Tyrosine phosphatase PTP-MEG2 negatively regulates vascular ...

Am J Physiol Cell Physiol 303: C548–C553, 2012. First published July 3, 2012; doi:10.1152/ajpcell.00415.2011.

Tyrosine phosphatase PTP-MEG2 negatively regulates vascular endothelial growth factor receptor signaling and function in endothelial cells Qin Hao,1 Buka Samten,2 Hong-Long Ji,1 Z. Joe Zhao,3 and Hua Tang1 1

Department of Biochemistry, University of Texas Health Science Center, Tyler, Texas; 2Department of Microbiology and Immunology, University of Texas Health Science Center, Tyler, Texas and 3Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Submitted 22 November 2011; accepted in final form 2 July 2012

Hao Q, Samten B, Ji HL, Zhao ZJ, Tang H. Tyrosine phosphatase PTP-MEG2 negatively regulates vascular endothelial growth factor receptor signaling and function in endothelial cells. Am J Physiol Cell Physiol 303: C548 –C553, 2012. First published July 3, 2012; doi:10.1152/ajpcell.00415.2011.—Protein tyrosine phosphorylation is a fundamental mechanism for diverse physiological processes, which is regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). In this study, we searched for protein substrates of PTP-MEG2 (also called PTPN9), a nonreceptor PTP, and investigated its function in endothelial cells (ECs). By using a PTP-MEG2 substrate-trapping DA mutant, we found that a couple of tyrosine-phosphorylated proteins were associated with the DA mutant but not wild-type PTP-MEG2 and that the association was enhanced by vascular endothelial growth factor (VEGF) in ECs. We further found that VEGF receptor 2 (VEGFR2) was coimmunopricipitated with the DA mutant but not wild-type PTP-MEG2. The VEGFinduced phosphorylation of VEGFR2 on Tyr1175, a critical autophosphorylation site for VEGFR2 signaling, was inhibited 70% by overexpression of wild-type PTP-MEG2 but was enhanced (2.2-fold) by the DA mutant of PTP-MEG2. We also found that PTP-MEG2 DA mutant preferentially associated with Janus kinase 1 (JAK1) but not with other JAK kinases (Tyk2 and JAK2) present in ECs and regulated JAK1 tyrosine phosphorylation. Lastly, the VEGF-induced signal transduction and the production of interleukin (IL)-6 were significantly enhanced by PTP-MEG2 knockdown in ECs, whereas the VEGF-induced IL-6 production was inhibited 50% by PTP-MEG2 overexpression. Thus we have indentified VEGFR2 as a PTP-MEG2 substrate, and our findings indicate that PTP-MEG2 is a negative regulator of VEGFR2 signaling and function in ECs. protein tyrosine phosphatase; endothelial cells; vascular endothelial growth factor receptor 2; Janus kinase 1; interleukin-6 PROTEIN TYROSINE PHOSPHORYLATION is a fundamental mechanism for diverse physiological processes (8). It is regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). However, by comparison with protein tyrosine kinases, relatively less is known about the regulation and the specific functions of most PTPs (11, 16). PTP-MEG2 (also called PTPN9) is a nonreceptor PTP originally cloned from human umbilical vein endothelial cells (HUVECs) and megakarocyte cDNA libraries and is distinct from other mammalian PTPs by virtue of a putative lipid-binding domain at the NH2 terminus (5). The NH2-terminal segment shares 28% sequence identity with cellular retinaldehyde binding protein and 24% identity with yeast Sec14p, a protein that has phosphatidylinositol transfer activity (5). It has been shown that the NH2-terminal

Address for reprint requests and other correspondence: H. Tang, Dept. of Biochemistry, Univ. of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, Texas 75708 (e-mail: [email protected]). C548

domain of PTP-MEG2 binds to phosphatidylinositol 3,4,5triphosphate and phosphatidylserine (9, 20). Moreover, removal of the NH2-terminal domain significantly activates PTPMEG2 (10, 13), suggesting that the NH2-terminal domain may negatively regulate the enzymatic activity of the COOH-terminal PTP domain. PTP-MEG2 is present in both cytosolic and membrane fractions of most tested cells (10, 18). However, in Jurkat T leukemia cells, PTP-MEG2 is exclusively present in membrane fractions and localizes to the secretory vesicle membrane and plasma membrane (9). The cytosolic PTPMEG2 has been shown to be inactive, while the membraneassociated PTP-MEG2 is apparently active in erythroid colonyforming cells (18), suggesting that the translocation of PTPMEG2 from cytosol to membranes may activate the enzyme. The function of PTP-MEG2 is not clear and may vary depending on cell types. It has been shown that PTP-MEG2 regulates erythroid cells growth and expansion (18) and modulates cytokine release by CD3 and CD28 in murine lymphocytes but not in neutrophils and macrophages (17). Moreover, PTP-MEG2 dephosphorylates insulin receptor and antagonizes insulin signaling in hepatocytes (2). In breast cancer cells, PTP-MEG2 directly dephosphorylates epidermal growth factor receptor and inhibits signaling transduction evoked by epidermal growth factor (19). Nevertheless, knockout of PTP-MEG2 causes multiple neurodevelopment defects and hemorrhages, resulting in ⬎90% late embryonic lethality (17). As PTPMEG2 is expressed in endothelial cells (ECs; Ref. 5), the hemorrhages manifested in PTP-MEG2-deficient mice suggest that PTP-MEG2 may play an important role in vascular development and integrity through the regulation of EC function. The role of PTP-MEG2 in ECs is not known so far. In this study, we have searched for the potential substrates of PTP-MEG2 in ECs by using a PTP-MEG2 substrate-trapping mutant. We have shown that PTP-MEG2 dephosphorylates vascular endothelial growth factor receptor-2 (VEGFR2) and negatively regulates the receptor downstream signal transduction and production of proinflammatory cytokine interleukin (IL)-6 evoked by vascular endothelial growth factor (VEGF) in ECs, indicating that PTP-MEG2 is a negative regulator of VEGFR2 signaling and function. MATERIALS AND METHODS

Reagent. Recombinant human VEGF-A165 (VEGF) and PTPMEG2 monoclonal antibody were from R&D Systems (Minneapolis, MN). Antibodies against phosphotyrosine (PY99), VEGFR2, and tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-VEGFR2 (Tyr1175), phospho-JAK1 (Tyr1022/1023), phospho-p38 (Thr180/Tyr182), phospho-ERK1/2 (Thr202/Tyr204), and JAK2 antibodies and reagents for chemiluminescence detection were

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NEGATIVE REGULATION OF VEGF SIGNALING BY PTP-MEG2

from Cell Signaling (Beverly, MA). Receptor PTP␣ (RPTP␣) antibody was from Millipore (Temecula, CA). Antibodies against JAK1, TYK2, p130Cas, and focal adhesion kinase (FAK) were from BD Transduction Laboratories (San Diego, CA). FuGENE 6 transfection reagent was from Roche Applied Science (Indianapolis, IN). Cell culture. HUVECs were from Lonza (Walkersville, MD) and cultured in EGM-2 medium and used for experiments within eight passages. HUVECs were normally starved 2 h in serum-free endothelial basal medium before treatment. Recombinant adenovirus and cell infection. PTP-MEG2 wild-type and the substrate-trapping mutant with Asp-470 mutation to Ala (MEG2DA) were prepared as described previously (18). The constructs were subcloned into adenovirus transfer vector pACCMV.pLpA, and recombinant adenovirus was generated by cotransfection of 293 cells with the pACCMV.pLpA construct and pJM17 adenovirus genome DNA by using Fugene 6 cell transfection reagent. The resulting recombinant viral clones were purified by soft agar plaque assay and then amplified in 293 cells according to a standard procedure (1). Positive clones were selected based on expression of PTP-MEG2 in infected 293 cells by Western blot. Subconfluent HUVECs were infected with the above replicationdefective recombinant adenovirus at a multiplicity of infection of 10 –20 plaque-forming units/cell. Immunoprecipitation, Western blotting, and subcellular fractionation. Immunoprecipitation and Western blot analysis were performed, and EC cytosolic and membrane fractions were prepared essentially as we described previously (15). PTP-MEG2 was immunoprecipitated with a polyclonal antibody no.144 as described previously (20). Immunofluorescence microscopy. HUVECs grown on glass coverslips in a sixwell plate were infected with replication-defective recombinant adenovirus expressing PTP-MEG2 wild type or vector alone. Cells were then fixed, and the subcellular localization of PTP-MEG2 was assessed with a PTP-MEG2 monoclonal antibody by using immunofluorescence microscopy as we described previously (14). Adobe Photoshop 7.0 software was used for image processing. Small interference RNA and small interference RNA transfection. AllStars nontargeting negative control small interference RNA (siRNA; no. 1027280) and the validated human PTP-MEG2 siRNA-1 (no. SI02759246), PTP-MEG2 siRNA-2 (no. SI02759253), and RPTP␣ siRNA (no. SI02658775) were from Qiagen (Valencia, CA). For siRNA transfection, HUVECs were seeded into different plates for 24 h to reach 50 –70% confluence, and then siRNA was transfected into HUVECs in a final concentration of 20 nM by using Lipofectamine 2000 and Opti-MEM reduced serum medium according to the manufacturer’s protocols (Invitrogen, Carlsbad, CA). The medium was replaced with fresh EGM-2 medium 12 h after transfection. The silencing effects of siRNAs were then confirmed by Western blot analysis.

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ELISA. IL-6 in cell culture medium was measured with DuoSet ELISA development kits following the manufacturer’s protocols (R&D Systems). Statistical analysis. Data are expressed as means ⫾ SE. A Student’s t-test was used for statistical analysis. P ⬍ 0.05 is considered statistically significant. RESULTS

Expression and localization of PTP-MEG2 in ECs. As shown in Fig. 1A, subcellular fractionation experiment revealed that endogenous PTP-MEG2 (68 kDa) was detected near equally in both cytosolic and membrane factions in HUVECs, and this intracellular distribution was not altered by overexpression of PTP-MEG2. We performed immunofluorescence microscopy with a PTP-MEG2 monoclonal antibody to access PTP-MEG2 localization in HUVECs overexpressing PTP-MEG2 wild type. Figure 1B shows that PTP-MEG2 displayed perinuclear (white arrow), plasma membrane (green arrow) and intracellular vesicular (blue arrow) staining in the cells. The plasma membrane localization of PTP-MEG2 suggests that it may regulate receptor signaling in ECs. Identification of PTP-MEG2 potential substrates in ECs. The identification of PTP-MEG2 substrates is an essential step towards understanding of PTP-MEG2 physiological function. However, the potential substrates of PTP-MEG2 in ECs have not yet been identified so far. As a first step, we performed substrate-trapping experiments in HUVECs. All the PTPs contain an invariant aspartic acid residue, which functions as a general acid in the phosphate ester hydrolysis reaction. Mutation of this aspartic acid ablates the ability of PTPs to dephosphorylate target substrates but leaves substrate binding intact. Thus the PTP-substrate complex is stabilized sufficiently to permit isolation by immunoprecipitation, and the substrates can subsequently be identified (4). As shown in Fig. 2, HUVECs were infected with recombinant adenovirus carrying PTP-MEG2 wild-type or the substrate-trapping mutant with Asp-470 mutation to Ala (MEG2-DA mutant; Ref. 18) and stimulated 5 min with or without VEGF. PTP-MEG2 was immunoprecipitated and then subjected to Western blot analysis with an anti-phosphotyrosine antibody PY99. We found that several tyrosine-phosphorylated proteins indicated by arrows were associated with the DA mutant but not wild-type PTP-MEG2 and that the association was enhanced by VEGF treatment (Fig. 2). These data suggest that tyrosine phosphorylation of these proteins is required for their associations with

Fig. 1. Expression and localization of protein tyrosine phosphatases (PTP)-MEG2 in endothelial cells (ECs). A: equal protein amounts of cytosolic (Cyto.) and membrane (Mem.) fractions from human umbilical vein endothelial cells (HUVECs) infected with recombinant adenovirus expressing vector alone (Vector) or PTP-MEG2 wild type (MEG2-WT) were assessed by Western blotting with a monoclonal PTP-MEG2 antibody. B: intracellular localization of PTP-MEG2 by immunofluorescence microscopy in ECs. HUVECs were infected with recombinant adenovirus expressing PTP-MEG2 wild type, and the cells were fixed and stained with a monoclonal PTP-MEG2 antibody. Arrows indicate the localization of PTP-MEG2 (10 ⫻ 60). Results represent 3 independent experiments. AJP-Cell Physiol • doi:10.1152/ajpcell.00415.2011 • www.ajpcell.org

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Fig. 2. Identification of PTP-MEG2 interacting proteins in ECs. HUVECs were infected 48 h with recombinant adenovirus carrying wild type (WT) or the DA substrate-trapping mutant (DA) of PTP-MEG2 and then starved 2 h and treated 5 min with or without VEGF (50 ng/ml). Lysates (500 ␮g) were immunoprecipiated (IP) with PTP-MEG2 polyclonal antibody and subjected to Western blot (WB) analysis with a monoclonal anti-phosphotyrosine antibody PY99 (pTyr) as indicated. The membrane was stripped and reprobed with PTPMEG2 (MEG2) antibody to show equal loading. Results represent Western blots of 2 independent experiments.

PTP-MEG2. It should be noted that a protein band ⬃230 kDa, presumably VEGFR2, was associated with the DA substratetrapping mutant of PTP-MEG2 and that the association was enhanced by VEGF treatment in ECs. To determine whether VEGFR2 interacts with the DA mutant of PTP-MEG2 in the presence of VEGF, we immunoprecipitated PTP-MEG2 from HUVECs overexpressing PTPMEG2 wild type or the DA mutant and subjected the immune complexes to Western blot with a VEGFR2 antibody. As shown in Fig. 3A, we found that VEGFR2 was coimmunoprecipitated with the DA mutant but not wild-type PTP-MEG2 in VEGF-treated ECs. These data suggest that the VEGF-induced phosphorylation of VEGFR2 may be required for its association with PTP-MEG2. We next determined the regulation VEGFR2 tyrosine phosphorylation at a critical autophosphorylation site Tyr1175 by PTP-MEG2 in HUVECs. It has been demonstrated that the phosphorylation of Tyr1175 is essential for VEGFR2 signaling (12). As shown in Fig. 3B, VEGF rapidly induced the phosphorylation of VEGFR2 on Tyr1175 detected by a phospho-specific antibody against the Tyr1175phosphorylated VEGFR2. Remarkably, the VEGF-induced

phosphorylation of VEGFR2 on Tyr1175 was inhibited 70% by overexpression of wild-type PTP-MEG2 but was enhanced (2.2-fold) by the DA mutant of PTP-MEG2 in HUVECs. These data indicate that VEGFR2 is a bona fide substrate of PTPMEG2 and PTP-MEG2 may be an important negative regulator of VEGFR2 signaling in ECs. Figure 2 shows that PTP-MEG2 substrate-trapping mutant also interacted with highly tyrosine-phosphorylated protein bands ⬃130 kDa (indicated as p130). We thus attempted to identify these proteins in HUVECs. Interestingly, we found that JAK1 was preferentially associated with the DA substratetrapping mutant of PTP-MEG2 compared with PTP-MEG2 wild type (Fig. 4A, 1st row). VEGF treatment did not affect the associations of JAK1 with PTP-MEG2 wild type or the DA mutant. In addition, JAK1 was not detected in the complexes precipitated with normal rabbit IgG (data not shown). In contrast, the DA substrate-trapping mutant did not prefer to associate with other JAK kinases present in HUVECs, including Tyk2 and JAK2 (Fig. 4B). Additionally, we found that neither wild type nor the DA mutant associated with focal adhesion molecules, such as p130Cas and FAK (125 kDa) in HUVECs (Fig. 4A, 2nd and 3rd rows). We next assessed the effect of PTP-MEG2 on tyrosine phosphorylation (activation) of JAK1. JAK1 contains Tyr1022/1023 in the activation loop, and its phosphorylation is obligatory for kinase activation (3). Using a phospho-specific antibody against Tyr1022/1023phosphorylated JAK1, we found that the phosphorylation of JAK1 on the tyrosine residues was readily detected in basal condition, which was slightly enhanced by a 5-min treatment of HUVECs with VEGF (Fig. 4C). The phosphorylation of JAK1 on Tyr1022/1023 in basal condition or induced by VEGF was suppressed ⬃45% by overexpression of PTPMEG2 wild type in HUVECs. In contrast, the phosphorylation of JAK1 on Tyr1022/1023 was significantly enhanced by the DA mutant of PTP-MEG2. These data suggest that JAK1 is likely a substrate of PTP-MEG2 in ECs. PTP-MEG2 negatively regulates the VEGF-induced signal transduction and IL-6 production in ECs. We knocked down PTP-MEG2 and assessed the effect on VEGF signaling and function in ECs. As shown in Fig. 5A, knockdown of PTPMEG2 enhanced the VEGF-induced phosphorylation (activation) of important downstream signal molecules, such as JAK1, p38, and ERK1/2 in ECs. In contrast, knockdown of RPTP␣ had a minor effect on the VEGF-induced signal transduction. We (7) have recently shown that VEGF induces production of proinflammatory cytokines including IL-6 and IL-8 in

Fig. 3. PTP-MEG2 associates and dephosphorylates VEGFR2 on a critical autophosphorylation site Tyr1175 in ECs. HUVECs were infected 48 h with recombinant adenovirus carrying vector alone (Vector), PTP-MEG2 wild type (MEG2-WT), or DA substrate-trapping mutant (MEG2-DA) and then starved 2 h and left untreated (control) or treated 5 min with VEGF (50 ng/ml). A: lysates (500 ␮g) were immunoprecipitated with PTP-MEG2 (MEG2) polyclonal antibody and subjected to Western blot analysis with antibodies against VEGFR2 or PTP-MEG2 as indicated. Lane “Lysates” refers to 50 ␮g protein lysates from HUVECs infected with vector alone. B: cell lysates were directly subjected to Western blot with antibodies against VEGFR2 [pY1175], VEGFR2, or PTP-MEG2. Results represent Western blots of 3 independent experiments. AJP-Cell Physiol • doi:10.1152/ajpcell.00415.2011 • www.ajpcell.org


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Fig. 4. Identification of JAK1 as a PTP-MEG2 potential substrate in ECs. HUVECs were infected 48 h with recombinant adenovirus carrying vector alone (V), PTP-MEG2 wild type, or the DA substrate-trapping mutant and then starved 2 h and left untreated (⫺ or control) or treated 5 min with 50 ng/ml VEGF (⫹ or VEGF). A and B: PTP-MEG2 was immunoprecipiated from 500 ␮g lysates and subjected to Western blot analysis with specific antibodies as indicated. Lane “Lysates” refers to 50 ␮g protein lysates from HUVECs infected with vector alone. C: cell lysates were directly subjected to Western blot with p-JAK1 (pY1022/ 1023) antibody that recognizes the phosphorylated and activated JAK1. The same blot was stripped and reprobed with JAK1 antibody to show equal loading. Results represent Western blots of 3 independent experiments.

ECs. We found that knockdown of PTP-MEG2 by two different siRNAs significantly enhanced IL-6 production induced by VEGF but not by porbol-12-myristate 13-acetate (PMA) in ECs (Fig. 5B). In contrast, knockdown of RPTP␣ generally had a minor effect on IL-6 production by VEGF and PMA in ECs. On the other hand, we found that overexpression of PTP-MEG2 significantly inhibited IL-6 production induced by VEGF but not by PMA in ECs (Fig. 5C). These data indicate that PTP-MEG2 negatively regulates the VEGF-induced signal transduction and function in ECs. DISCUSSION

In this study, we have investigated the function of PTPMEG2 in EC biology. The major findings obtained from this study are that PTP-MEG2 interacts with and regulates the tyrosine phosphorylation and activation of VEGFR2 and that PTP-MEG2 functions as a negative regulator of VEGFR2 signaling and function in ECs. Our novel findings may provide new insights into the mechanism of PTP-MEG2 action in vascular development and integrity. PTP-MEG2 is a nonreceptor PTP originally cloned from HUVECs and megakarocyte cDNA libraries (5). PTP-MEG2 is distinct from other mammalian PTPs by virtue of a putative lipid-binding domain at the NH2 terminus, which can bind to phosphatidylinositol 3,4,5-triphosphate and phosphatidylserine (9, 20). Consistent with earlier reports (10, 18), we found that PTP-MEG2 was detected in both cytosolic and membrane factions of HUVECs by subcellular fractionation experiment. Immunofluorescence microscopy further revealed that PTPMEG2 displayed perinuclear, plasma membrane, and intracellular vesicular staining in the cells. The plasma membrane localization of PTP-MEG2 suggests that it may regulate receptor signaling in ECs. It has been shown that PTP-MEG2 deficiency causes multiple neurodevelopment defects and hemorrhages, resulting in ⬎90% late embryonic lethality (17). The hemorrhages manifested in PTP-MEG2-deficient mice suggest

that PTP-MEG2 may play an important role in vascular development and integrity through regulation of EC biology. However, the role and mechanism of PTP-MEG2 in the aspects of EC biology are not known so far. This could be in part due to the relative difficulty to identify PTP-MEG2 substrates. Indeed, we found that no clear tyrosine-phosphorylated protein band was detected in the immune complexes of PTP-MEG2 from ECs in the presence and absence of VEGF. All the PTPs contain an invariant aspartic acid residue, which functions as a general acid in the phosphate ester hydrolysis reaction. It has been demonstrated that mutation of this aspartic acid ablates the ability of PTPs to dephosphorylate target substrates but leaves substrate binding intact. Thus the PTP-substrate complex is stabilized sufficiently to permit isolation by immunoprecipitation, and the substrates can subsequently be identified (4). By using the PTP-MEG2 substrate-trapping mutant with Asp-470 mutation to Ala (MEG2-DA mutant; Ref. 18), we found that a couple of tyrosine-phosphorylated proteins were associated with the DA mutant and that the association were enhanced by VEGF treatment. These data indicate that the DA phosphatase domain likely binds the phosphotyrosine residues of the associate proteins but cannot catalyze the dephosphorylation reaction. Among the associated proteins, we found that VEGFR2 (230 kDa) interacted with the DA mutant but not wild-type PTP-MEG2 in VEGF-treated ECs. We further showed that the VEGF-induced phosphorylation of VEGFR2 on Tyr1175, a critical autophosphorylation site for VEGFR2 signaling (12), was inhibited 70% by overexpression of wild-type PTP-MEG2 but was enhanced (2.2-fold) by the DA mutant of PTP-MEG2 in HUVECs. These data indicate that VEGFR2 is a protein substrate of PTP-MEG2. PTP-MEG2 may bind VEGFR2 directly or indirectly through tyrosine-phosphorylated proteins such as the p130 protein that has an abundant tyrosine phosphorylation in the immune complexes of PTP-MEG2 DA mutant. Moreover, knockdown of PTP-MEG2 enhanced

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Fig. 5. PTP-MEG2 negatively regulates the VEGF-induced signal transduction and IL-6 production in ECs. A: HUVECs were transfected with 20 nM nontargeting control siRNA (con.), PTP-MEG2 siRNA-I (M) or receptor PTP␣ (RPTP␣) small interfering (si)RNA (R) and grown for 72 h and then left untreated (control) or treated 10 min with VEGF (50 ng/ml). Cell lysates were directly subjected to Western blot with phospho-specific antibodies against JAK1, p38, or ERK1/2 as indicated (left) or with PTP-MEG2 (MEG2) or RPTP␣ antibodies (right). Same blot was stripped and reprobed with actin or tubulin antibodies to show equal loading. B: HUVECs were transfected with 20 nM nontargeting control siRNA, PTP-MEG2 siRNA-I or -II (MEG2-siRNA-I or -II) or RPTP␣ siRNA and grown for 72 h and then left untreated (control) or treated 4 h with VEGF (50 ng/ml) or PMA (50 nM). C: HUVECs were infected 48 h with recombinant adenovirus expressing vector alone (vector) or human PTP-MEG2 wild type (MEG2-WT) and then left untreated (control) or treated 4 h with VEGF (50 ng/ml) or PMA (50 nM). IL-6 released into the medium in B and C was measured by ELISA and is presented as pg/ml from 1 ⫻ 106 cells/ml. Data are means ⫾ SE (n ⫽ 3). **P ⬍ 0.01 vs. control siRNA or vector control. PTP-MEG2 knockdown or overexpression or RPTP␣ knockdown was confirmed by Western blot analysis with indicated antibodies.

the VEGF-induced phosphorylation (activation) of important downstream signal molecules, such as JAK1, p38, and ERK1/2 in ECs. We have recently shown that VEGF, via VEGFR2, induces production of proinflammatory cytokines including IL-6 and IL-8 in ECs (7). We found that knockdown of PTP-MEG2 also significantly enhanced IL-6 production induced by VEGF in ECs. On the other hand, overexpression of PTP-MEG2 significantly inhibited IL-6 production induced by VEGF in the cells. Taken together, our findings indicate that PTP-MEG2 dephosphorylates VEGFR2 and negatively regulates VEGFR2 signaling and function in ECs. Another interesting finding from the present study is that JAK1 may be a potential substrate of PTP-MEG2 in ECs. We found that PTP-MEG2 substrate-trapping mutant interacted with highly tyrosine-phosphorylated protein bands ⬃130 kDa. We further found that the DA substrate-trapping mutant preferentially associated with JAK1 but not other JAK kinases present in HUVECs, including Tyk2 and JAK2. Additionally, neither PTP-MEG2 wild type nor the DA mutant associated with focal adhesion molecules, such as p130Cas and FAK (125 kDa) in HUVECs. JAK1 contains Tyr1022/1023 in the activation loop, and its phosphorylation is obligatory for kinase activation (3). We found that there was a certain amount JAK1 phosphorylation on Tyr1022/1023 in basal condition, which was slightly enhanced by VEGF treatment. Furthermore, the

phosphorylation of JAK1 on Tyr1022/1023 in basal condition or induced by VEGF was suppressed ⬃45% by overexpression of PTP-MEG2 wild type in ECs. In contrast, the phosphorylation of JAK1 on Tyr1022/1023 was significantly enhanced by the DA mutant of PTP-MEG2. These results suggest that JAK1 is likely a protein substrate of PTP-MEG2. Characterization of JAK1 association with and regulation by PTP-MEG2 merits further investigation. In summary, we have indentified VEGFR2 as a PTP-MEG2 substrate in ECs by using a PTP-MEG2 substrate-trapping mutant. PTP-MEG2 functions as a negative regulator of VEGFR2 signaling and function in ECs. GRANTS This work was supported by a departmental fund (to H. Tang) and National Heart, Lung, and Blood Institute Grant R01-HL-079441 (to Z. J. Zhao). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: Q.H., B.S., Z.J.Z., and H.T. performed experiments; Q.H. and H.T. analyzed data; Q.H., B.S., H.-L.J., Z.J.Z., and H.T. interpreted results of experiments; Q.H. and H.T. prepared figures; Q.H. and H.T. drafted manuscript; Q.H., B.S., H.-L.J., Z.J.Z., and H.T. approved final version of manuscript; Z.J.Z. and H.T. conception and design of research; H.T. edited and revised manuscript.


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