shown are representative immunoblots of three independent experiments.

 



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  		FIG. 5.
H2O2 predominantly inactivates the SFKs localized to focal adhesions and the plasma membrane of ECs. HUVECs grown on glass coverslips were left untreated (control) or treated with H2O2 (250 µM) for 5 or 60 min, washed twice, and fixed. The activated SFKs were viewed (10 x 60) by staining the fixed cells with the phospho-Src (Tyr-418) antibody. Arrows indicate localization of the activated SFKs. Results shown are representative of three independent experiments.

 
H2O2 Predominantly Inactivates the SFKs Localized to Focal Adhesions and the Plasma Membrane—Although the H2O2-induced inhibition in the total activities of SFKs was reversible (Fig. 4A), the recovery of tyrosine phosphorylation of SFK-bound substrates was only observed in certain protein bands (Fig. 4B). These data suggest that SFKs localized at different cellular compartments may be regulated differently by H2O2. We performed immunofluorescence microscopy using a confocal microscope to access the localization of activated SFKs in HUVECs. In agreement with early reports (38-40), we found that the activated SFKs detected with the phospho-Src (Tyr-418) antibody were mainly localized to cell peripheral focal adhesions (Fig. 5A, solid arrow). The signal was also detected at plasma membrane (Fig. 5A, broken arrow). Remarkably, the staining signals of the activated SFKs localized at cell periphery were virtually abolished by 5 min of treatment of HUVECs with H2O2 (Fig. 5B), which correlated well with a profound reduction in the tyrosine phosphorylation of focal adhesion proteins p130Cas and paxillin (Fig. 1C). At this moment, the activated SFKs were mainly detected as a granular cytoplasmic staining, likely associated with endosomes as shown previously (41). By 1 h after H2O2 treatment, the activated SFKs were mainly detected as big clusters in cytoplasm (Fig. 5C). These data were highly reproducible in HAECs (data not shown). These findings indicate that H2O2 rapidly and sustainedly inactivates SFKs localized to cell peripheral focal adhesion sites and the plasma membrane in HUVECs.

H2O2 Does Not Directly Affect the Catalytic Activities of SFKs in Vitro—To determine whether H2O2 directly inactivates SFKs in vitro, purified recombinant full-length human Src or Fyn was incubated with various concentrations of H2O2, and the kinase activity was measured with a specific substrate peptide. As shown in Fig. 6, H2O2 (0-1 mM) did not significantly affect the catalytic activity of Src or Fyn in vitro.



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  		FIG. 6.
H2O2 does not directly affect the catalytic activities of SFKs in vitro. Purified recombinant human full-length Src (6 units) or Fyn (25 ng) was incubated with various concentrations of H2O2 (0-1000 µM) for 20 min at 30 °C. The kinase activity was measured with a SFK-specific substrate peptide as described under "Experimental Procedures." Results shown are representative of three independent experiments.

 
Inhibition of PTPs Is Involved in the H2O2-induced Inactivation of SFKs in Vivo—Because PTPs contain a catalytically essential cysteine residue in the signature active site motif, HCXXGXXR(S/T), which can be reversibly oxidized by ROS to inactivate PTPs (42), it has been proposed that the inhibition of PTPs by ROS could regulate their counterpart protein tyrosine kinases and cellular signaling. As shown in Fig. 7A, SHP-2, a non-receptor PTP, was directly inactivated by H2O2 in a dose-dependent manner with an IC50 of 75 µM. SHP-2 was also directly inhibited by other PTP inhibitors such as Na3VO4 (43) and phenylarsine oxide (PAO) (44), but not by the serine/threonine phosphatase inhibitor NaF (45) (data not shown). Fig. 7B shows the effect of H2O2 on total activities of PTPs in HUVECs. The total activities of PTPs in HUVECs were inhibited ~50% by H2O2 at 5 min, gradually recovered to basal level at 20 min, and increased to a higher level (1.62-fold) at 60 min. We next determined whether SFKs can be inactivated by other PTPs inhibitors. As shown in Fig. 7, C and D, SFKs were markedly inactivated by H2O2, Na3VO4, and PAO, but not by NaF, in HUVECs. In addition, we found that protein kinase C and calcium did not play a role in the inactivation of SFKs by H2O2 (data not shown). These data indicate that PTPs can be reversibly inhibited by H2O2 in HUVECs and that the inhibition of PTPs may be involved in the H2O2-induced inactivation of SFKs.

It has been shown that phosphorylation of a conserved tyrosine (Tyr-529 in human Src) in the C-terminal tail causes formation of an inactive conformation of SFKs, thus inhibiting SFKs (13, 14). We determined the effect of H2O2 on the phosphorylation of the conserved tyrosine using a phospho-Src (Tyr-529) antibody (BIOSOURCE) that recognizes SFKs when phosphorylated on Tyr-529 or the equivalent sites (46, 47). As shown in Fig. 8A, the phosphorylation of Src Tyr-529 was slightly increased by H2O2 at 5 and 10 min (1.3-1.4-fold) and then gradually returned to basal level at 30 min and to a level even less than control (85%) at 60 min in HUVECs. However, H2O2 did not affect the phosphorylation of Tyr-529 in E6 fibroblasts and 293 cells (Fig. 8, B and C). Similar findings were observed in HUVECs, E6 fibroblasts, and 293 cells when another phospho-specific antibody against Tyr-528-phosphorylated Fyn or Tyr-530-phosphorylated Src (BD Pharmingen) was employed (data not shown). Because SFKs were inactivated to a similar extent in ECs, fibroblasts, and 293 cells, the slight increase in the phosphorylation of Tyr-529 observed only in HUVECs may not be the key mechanism for the H2O2-induced inactivation of SFKs.

H2O2 Suppresses the Responses of ECs to Growth Factors and Cytokines—Because SFKs play key roles in the signal transduction of growth factors (3), we determined the effect of H2O2 on the mitogenic responses of ECs to growth factors. As shown in Fig. 9A, top panel, pretreatment of HUVECs with H2O2 (250 µM) for 3 min virtually abolished the activation of extracellular signal-regulated kinase (ERK1/2) by platelet-derived growth factor (PDGF). Furthermore, PDGF alone slightly, if at all, activated SFKs by enhancing the phosphorylation of Tyr-418 in HUVECs. However, PDGF was unable to rescue the H2O2-induced inactivation of SFKs (Fig. 9A, middle panel), suggesting that different mechanisms may be involved in the regulation of SFKs by PDGF and H2O2. In addition, we found that the responses of HUVECs to epidermal growth factor were also blunted by H2O2 pretreatment (data not shown).

Associated with the induction of cell adhesion molecules, EC inflammatory activation by cytokines is a major feature during the development of EC-related cardiovascular diseases (48). It has been shown that the expression of VCAM-1 by thrombin and TNF-{alpha} is dependent on nuclear factor-{kappa}B (NF-{kappa}B) in ECs (49, 50). Because SFKs are essential for the activation of NF-{kappa}B (51, 52), we assessed the effect of H2O2 on the expression of VCAM-1 by thrombin and TNF-{alpha} in HUVECs. As shown in Fig. 9, B and C, the expression of VCAM-1 induced by thrombin and TNF-{alpha} was suppressed by H2O2 in a dose-dependent manner. In particular, the expression of VCAM-1 by thrombin or TNF-{alpha} was almost completely suppressed by 250 or 350 µM H2O2, respectively. Moreover, PAO (44), which inactivated SFKs (Fig. 7D), also markedly inhibited the expression of VCAM-1 by thrombin and TNF-{alpha} in HUVECs (Fig. 9, B and C). These findings suggest that ROS (H2O2) may be protective through suppression of EC responses to cytokines via inactivation of SFKs, thus preventing endothelial inflammation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have reported a novel finding that H2O2 at a low to moderate level (50-250 µM) markedly inactivates SFKs temporally and spatially in vivo, but not in vitro, using multiple approaches. We have further shown that SFKs localized to EC peripheral focal adhesion sites and the plasma membrane are rapidly and permanently inactivated by H2O2, which results from a profound reduction in phosphorylation of the activation loop conserved tyrosine (Tyr-418 in human Src). The inactivation of SFKs correlates well with the H2O2-induced resistance of ECs to growth factors and cytokines. Because SFKs play initiating and key roles in cell signaling by a diverse set of cell surface receptors (1), our findings of the rapid inactivation of SFKs by H2O2, a stable form of ROS, may provide new insights into the mechanism of the protective effects of ROS.

ROS, including H2O2, , and , are constantly produced in the human body in physiological conditions, such as metabolism and activation of cell surface receptors, to maintain cell survival (19-21) and in pathophysiological settings like inflammation, infection, tissue injury, and hypercholesterolemia (22-24). Although the exact concentrations of ROS in these settings are not clear, it has been shown that the oxidant generation induced by TNF-{alpha} (100 units/ml), detected with an oxidant-sensitive dye, is comparable with that by 500 µM H2O2 in culture of human dermal microvascular endothelial cells (19). The first finding obtained from our study is that SFKs can be rapidly inactivated by H2O2 in HAECs, HUVECs, 293 cells, and E6 fibroblasts as well as Chinese hamster ovary-K1 cells. The enzymatic activities of SFKs are positively regulated by phosphorylation of a conserved tyrosine (Tyr-418 in human Src) in the activation loop (5-7). We found that the phosphorylation of Tyr-418 was suppressed by H2O2 in a time- and dose-dependent manner in all the cells we examined. In HAECs and HUVECs, H2O2 over 50 µM was required to induce a reduction in the phosphorylation of Tyr-418, whereas H2O2 over 100 µM was required in E6 fibroblasts and 293 cells. These data suggest that the primary human ECs are more sensitive to H2O2 than fibroblasts and 293 cells on the inactivation of SFKs. Furthermore, the phosphorylation of Src Tyr-418 was almost abolished by 250 µM H2O2 in all cells examined, which correlated with a profound inhibition in the catalytic activities of SFKs. Thus, it is likely that the reduction in phosphorylation of the activation loop conserved tyrosine may be involved in the H2O2-induced inactivation of SFKs in vivo. Immunofluorescence study revealed that, consistent with earlier reports (38-40), the activated SFKs were predominantly localized to cell peripheral focal adhesion sites and the plasma membrane at resting HUVECs. Remarkably, we found that the activated SFKs localized at these sites were completely inactivated by H2O2 at 5 min in HUVECs, and the inactivation cannot be recovered even by a prolonged (1 h) H2O2 treatment. These findings were further supported by the notion that H2O2 induced a great reduction in the tyrosine phosphorylation of focal adhesion proteins p130Cas and paxillin. Interestingly, along with a complete inhibition of SFKs localized at the cell periphery, the SFKs associated with endosomes (41) in cytoplasm were activated by H2O2 in a time-dependent manner through an enhanced phosphorylation of Src Tyr-418. By 1 h after H2O2 treatment, the activated SFKs were mainly detected as big and bright clusters in the cytoplasm of HUVECs. Thus, the activation of cytoplasmic SFKs may compensate for the lost activities of SFKs localized to focal adhesions and the plasma membrane and account for the H2O2-induced reversible inhibition in total phosphorylation of Src Tyr-418 and total catalytic activities of SFKs. However, this compensation did not fully restore the function of SFKs, because the recovery of tyrosine phosphorylation of SFK-bound substrates was only observed in certain protein bands by 1 h after H2O2 treatment in HUVECs. Indeed, we found that the activation of ERK by PDGF and epidermal growth factor and the induction of VCAM-1 by thrombin and TNF-{alpha}, which require a proper localization of the activated SFKs and intact focal adhesion complexes (3, 4, 53), were greatly inhibited by H2O2 in HUVECs.



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  		FIG. 7.
Inhibition of PTPs is involved in the H2O2-induced inactivation of SFKs in vivo. A, dose-dependent inhibition of SHP-2 phosphatase activity by H2O2 in vitro. B, time-dependent effect of H2O2 (250 µM) on total activities of PTPs in HUVECs. C and D, inactivation of SFKs by PTP inhibitors in HUVECs. HUVECs were treated with H2O2 (250 µM) for 5 min or Na3VO4 for 1 or 5 min (C) or for 5 min with H2O2 (250 µM), phenylarsine oxide (PAO, 25 µM), or NaF (10 mM) (D). Lysates were immunoblotted (IB) with Src (pY418) antibody that recognizes the activated SFKs. The same blot was stripped and reprobed with SRC-2 antibody against the C-terminal sequence of SFKs to show the equal loading. Results shown are representative immunoblots of three independent experiments.

 



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  		FIG. 8.
Effect of H2O2 on phosphorylation of the C-terminal conserved tyrosine (Tyr-529 in Src). HUVECs (A), E6 fibroblasts (B), or 293 cells (C) were serum-starved and treated with H2O2 (250 µM) for the indicated time periods. Lysates were subjected to immunoblotting (IB) with Src (pY529) antibody that recognizes Tyr-529-phosphorylated Src and other SFK members when phosphorylated at the equivalent sites. The same blot was stripped and reprobed with the SRC-2 antibody to show the equal loading. Results shown are representative immunoblots of three independent experiments.

 
Previous studies on the regulation of SFKs by H2O2 have yielded contradictory results. Cunnick et al. (54) reported that, consistent with our findings, Src kinase activity was inhibited by H2O2 (500-2000 µM) in HeLa cells. In contrast, several other studies showed that treatment of cells with an extremely high dose of H2O2 (5 mM) augmented the phosphorylation of Lck on Tyr-394 and activated the kinase (7, 10). However, these studies did not show what compartment of Lck was activated, and the mechanism was not clear. Herein, we demonstrated that SFKs at different cellular compartments were regulated differently by H2O2 when a low to moderate concentration (50-250 µM) of H2O2 was employed. We showed that SFKs localized to cell peripheral focal adhesion sites and the plasma membrane were rapidly and permanently inactivated by H2O2 in HUVECs, whereas the cytoplasmic SFKs associated with endosomes were gradually activated by H2O2. Based on our findings, the discrepancy between these previous studies and our results may be reconciled with the following interpretation. First, H2O2 at an extreme high level (5 mM) may override the normal regulation of Lck and activate the kinases. Second, it is possible that Lck localized at the cell periphery is inactivated by 5 mM H2O2 but the cytoplasmic Lck is hyperphosphorylated on Tyr-394 and overactivated, leading to an increase in total phosphorylation of Lck on Tyr-394 and an elevated kinase activity.



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  		FIG. 9.
H2O2 suppresses the responses of ECs to growth factors and cytokines. A, HUVECs were left untreated (-) or pretreated for 3 min with H2O2, followed with (±) or without (+) a wash to remove H2O2 and then stimulated with PDGF (50 ng/ml) for 5 min. Lysates were immunoblotted with antibodies against phospho-ERK1/2 (Thr-202/Tyr-204) or phospho-Src (Tyr-418). The same blot was stripped and reprobed with the ERK1/2 antibody to show the equal loading. B and C, HUVECs were left untreated (-) or pretreated for 30 min with various concentrations of H2O2 as indicated or PAO (100 nM) and then incubated without (-) or with thrombin (3 units/ml) (B) or TNF-{alpha} (10 ng/ml) (C) for 4 h. Lysates were immunoblotted with VCAM-1 antibody. The same blots were stripped and reprobed with tubulin antibody to show the equal loading. Results shown are representative immunoblots of three independent experiments.

 
We found that the H2O2-induced rapid inactivation of SFKs was mimicked by two other PTP inhibitors, Na3VO4 (43) and PAO (44). These findings suggest that inhibition of PTPs may be involved in the inactivation of SFKs by H2O2 in vivo. PTPs contain a catalytically essential cysteine residue in the signature active site motif, HCXXGXXR(S/T), which can be reversibly oxidized by ROS to inactivate PTPs (42). Several lines of evidence, including our results (Fig. 7), have demonstrated that PTPs can be directly inhibited by H2O2 in vitro and reversibly inhibited by H2O2 in intact cells (55, 56). Our data also suggest that inhibition of PTPs does not always lead to an increase in the tyrosine phosphorylation of cellular proteins; we found that H2O2 inactivated SFKs and suppressed the tyrosine phosphorylation of two major protein bands of 130- and 70-kDa in HAECs and E6 fibroblasts, which may at least represent p130Cas and paxillin. SFKs are negatively regulated by phosphorylation of a conserved tyrosine (Tyr-529 in human Src) in the C-terminal tail, which causes formation of an inactive conformation of SFKs (13, 14). Dephosphorylation of this site has been shown to be mediated by RPTP{alpha} and CD45, resulting in the activation of SFKs (15). It is conceivable that inhibition of PTPs may augment phosphorylation of the C-terminal tyrosine and render SFKs to an inactive form. However, the hypothesis seems not to be a key mechanism for the H2O2-induced inactivation of SFKs localized at cell periphery. We found that the phosphorylation of Src Tyr-529 was only slightly increased (~1.35-fold) by H2O2 at 5 and 10 min in HUVECs, which did not correlate well with a profound inactivation of SFKs by H2O2. Furthermore, the phosphorylation of Tyr-529 was not altered by H2O2 in E6 fibroblasts and 293 cells, in which SFKs were equally well inactivated by H2O2 compared with HUVECs. It appears likely that other mechanisms may be involved. SFKs are activated through phosphorylation of the activation loop conserved tyrosine (Tyr-418 in human Src) (5-7). This phosphorylation has been traditionally thought to occur as an intermolecular event carried out by the kinase itself (8, 9). Because H2O2 does not directly inhibit SFKs in vitro (Fig. 6), the fact of a rapid and profound reduction in the phosphorylation of Src Tyr-418 by H2O2 in vivo argues strongly against the theory that the phosphorylation of Src Tyr-418 is merely catalyzed by the kinase itself in cells. Indeed, a set of evidence has indicated that phosphorylation of the activation loop tyrosine can be achieved by a kinase other than SFKs in several types of cells (7, 10). Therefore, the simplest interpretation of our results is that H2O2 may inhibit the novel kinase through oxidation and inhibition of PTPs and suppress phosphorylation of the activation loop tyrosine in SFKs, leading to inactivation of SFKs in H2O2-treated cells. The hypothesis merits further investigation.

Another interesting finding from this study is that H2O2 induces insensitivity of ECs to growth factors and cytokines and protects endothelial inflammatory activation by cytokines. Conventional thought has generally regarded ROS as harmful to the vasculature, leading to such pathological processes as atherosclerosis, coronary ischemia, retinopathy, restenosis, and hypertension (16, 24, 25). However, controlled clinical trials have failed to show a consistent benefit of antioxidants on these cardiovascular diseases (26-28). If oxidants are toxic to the human body, why have clinical trials of antioxidants produced such mixed and mostly negative outcomes? Although the mechanism is not yet clear, a growing body of evidence suggests that ROS may be both protective and deleterious depending on concentration (24, 29). In the present study, we found that acute (3 min) pretreatment of HUVECs with a subtoxic concentration of H2O2 (250 µM) abolished the cell responses to PDGF and epidermal growth factor. Moreover, H2O2 at 100-350 µM markedly suppressed the expression of VCAM-1 by thrombin and TNF-{alpha} in HUVECs. These data indicate that ROS could blunt signal transduction of the activated receptors in ECs. The hypothesis is supported by a finding that HepG2 cells that overexpressed mitochondrial catalase are more resistant to H2O2 but develop increased sensitivity to TNF-{alpha} (57). Because SFKs play initiating and key roles in cell signaling by a diverse set of cell surface receptors (1), our data suggest that the rapid and profound inactivation of SFKs by H2O2 may be at least one of the mechanisms for the protective effect of H2O2 on vascular cells. The production of cytokines and ROS is always accompanied with inflammation. At the sites of inflammation caused by infection or injury, high concentration of ROS may harm the vasculature and cause endothelial damage. On the other hand, ROS can be easily diffused and diluted to joint areas or tissues. Before being destroyed by the antioxidant system, the diluted ROS, on the basis of present findings, will inactivate SFKs and render the ECs resistant to cytokines in these adjoining areas, thus protecting development of a spread inflammation caused by cytokines. It is possible that an elevated antioxidant system may protect cells at the inflammation sites, but it may sacrifice the vascular protective effect of ROS for nearby vascular cells. The imbalance between these two opposite effects of ROS may impact outcomes in the antioxidant trials.

In summary, we have obtained substantial evidence indicating that ROS inactivate SFKs temporally and spatially in vivo, but not in vitro, which may offer new insights into the mechanism of the protective effect of ROS in vivo.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL-69806 (to H. T.) and HL-076309 (to Z. J. Z) and American Heart Association Grant 0130038N (to H. T.). 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. Back

§ To whom correspondence should be addressed: Dept. of Biochemistry, The University of Texas Health Center at Tyler, 11937 U. S. Highway 271, Tyler, TX 75708. Tel.: 903-877-7938; Fax: 903-877-2881; E-mail: hua.tang{at}uthct.edu.

1 The abbreviations used are: SFKs, Src family tyrosine kinases; SH, Src homology; ROS, reactive oxygen species; PTPs, protein tyrosine phosphatases; EC, endothelial cells; HAECs, human aortic endothelial cells; HUVECs, human umbilical vein endothelial cells; PAO, phenylarsine oxide; PDGF, platelet-derived growth factor; VCAM-1, vascular cell adhesion molecule-1; PBS, phosphate-buffered saline; TNF-{alpha}, tumor necrosis factor-{alpha}; ERK, extracellular signal-regulated kinase; p130Cas, p130Crk-associated substrate. Back


   ACKNOWLEDGMENTS
 
We thank Jan Sap for providing E6 fibroblasts.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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