Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration*
© Bhattacharya et al. 2008
Received: 09 January 2008
Accepted: 31 March 2008
Published: 31 March 2008
Vascular endothelial growth factor receptor-2 (VEGFR-2, KDR), a receptor tyrosine kinase, regulates mitogenic, chemotactic, hyperpermeability, and survival signals in vascular endothelial cells in response to its ligand vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF). SHP-1 is a protein tyrosine phosphatase known to negatively regulate signaling from receptors such as EGF receptor, IL3 receptor, erythropoietin receptor and also KDR. However, the mechanism by which SHP-1 executes KDR dephosphorylation, the targeted tyrosine residue(s) of KDR and also overall downstream signaling or phenotypic change(s) caused, is not defined.
Here, we have demonstrated that KDR and SHP-1 are constitutively associated and upon VEGF treatment, the phosphatase activity of SHP-1 is stimulated in a c-Src kinase dependent manner. Knockdown of SHP-1 by siRNA or inhibition of c-Src by an inhibitor, results in augmented DNA synthesis perhaps due to increased phosphorylation of at least three tyrosine residues of KDR 996, 1059 and 1175. On the other hand, neither tyrosine residue 951 of KDR nor VEGF-mediated migration is affected by modulation of SHP-1 function.
Taken together our results define the tyrosine residues of KDR that are regulated by SHP-1 and also elucidates a novel feed back loop where SHP-1 is activated upon VEGF treatment through c-Src and controls KDR induced DNA synthesis, eventually leading to controlled angiogenesis.
Angiogenesis, the sprouting of new blood vessels from pre-existing endothelium is a fundamental feature of both normal physiology and pathologic states including coronary heart disease, diabetes, retinopathy and cancer [1–4]. The growth factor VEGF-A is a key regulator of physiologic and pathologic angiogenesis . VEGF was identified due to its ability to induce vascular hyperpermeability but has since been recognized as a potent inducer of endothelial proliferation, migration and survival. VEGF also acts as a proinflammatory cytokine and induces the expression of a number of molecules implicated in regulating angiogenesis [6, 7].
The effects of VEGF and its family of proteins are mediated by three structurally related receptor tyrosine kinases namely VEGFR1/Flt-1, VEGFR-2/Flk-1/KDR, VEGFR3/Flt-4 [8–12]. Among these, KDR has emerged as the main receptor mediating VEGF effects such as endothelial cell proliferation, migration and proinflammatory activation. In contrast, Flt-1 is thought to mediate inhibitory and/or decoy effects in endothelial cells [13, 14]. Flt-4 is mainly expressed in lymphatics and regulates lymphangiogenesis . The importance of VEGF/KDR axis is accentuated by the fact that increased levels of both ligand and receptor are found in tumor cells as well as stroma [15–19].
Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP) -1 and -2 are non-receptor protein tyrosine phosphatases (PTPs). Expression of SHP-1 is restricted to hematopoietic cells whereas SHP-2 is more widely expressed . SHP-1 has been proposed to be a candidate tumor suppressor gene in lymphoma, leukaemia and other cancers . Evidence for the differing roles of SHP-1 and SHP-2 in cell signaling has come from the study of mice lacking functional SHP-1 or SHP-2. The SHP-1 gene mutated motheaten (me) mice display severe haematopoietic disruption with chronic inflammation and systemic autoimmunity and die from hemorrhagic pneumonitis [22, 23]. Thus the results provide strong evidence for a major role of this phosphatase in the negative regulation of cell function. Targeted disruption of the SHP-2 gene results in embryonic lethality of homozygous mutant mice however generation of chimeric mice from homozygous SHP-2 mutant ES cells and wild-type embryos determined a role for SHP-2 in blood cell development [24, 25]. Other studies have demonstrated a role for SHP-2 in positively regulating signaling downstream of the insulin receptor, platelet derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) (reviewed in . The SHP-1 enzyme contains two tandem SH2 domains at the N-terminus, followed by the catalytic domain and a C-terminal tail. The C-terminal tail contains multiple sites of tyrosine and serine phosphorylation and this part of the protein has been proposed to have an important regulatory function .
A number of studies have shown that SHP-1 negatively regulates signaling of receptors such as the EGF receptor, IL3 receptor, erythropoietin receptor and KDR [27–29]. Studies have also suggested that SHP-1 is a negative regulator of angiogenesis in vivo because SHP-1 deficient mice are resistant to TIMP-2 inhibition . SHP-1 has also been shown to co-precipitate with KDR upon stimulation with VEGF and overexpression of SHP-1 resulted in impairment of VEGF mediated KDR autophosphorylation and ERK activation . However, the signaling mechanism by which this inhibition takes place remains to be elucidated. Here, we have clearly defined the respective tyrosine residues of KDR that are regulated by SHP-1 and hence affect downstream signaling. We have also demonstrated that KDR and SHP-1 are constitutively associated and upon VEGF treatment the phosphatase activity of SHP-1 is stimulated in a c-Src kinase dependent manner. Tyrosine phosphorylation and activation of SHP-1 is regulated by c-Src. Knockdown of SHP-1 by siRNA or inhibition of c-Src results in amplified proliferation that may be due to increased phosphorylation of at least three tyrosine residues on KDR such as 996, 1059  and 1175 . Interestingly, VEGF-mediated migration is not affected as well as tyrosine residue 951 on KDR is unchanged . Overall, our results define the residues on KDR that are regulated by SHP-1 and also elucidate a novel feed back loop by which SHP-1 is activated upon VEGF treatment through c-Src kinase and attenuates KDR mediated DNA synthesis.
SHP-1 Co-Precipitates with KDR and Src
Activation of SHP-1 Upon VEGF Stimulation
SHP-1 is Required for Modulating the VEGF-Mediated DNA Synthesis
Knockdown of SHP-1 Interferes With VEGF Induced Signaling
Modulation of KDR Phosphorylation and DNA Synthesis by c-Src
Modulation of KDR Phosphorylation by SHP-1 Tyrosine Mutants
VEGF is a critical regulator of angiogenesis, primarily through the signaling mediated by KDR. Although the signaling pathways via KDR have been extensively studied, the role of key proteins that modulate and 'fine-tune' VEGF signaling remains to be elucidated. In the present study, we have defined the specific tyrosine residues on KDR that are dephosphorylated by SHP-1 and modulate VEGF-mediated signaling. We also show that this dephosphorylation of tyrosine residues on KDR occurs upon stimulation with VEGF resulting in the activation of c-Src kinase and tyrosine phosphorylation of SHP-1. Our results are supported by the fact that the inhibition of c-Src kinase resulted in increased immunostaining of phospho-KDR with a concomitant increase in HUVEC proliferation and pERK. Our data clarifies a previously unexplained increase in phospho-KDR levels upon treatment with PP2 as described by Labrecque et al .
SHP-1 and KDR and KDR and c-Src co-immunoprecipitated with each other and the association between SHP-1 and KDR remained unchanged with or without VEGF treatment. Prior studies have indicated that VEGF induces association of SHP-1 with KDR. However our data consistently suggested that SHP-1 associates with KDR regardless of VEGF treatment. We presumed that in the culture conditions even after overnight starvation KDR remains phosphorylated at low levels leading to association with SHP-1. More importantly our data suggest that it is perhaps not the association but the activation of the tyrosine phosphatase activity of SHP-1 that is critical in regulating KDR-mediated downstream signaling. Nonetheless, the association of KDR with c-Src increased upon treatment with VEGF in HUVEC. Our results are in complete accordance with prior studies that have reported preferential association of c-Src with KDR upon stimulation with VEGF .
Knockdown of SHP-1 by siRNA in HUVEC caused a significant and consistent increase in proliferation after treatment with VEGF. These results suggest that the phosphatase activity of SHP-1 potentiates upon VEGF treatment. Previous reports suggested that the activity of SHP-1 is regulated by phosphorylation of the tyrosine and serine residues in the C terminus of the protein . SHP-1 is phosphorylated on serine residues, constitutively in resting murine T cells. In human platelets it is phosphorylated on both tyrosine and serine residues in response to thrombin . This is PKC dependent and correlates with an increase in the activity of SHP-1. In platelets, PKCα is constitutively bound to SHP-1 and PKCα dependent serine phosphorylation at 591 led to a decrease in phosphatase activity. However our results suggest that serine phosphorylation of SHP-1 decreases upon VEGF stimulation. SHP-1 is also phosphorylated on Tyr536 by the insulin receptor tyrosine kinase and this induces activation of its phosphatase activity . A recent report by Frank et al., showed that c-Src is able to tyrosine phosphorylate SHP-1 in vitro, leading to an increase in the activity of the phosphatase . Phosphorylation of SHP-1 at tyrosine 538 was required for optimal phosphatase activity of SHP-1 . Therefore our observation of increased KDR phosphorylation upon mutation of Y538 of SHP-1 compared to the control in VEGF stimulated cells is substantiated by the previous reports. We also observed an increased c-Src dependent tyrosine phosphorylation of SHP-1 and inhibition of c-Src kinase activity by PP2 caused a significant increase in DNA synthesis as well as phospho-KDR (Y996) staining on HUVEC. It should be mentioned here that the relative increase in DNA synthesis after inhibiting c-Src kinase compared to knockdown of SHP-1 was much more potent. This is probably because c-Src negatively regulates a number of other proteins including caveolin-1 and dynamin that may play a role in proliferation [35, 40]. We also identified specific tyrosine residues on KDR that were phosphorylated more in SHP-1 knockdown and PP2 treated cells. Migration, as determined by the wound healing assay or phospho-KDR (Y951) a marker for migration, was not significantly affected while proliferation markers like phospho-Y996, Y1059 and Y1175 were significantly more phosphorylated. An increase in phospho-ERK was also observed in SHP-1 siRNA treated cells. This finding is supported by a previous study that showed activation of ERK1/2 after using a dominant-negative inhibitor of SHP-1 .
It has been previously shown that in 293T cells Tyr996 of KDR perhaps remains phosphorylated as no difference is observed in the phosphopeptide map after addition of VEGF (12). However, in our experiments we repeatedly find Tyr996 of KDR to be phosphorylated in a VEGF dependant manner. This has been confirmed by Western blotting as well as confocal microscopy. We also confirmed specificity of the antibody by staining with or without bFGF treatment. We found clear, distinct membrane staining after treatment with VEGF but not bFGF. This leads us to speculate that HUVEC being endothelial cells differ from 293T in the way they respond to VEGF or that Tyr996 might not be autophosphorylated and could be phosphorylated by some other kinase upon activation with VEGF.
However, the question remains why SHP-1 with the help of c-Src kinase controls KDR-mediated proliferation in HUVEC. In order for proper vascular remodeling to occur, fine-tuning of proliferative and/or anti-proliferative cues controlling KDR-induced downstream signaling is expected to be required and with VEGF as a potential endothelial mitogen, it is a logical candidate. In this regard, other than its role in vascular hyperpermeability , VEGF-mediated c-Src kinase activation, leads to a better organization of vascular structure by controlling KDR function. Future studies are in progress to resolve the current hypothesis. Nonetheless, our study illustrates a new role for c-Src as well as SHP-1 in VEGF-induced angiogenesis.
The main conclusions are: 1. KDR, SHP-1 and c-Src are part of the same immnocomplex. 2. The phosphatase activity of SHP-1 increases after VEGF treatment and is Src dependent. 3. Inhibition of Src in endothelial cells causes increased tyrosine phosphorylation of VEGFR-2 and pERK ultimately leading to increased proliferation. 4. We identified the specific tyrosine residues on KDR that are modulated by SHP-1. 5. We identified Y538 on SHP-1 to be the major site that regulates its phosphatase activity and hence is responsible for de-phosphorylation of tyrosine residues on VEGFR-2 subsequent to VEGF stimulation.
VEGF-A was obtained from R&D systems, Minneapolis, MN. [3H] Thymidine was from Amersham Biosciences. The antibodies to KDR, c-Src, phosphoKDR (996) and phosphoKDR (951) were from Santa Cruz Biotechnology (Santa Cruz, CA); phosphoKDR (1059) was from Upstate and phospho KDR (1175) from Cell Signaling. PP2 and PP3 were from EMD Biosciences (San Diego, CA). The antibodies to SHP-1 were from Santa Cruz Biotechnology (Santa Cruz, CA) and BD-Biosciences (San Jose, CA).
Anti-KDR monoclonal antibody used for immunoflourescence was from Sigma-Aldrich, St. Louis, MS. AlexaFlour 488 anti-mouse or AlexaFlour 546 anti-rabbit secondary antibody was from Molecular probes, Eugene, OR. 2 × 104 HUVEC were seeded on collagen coated Lab-Tek chamber slides. After 24 h cells were serum starved. The next day cells were treated with or without VEGF 10 ng/ml. Slides were washed in PBS, fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.2% TRITON X-100 at room temperature. Slides were washed in PBS, blocked in 10% goat serum and stained with respective primary antibodies in 1% goat serum for 2 hrs. Slides were washed in PBS and incubated for 1 h in respective secondary antibody at a dilution of 1:200 followed by post-fixing in 4% PFA and mounting in Vectashield, Vector Labs, CA. Confocal microscopy was performed using a Zeiss LSM 510 confocal laser scan microscope with C-Apochromat 63×/NA 1.2 water-immersion lens. Absence of signal crossover was established using single-labeled samples.
Immunoprecipitation and Western Blot Analysis
Serum starved HUVEC were treated with or without VEGF 10 ng/ml and/or PP2 or PP3 5 uM. Cell lysates in RIPA buffer supplemented with protease inhibitor cocktail were prepared from HUVEC. The lysates were collected after centrifugation at 14000 × g for 10 min at 4°C. 500 ug of lysate protein was incubated with 1 mg respective antibody for 1 h and 50 ml of proteinA/G-conjugated agarose-beads for an additional hour at 4°C. Beads were washed with RIPA buffer three times and immunoprecipitates were resuspended in 2× SDS sample buffer.
HUVEC were transfected with the following plasmids control pCDNA (C) or SHP-1 wildtype (WT) or SHP-1 Y538 or SHP-1 Y543 or SHP-1 Y566 mutants using nucleofector based electroporation. The above plasmids were a kind gift from Dr. Z. Yu (National Research Council, Canada). After 48 hrs. the cells were starved overnight in EBM medium without serum. Subsequently the cells were stimulated with VEGF 10 ng/ml for 5 min. Cell lysates were collected and immunoblotted with respective antibodies. Please note that immunoprecipitation or western blot as shown in Fig. 1 through Fig. 5 was performed exactly as described here but without any overexpression of SHP-1, KDR or Src rather on endogenous proteins from HUVEC lysates. Experiments were repeated at least three times.
Cell Proliferation Assay
HUVECs (2 × 104) were seeded in 24-well plates and cultured for 24 h in EGM. After 24 h the cells were serum starved and pre-treated with or without PP2 or PP3 at 5 mM for 1 h respectively before stimulation with VEGF 10 ng/ml. After culture for 20 h, 1 mCi of [3H]thymidine was added to each well; 4 hrs later, cells were washed with chilled PBS, fixed with 100% cold methanol and collected for measurement of trichloroacetic acid-precipitable radioactivity. Experiments were repeated at least three times each time in triplicate.
1 × 105 HUVEC were seeded in 60 mm plates and cultured for 24 h in EGM. The next day cells were washed with OPTI-MEM reduced serum medium and transfected with 50 nM SHP-1 siRNA obtained from Santa Cruz Biotechnology (sc29478) using oligofectamine (Invitrogen). This siRNA is a pool of 3 sequences. Sense Strand (A):CUGGUGGAGCAUUUCAAGATT, (B):CGCAGUACAAGUUCAUCUATT and (C): CAACCCUUCUCCUCUUGUATT. After 4 hrs antibiotic free EGM was added and cell lysates were prepared 48 hrs after transfection.
SHP-1 Phosphatase Assay
Phosphatase assay was performed according to manufacturers protocol (Stratagene, Signal Scout Phasphatase Profiling System). Briefly, HUVEC were pretreated for 1 h with 5 mm PP2 or PP3 and then with or without 10 ng/ml VEGF for 5 min. Cells were lysed (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton ×, 5 mM DTT), lysate pre-cleared and protein quantity determined by Bradford assay. Equal amount of protein from each sample was then immunoprecipitated with a SHP-1 antibody using the "catch and release column" from Upstate. One part was then subjected to western blot and another part was treated as follows; 80 ul complete assay buffer (14 mM HEPES pH 7.4, 30 mM NaCl, 1.5 mM EDTA, 5 mM DTT) was added and eluted from the column. 120 ul pNPP substrate (20 mM) was then added to each sample and absorbance at 405 nm was read after 30 min incubation at 30°C on a TECAN Spectra Flour Plus. Data presented were normalized with respect to negative control. Three independent experiments were performed.
Wound Healing Migration
Monolayers of HUVEC transfected with scrambled control or SHP-1 siRNA were scratched with a universal blue pipette tip and incubated for 12 hrs in the presence of 10 ng/ml VEGF. Thymidine (10 mM; Sigma-Aldrich) was included during the incubation to inhibit cell proliferation. Migration of cells across the scratched area was recorded by time-lapse microscopy (Apotome, Carl Zeiss) using AXIO Vision software. Cells were counted from five fields per well.
All values are expressed as means ± SD. Statistical significance was determined using two-sided Student's t test, and a value of P < 0.05 was considered significant. For Figure 5B, ANOVA was calculated using Tukey's Studentized Range (HSD) Test. Comparisons significant at the 0.05 level are shown (Supplemental data).
Abbreviations used are
Src homology 2 domain containing protein tyrosine phosphatase-1
Src homology 2 domain containing protein tyrosine phosphatase-2
Vascular endothelial growth factor
Vascular endothelial growth factor receptor-2
Human umbilical vein endothelial cells
Mitogen activated protein kinase
* We would like to thank Jim Tarara for help with the confocal microscopy experiments. This work is partly supported by NIH grants HL072178, HL70567 and CA78383 and also a grant from American Cancer Society to DM.
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