3385Research Article

IntroductionAngiogenesis performs a crucial role in physiological conditions,

including embryonic development, wound healing and organ

regeneration, as well as in pathological processes such as diabetes

retinopathies, atherosclerosis, tumorigenesis and tumor metastasis

(Carmeliet, 2003; Ferrara et al., 2003; Folkman, 1971; Folkman, 1995;

Sueishi et al., 1997). Vascular endothelial growth factor (VEGF), also

known as vascular permeability factor (VPF), is an important

mediator of these angiogenic processes in both normal and diseased

conditions. As one of the most important cytokines, VEGF is widely

expressed by a number of human and animal tumors and has the

ability to regulate most of the steps in the angiogenic signal cascade

(Dvorak, 1990; Dvorak et al., 1999; Dvorak et al., 1979; Ferrara,

1999; Risau, 1997). The VEGF receptor family consists of three

members, namely VEGFR-1, VEGFR-2 and VEGFR-3 (Shibuya,

2008). VEGFR-2, the major positive signal transducer for both

physiological and pathological angiogenesis (Mukhopadhyay et al.,

2004; Shibuya, 2006), is selectively expressed on vascular endothelial

cells. The binding of VEGF to its receptors induces dimerization and

subsequent receptor phosphorylation, which then leads to the

activation of several intracellular downstream signaling pathways

promoting angiogenesis (Olsson et al., 2006; Sakurai et al., 2005;

Takahashi et al., 2001; Zachary, 2003).

The role of the neurotransmitter dopamine in modulating VEGF-

induced angiogenesis has been well defined (Basu et al., 2001; Sarkar

et al., 2004). Dopamine is produced in a wide variety of animals

including both vertebrates and invertebrates, and activates the five

types of dopamine receptors – D1, D2, D3, D4 and D5 – and their

variants (Neve et al., 2004). Dopamine has a role in the management

of Parkinson’s disease and is also involved in the etiopathogenesis

of several neuropsychiatric diseases such as schizophrenia (Egan and

Weinberger, 1997; Goldstein and Deutch, 1992; Graybiel et al., 1990;

Olanow and Tatton, 1999). Dopamine, when applied to patients with

septic shock, can effectively maintain the circulatory stability and

promote restoration of renal function (Dellinger et al., 2008).

Although it is known that dopamine can inhibit different signaling

steps involved in VEGF-mediated angiogenesis, beginning at

VEGFR-2 phosphorylation (Basu et al., 2001; Bhattacharya et al.,

2008b; Sarkar et al., 2004), the pathophysiological implication of

dopamine in the context of VEGF-induced angiogenesis is novel and

requires further investigation. Here, we examine how dopamine

specifically regulates one of the earliest steps of the angiogenic

process, the VEGF-induced phosphorylation of VEGFR-2, and

define a novel role of Src-homology-2-domain-containing protein

tyrosine phosphatase 2 (SHP-2) in the dopamine-mediated anti-

angiogenic pathway.

ResultsDopamine modulates individual tyrosine phosphorylation ofVEGFR-2We previously demonstrated that total tyrosine phosphorylation of

VEGFR-2 was inhibited upon dopamine pretreatment (Basu et al.,

Vascular endothelial growth factor (VEGF)-induced receptor

phosphorylation is the crucial step for initiating downstream

signaling pathways that lead to angiogenesis or related

pathophysiological outcomes. Our previous studies have shown

that the neurotransmitter dopamine could inhibit VEGF-

induced phosphorylation of VEGF receptor 2 (VEGFR-2),

endothelial cell proliferation, migration, microvascular

permeability, and thus, angiogenesis. In this study, we address

the mechanism by which VEGFR-2 phosphorylation is

regulated by dopamine. Here, we demonstrate that D2

dopamine receptor (D2DR) colocalizes with VEGFR-2 at the

cell surface. Dopamine pretreatment increases the translocation

and colocalization of Src-homology-2-domain-containing

protein tyrosine phosphatase (SHP-2) with D2DR at the cell

surface. Dopamine administration leads to increased VEGF-

induced phosphorylation of SHP-2 and this increased

phosphorylation parallels the increased phosphatase activity of

SHP-2. Active SHP-2 then dephosphorylates VEGFR-2 at

Y951, Y996 and Y1059, but not Y1175. We also observe that

SHP-2 knockdown impairs the dopamine-regulated inhibition

of VEGF-induced phosphorylation of VEGFR-2 and,

subsequently, Src phosphorylation and migration. Our data

establish a novel role for SHP-2 phosphatase in the dopamine-

mediated regulation of VEGFR-2 phosphorylation.

Supplementary material available online at

http://jcs.biologists.org/cgi/content/full/122/18/3385/DC1

Key words: Dopamine, Dopamine D2-receptor (D2DR), SHP-2,

Vascular endothelial growth factor receptor 2 (VEGFR-2),

Phosphorylation, Migration, Angiogenesis

Summary

Dopamine regulates phosphorylation of VEGFreceptor 2 by engaging Src-homology-2-domain-containing protein tyrosine phosphatase 2Sutapa Sinha, Pawan Kumar Vohra, Resham Bhattacharya, Shamit Dutta, Shirshendu Sinha andDebabrata Mukhopadhyay*Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, Rochester, MN 55905, USA*Author for correspondence (mukhopadhyay.debabrata@mayo.edu)

Accepted 3 July 2009Journal of Cell Science 122, 3385-3392 Published by The Company of Biologists 2009doi:10.1242/jcs.053124

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2001), but the specific tyrosine residues were not identified. Here,

we found that phosphorylation of the specific tyrosine residues

Y951, Y996, Y1059 and Y1175 on VEGFR-2 were significantly

inhibited when HUVEC cells were pretreated with 10 μM dopamine

or quinpirole (D2-receptor agonist) before the administration of

VEGF (Fig. 1) for 10 minutes. A dose-response experiment showed

that a 10 μM concentration of dopamine was more effective than

1 μM (Basu et al., 2001) to inhibit VEGFR-2 phosphorylation

(supplementary material Fig. S1A). These results also suggested

that the dopamine-mediated effect on VEGFR-2 phosphorylation

was through D2DR.

Whole-cell lysates from serum-starved HUVECs were

immunoprecipitated with VEGFR-2 antibody and immunoblotted

with antibody against phosphorylated (-P) VEGFR-2(951) to show

the specificity of the phospho-antibody against VEGFR-2-P(supplementary material Fig. S1B).

D2DR coprecipitates with VEGFR-2 and SHP-2As dopamine modulates VEGFR-2 tyrosine phosphorylation, we

posited that certain phosphatases might be involved in this modulation.

Therefore, we performed coprecipitation experiments to identify

specific protein tyrosine phosphatases that are involved in the

dopamine-mediated VEGFR-2 dephosphorylation. Whole-cell lysates

from serum-starved HUVECs were immunoprecipitated with D2DR

antibody and immunoblotted with antibodies against VEGFR-2 and

SHP-2. We found constitutive association between D2DR, VEGFR-

2 and SHP-2. The association between D2DR and VEGFR-2

decreased upon VEGF (10 ng/ml) induction but increased

significantly with dopamine (10 μM) pretreatment, followed by

VEGF induction (P=0.05) (Fig. 2A). Interestingly, the association

between D2DR and SHP-2 markedly increased upon VEGF treatment

and remained unchanged even with dopamine pretreatment (Fig. 2A).

We also found that VEGFR-2 associated with SHP-2 and that this

association increased upon treatment with VEGF for 10 minutes;

however, dopamine pretreatment had no additive effect on the

VEGF-induced association of VEGFR-2 and SHP-2. (supplementary

material Fig. S2). These results were further corroborated using

confocal microscopy. Cells were stained without permeabilization and

we found that VEGFR-2 and D2DR remained associated with each

other at the cell surface when cells were untreated. This localization

and association at the cell surface markedly decreased (P=0.021) upon

stimulation with VEGF and again increased significantly (P=0.004)

with dopamine pretreatment followed by VEGF induction (Fig. 2B).

In untreated cells, little SHP-2 staining and colocalization with D2DR

were observed at the cell surface. However, after 10 minutes of VEGF

treatment, SHP-2 appeared at the surface, indicating that SHP-2

translocated from the cytoplasm to the cell surface and became

associated with D2DR at the surface (P=0.036). Dopamine

pretreatment caused a significant induction in the VEGF-stimulated

colocalization of SHP-2 with D2DR (P=0.009) at the cell surface

(Fig. 2C).

VEGFR-2, SHP-2 and D2DR in membrane fractionsTo further support our immunoprecipitation and confocal data,

sucrose density gradient and cell surface biotinylation experiments

were performed. To isolate endothelial cell low-density membrane

fractions, HUVEC homogenates were subjected to a sucrose density

gradient and 12 fractions were recovered. Eight fractions were

subjected to western blot analysis. Under these conditions (see the

Materials and Methods), low-density membranes were sedimented

at the 5% and 35% sucrose interface (fractions 3-6; Fig. 2D). In the

absence of any treatment, SHP-2 was not detected in the low-density

membrane fraction. With VEGF induction, colocalization of SHP-2

with VEGFR-2 was observed in the low-density membrane fraction

(fraction 4; Fig. 2D). Dopamine pretreatment followed by VEGF

induction resulted in further enrichment of both VEGFR-2 and SHP-

2 in the low-density fraction (fraction 4). For the detection of

dopamine receptor D2, the immunoprecipitation experiment was done

with D2DR antibody and seven fractions were analyzed (fractions

2-8; Fig. 2D). We observed expression of D2DR in all fractions, with

the moderate enrichment of D2DR in fractions 3 and 4 with dopamine

treatment.

Cell-surface-bound VEGFR-2 and D2DR were also measured

with and without dopamine or VEGF treatment via biotinylation.

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Fig. 1. Effect of dopamine and quinpirole pretreatmenton VEGF-induced specific tyrosine phosphorylation ofVEGFR-2. A significant decrease in VEGF-inducedphosphorylation of VEGFR-2 at Y1175, Y996, Y951 andY1059 is seen in HUVECs pretreated with eitherdopamine (10 μM) or quinpirole (10 μM) for 15 minutesbefore VEGF (10 ng/ml) treatment. Con, HUVECswithout any VEGF or dopamine treatment; +V, HUVECstreated with only VEGF (10 ng/ml) for 10 minutes; D-V,HUVECs pretreated with only 10 μM dopamine for 15minutes; D+V, HUVECs pretreated with 10 μMdopamine for 15 minutes and then treated with VEGF(10 ng/ml) for 10 minutes; Q-V, HUVECs treated withonly 10 μM quinpirole for 15 minutes; Q+V, HUVECspretreated with 10 μM quinpirole for 15 minutes and thentreated with VEGF (10 ng/ml) for 10 minutes. TotalVEGFR-2 was used as the loading control. Results fromthe blots are summarized graphically on the right. Foldchange for each treatment in the blots is quantified byNIH image densitometry and is shown in the bar graph.The figures are representative of three separateexperiments with similar results.

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3387SHP-2 in D2DR and VEGFR-2 crosstalk

As shown in Fig. 2E, after the addition of VEGF, the

concentration of biotinylated VEGFR-2 decreased. However,

upon dopamine pretreatment, increased VEGFR-2 and decreased

D2DR levels were observed at the cell surface (Fig. 2E). Fig. 2E

also indicated an increased surface recruitment of SHP-2 upon

dopamine pretreatment followed by VEGF induction for 10

minutes.

Knockdown of SHP-2 interferes with the inhibitory role ofdopamine towards VEGFR-2 phosphorylationPrevious reports (Gallicchio et al., 2005; Mitola et al., 2006) showed

that the tyrosine phosphatase SHP-2 limited VEGF-induced

VEGFR-2 activation, and we observed an increased association of

SHP-2 with both VEGFR-2 and D2DR in HUVECs after dopamine

treatment. We used SHP-2 siRNA to knock down SHP-2 and

Fig. 2. (A) D2DR coprecipitates with VEGFR-2 and SHP-2.Serum-starved (0.1% serum, for 24 hours) HUVECs werepretreated with dopamine (10 μM) for 15 minutes before VEGF(10 ng/ml) stimulation and then the lysates wereimmunoprecipitated with D2DR antibody and immunoblottedwith antibodies against VEGFR-2 and SHP-2. Con, HUVECswithout VEGF or dopamine treatment; +V5, HUVECs treatedwith only VEGF (10 ng/ml) for 5 minutes; +V10, HUVECstreated with only VEGF (10 ng/ml) for 10 minutes; D+V5,HUVEC pretreated with 10 μM dopamine for 15 minutes andthen treated with VEGF (10 ng/ml) for 5 minutes; D+V10,HUVECs pretreated with 10 μM dopamine for 15 minutes andthen treated with VEGF (10 ng/ml) for 10 minutes. The figuresare representative of three separate experiments with similarresults. Results from the blots are summarized graphically on theright. (B) D2DR colocalizes with VEGFR-2. Serum-starvedHUVECs were pretreated with dopamine for 15 minutes and thenstimulated with VEGF (10 ng/ml) for 10 minutes and stained withD2DR (green) and VEGFR-2 (red) antibodies. The D2DRlocalizes with VEGFR-2 at the cell surface. The intensity ofcomplex formation is shown in the lower chamber (arrow).(a) VEGFR-2 and D2DR colocalize at the cell surface without anyVEGF or dopamine treatment. (b) Colocalization of VEGFR-2and D2DR decreases with VEGF stimulation. (c) Pretreatmentwith dopamine followed by VEGF induction results in an increasein the colocalization of D2DR and VEGFR-2 at the cell surface.The figures are representative of three separate experiments withsimilar results. Quantification of surface colocalization is shownin bar graph on the right (mean ± s.d.). (C) D2DR colocalizes withSHP-2. Serum-starved HUVECs were pretreated with dopaminefor 15 minutes and then stimulated with VEGF (10 ng/ml) for 10minutes and stained with D2DR (green) and SHP-2 (red)antibodies. The intensity of complex formation is shown in thelower chamber (arrowhead). (a) Intense D2DR and minor SHP-2staining were observed at the cell surface without any VEGF ordopamine treatment. (b) Upon VEGF induction, SHP-2translocated more from the cytosol to the cell surface andlocalized with D2DR. (c) Pretreatment with dopamine, followedby VEGF induction, leads to a marked increase in colocalizationof D2DR with SHP-2 at the cell surface. The figures arerepresentative of three separate experiments with similar results.Quantification of surface colocalization is shown in bar graph onthe right (mean ± s.d.). (D) Cofractionation of VEGFR-2 andSHP-2 in the light density membrane fraction of HUVECs. Celllight density membrane fractions were purified using thehyperosmotic carbonate method. Equal volumes of each fractionwere separated by SDS-PAGE electrophoresis, immunoblotted,and tested for VEGFR-2 and SHP-2. Presence of D2DR wasmonitored by immunoprecipitation with an anti-D2DR antibody.Dopamine pretreatment causes increased colocalization ofVEGFR-2 and SHP-2 to the low-density domain. (E) Effect ofdopamine treatment on biotinylated VEGFR-2 and D2DR.Serum-starved HUVECs were pretreated with dopamine for 15minutes, then stimulated with VEGF (10 ng/ml) for 10 minutes,and then subjected to cell surface biotinylation following themanufacturer’s instructions. After VEGF induction, lessbiotinylated VEGFR-2 is detected on the cell surface. However,with dopamine pretreatment, increased levels of VEGFR-2 anddecreased levels of D2DR are found. From the biotinylationexperiment, cell-surface-bound proteins were also recorded.VEGF induction increases surface recruitment of SHP-2 proteinand dopamine pretreatment significantly enhances thislocalization.

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observed the effect of dopamine on VEGF-induced phosphorylation

of VEGFR-2. We found that dopamine could not block the VEGF-

induced phosphorylation of the tyrosine residues Y951, Y996 and

Y1059 in SHP-2 siRNA-treated HUVECs (Fig. 3), but dopamine-

mediated dephosphorylation of Y1175 was not affected by SHP-2

knockdown (Fig. 3). Our data suggest that dopamine selectively

regulates the dephosphorylation of VEGFR-2 Y951, Y996 and

Y1059, but not Y1175, by engaging the protein tyrosine phosphatase

SHP-2.

SHP-2 phosphorylation and phosphatase activity upondopamine pretreatment in HUVECsCatalytic activity of SHP-2 is crucial for its ability to modulate

different signaling cascades. Previous reports have shown that the

phosphatase activity of SHP-2 increases upon tyrosine

phosphorylation (Tang et al., 1999; Vogel et al., 1993). There are

two important phosphorylation sites in SHP-2, Y542 and Y580

(Bennett et al., 1994), and the phosphorylation at Y542 (the major

phosphorylation site) (Bennett et al., 1994) has been proposed to

relieve the basal inhibition of SHP-2 phosphatase activity (Lu et

al., 2001). Compared with untreated cells, the total phosphorylation

of SHP-2 significantly increased in cells treated with VEGF for

both 5 and 10 minutes (Fig. 4A). When pretreated with dopamine,

the phosphorylation of SHP-2 was even higher than that observed

in cells treated with VEGF alone (Fig. 4A), translating into a 1.5-

fold upregulation in the VEGF-induced phosphorylation of Y542

(data not shown). Therefore, dopamine treatment promotes the

VEGF-induced total and Y542 phosphorylation of SHP-2. We did

not observe any effect of dopamine on the phosphorylation of SHP-

2 at Y580. Total phosphorylation of VEGFR-2 at these time points

is also indicated (Fig. 4A, upper panel; supplementary material Fig.

S3).

We also measured the SHP-2 phosphatase activity in response

to dopamine. As shown in Fig. 4B, SHP-2 phosphatase activity was

markedly increased (P=0.01) after a 15 minute dopamine

pretreatment when compared with VEGF or dopamine treatment

alone. In summary, dopamine treatment in endothelial cells resulted

in an upregulation of VEGF-induced phosphorylation of SHP-2,

increased phosphatase activity and decreased phosphorylation of

VEGFR-2 at Y951, Y996 and Y1059.

Role of Fyn in dopamine-mediated regulation of SHP-2 andVEGFR-2 phosphorylationNext, we looked at whether Fyn kinase or Src tyrosine kinase was

involved in phosphorylation of SHP-2 or VEGFR-2. There have

been reports describing a role for Fyn kinase upstream of SHP-2

and in the regulation of SHP-2 phosphorylation and phosphatase

activity (Samayawardhena et al., 2006; Tang et al., 1999). We first

examined the status of Fyn phosphorylation in HUVECs in the

presence or absence of dopamine and VEGF treatment. Dopamine

pretreatment in HUVECs did not cause any change in VEGF-

induced Fyn phosphorylation, and thus, its kinase activity

(supplementary material Fig. S5A). To rule out the role of Fyn in

dopamine-mediated regulation of VEGFR-2 dephosphorylation, we

then transfected HUVECs with Fyn siRNA. This knockdown of

Fyn did not affect the phosphorylation of SHP-2 at Y542 nor on

the phosphorylation of VEGFR-2 at Y1175 and Y951 after

dopamine pretreatment and VEGF stimulation (supplementary

material Fig. S5B).

Src regulates the phosphorylation SHP-2 at Y542HUVECs were pretreated with either 5 μM PP2 (a Src tyrosine

kinase inhibitor) (Bhattacharya et al., 2008a) or PP3 (an inactive

analog of PP2) for 1 hour in the presence or absence of dopamine

and VEGF. Cells were also pretreated with 10 μM SU6656 (a Src

tyrosine kinase inhibitor) for 1 hour before treatment with either

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Fig. 3. Effect of dopamine on VEGFR-2 phosphorylation in SHP-2-knockdown cells. HUVECs were transfected with a scrambled control (Consi)or SHP-2 siRNA (SHP-2si) using Oligofectamine. After 48 hours, cells wereserum-starved and pretreated with dopamine for 15 minutes and thenstimulated with VEGF for 10 minutes. In SHP-2-knockdown cells, dopaminecannot inhibit the VEGF-induced phosphorylation of VEGFR-2 at Y951,Y996 and Y1059, but successfully blocks Y1175 phosphorylation. TotalVEGFR-2 was used as the loading control. The figures are representative ofthree separate experiments with similar results.

Fig. 4. Effect of dopamine on SHP-2 phosphorylation and phosphataseactivity. (A) Dopamine leads to an increase in VEGF-induced tyrosinephosphorylation of SHP-2. Serum-starved HUVEC were pretreated withdopamine (10 μM) for 15 minutes before VEGF (10 ng/ml) treatment for 5 or10 minutes. Cell lysates were then immunoprecipitated with the Tyr-Pantibody and immunoblotted with antibodies against VEGFR-2 and SHP-2.The figures are representative of three separate experiments with similarresults. (B) SHP-2 phosphatase assay using pNPP. Serum-starved HUVECswere pretreated with 10 μM dopamine for 15 minutes followed by treatmentwith VEGF (10 ng/ml) for 10 minutes. Lysates were then immunoprecipitatedwith antibody against SHP-2. One half was run on a gel and the other half usedfor the phosphatase assay. SHP-2 phosphatase activity is markedly increased(P=0.01) after dopamine pretreatment, when compared with VEGF treatmentalone. Data represent an average of three independent determinations (± s.d.)normalized against the negative control.

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3389SHP-2 in D2DR and VEGFR-2 crosstalk

dopamine or VEGF. Without PP2, PP3 or SU6656 treatment, we

observed significant VEGF-induced phosphorylation of SHP-2 at

Y542 in the presence or absence of dopamine (Fig. 5A). Fig. 5A

clearly shows that VEGF-induced phosphorylation of SHP-2 at

Y542 was inhibited with PP2 treatment but not with PP3 even in

dopamine-pretreated cells. VEGF stimulated phosphorylation of

SHP-2 at Y542 was also blocked by the Src inhibitor SU6656 in

the presence or absence of dopamine (supplementary material Fig.

S4A). To further support our data we also checked the

phosphorylation of SHP-2 at Y542 after Src knockdown with

siRNA. We observed decreased phosphorylation of SHP-2 at Y542

after 24 hours of siRNA treatment with or without VEGF or

dopamine treatment (supplementary material Fig. S4B). These

findings suggest that although Src kinase is downstream of SHP-

2, it regulates the phosphorylation of SHP-2 in a feedback

mechanism that is independent of dopamine pretreatment.

Effect of dopamine on Src phosphorylation at Y418We have already shown (Bhattacharya et al., 2008b) that dopamine

inhibits the association of VEGFR-2 and Src by decreasing VEGFR-

2 phosphorylation, and that this resulted in impaired Src

phosphorylation at Y418 (the activation domain). We found that

after SHP-2 knockdown, the basal level and the VEGF-induced

phosphorylation of Src at Y418 increased compared with levels in

control siRNA-treated cells (Fig. 5B). Dopamine pretreatment led

to a significant upregulation of phosphorylation of Src at Y418 in

SHP-2-knockout cells (Fig. 5B). Therefore, we concluded that SHP-

2 has a prominent role in dopamine-mediated phosphorylation of

VEGFR-2, which ultimately affects Src phosphorylation at Y418

and its kinase activity.

Effect of dopamine on migration in SHP-2-knockdownHUVECsPhosphorylation of different tyrosine residues on VEGFR-2 results

in the activation of different signaling pathways. VEGF-dependent

phosphorylation at Y1175 leads to DNA synthesis and endothelial

cell proliferation (Sakurai et al., 2005). Phosphorylation of Y951

and Y1059 are essential for VEGF-induced migration and

proliferation in HUVECs (Zeng et al., 2001). Here, we show that

dopamine was unable to block the VEGF-induced Y951

phosphorylation in SHP-2-knockdown cells (Fig. 3). Because

VEGF-induced Y951 phosphorylation is responsible for HUVEC

migration (Zeng et al., 2001), we performed a Boyden chamber

migration assay with SHP-2 siRNA-transfected HUVEC cells in

the presence or absence of dopamine and VEGF treatments. SHP-

2 knockdown in HUVECs led to an increase in the basal level and

the VEGF-induced level of cell migration compared with levels in

control cells (Fig. 6). Dopamine significantly inhibited the VEGF-

induced HUVEC migration (P<0.001) (Fig. 6) in control siRNA

treated cells. However, dopamine could not affect the VEGF-

induced migration (P=0.304) in SHP-2-knockdown cells (Fig. 6).

Collectively, these data suggest that through SHP-2, dopamine

modulates the phosphorylation of VEGFR-2 at Y951, Y996 and

Y1059, but not at Y1175. The phosphorylation of VEGFR-2 at Y951

regulates Src kinase activity and thereby influences VEGF-mediated

migration in endothelial cells (Fig. 7).

DiscussionDopamine and its related molecules have been reported to inhibit

different types of tumor growth in mouse models (Basu and

Dasgupta, 2000; Sarkar et al., 2008; Wick, 1978; Wick, 1979). This

probably occurs through the inhibition of angiogenesis, because we

have previously shown that dopamine can inhibit VEGF-regulated

angiogenesis in vitro and in vivo (Basu et al., 2001). Furthermore,

a pharmacological dose of dopamine can inhibit VEGF-mediated

microvascular permeability, proliferation and migration of

endothelial cells (Basu et al., 2001; Bhattacharya et al., 2008b). It

has also been shown that the neurotransmitter dopamine, when

acting through the D2 receptor, blocks VEGF-induced focal

Fig. 5. (A) Effect of PP2 and PP3 on SHP-2 Y542 phosphorylation. Serum-starved HUVECs were pretreated with PP2 or PP3 (5 μM) for 1 hour beforetreatment with either 10 μM dopamine or VEGF (10 ng/ml). PP2, a Src kinaseinhibitor, significantly inhibits phosphorylation of SHP-2 at Y542. Total SHP-2 was used as a loading control. The phosphorylation of Src at Y418 issignificantly inhibited by treatment with 5 μM PP2. The figures arerepresentative of three separate experiments with similar results. (B) Effect ofdopamine on Src phosphorylation at Y418 after SHP-2 knockdown. 48 hoursafter siRNA transfection, HUVECs were serum starved and pretreated withdopamine for 15 minutes, followed by VEGF stimulation for 10 minutes.Dopamine pretreatment does not inhibit Src phosphorylation at Y418 in theabsence of SHP-2. β-actin was used as a loading control. The figures arerepresentative of three separate experiments with similar results.

Fig. 6. Effect of dopamine on migration in SHP-2-knockdown HUVECs. Cellswere transfected with the scrambled control or SHP-2 siRNA usingOligofectamine. After 48 hours, cells were serum starved and a migrationexperiment was carried out as described in the Materials and Methods. AfterSHP-2 knockdown, dopamine does not inhibit the VEGF-induced HUVECmigration. Data represent an average of three independent determinations (±s.d.), each in triplicate.

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adhesion kinase (FAK) and mitogen-activated protein kinase

(MAPK) phosphorylation in endothelial cells (Sarkar et al., 2004).

Recently, it has been reported that dopamine, by acting through

D2DR, can regulate the mobilization of bone-marrow-derived

endothelial progenitor cells and tumor growth (Chakroborty et al.,

2008). However, the basic molecular mechanism involved in this

regulation is still unclear. Moreover, phosphorylation of different

VEGFR-2 tyrosine residues lead to activation of different

downstream signaling pathways (Sakurai et al., 2005; Zeng et al.,

2001). It is therefore important to understand the molecular

mechanism by which dopamine regulates the phosphorylation of

each residue. Our aim was to define the crucial molecular signaling

partners engaged by dopamine to bring about the inhibition of

VEGF-induced VEGFR-2 phosphorylation and the subsequent

blockade of angiogenesis.

The involvement of different phosphatases, including DEP-1,

SHP-1 and SHP-2 have been described in the regulation of VEGF-

induced VEGFR-2 phosphorylation and subsequent downstream

signaling (Bhattacharya et al., 2008a; Gallicchio et al., 2005; Grazia

Lampugnani et al., 2003; Mitola et al., 2006; Seo et al., 2003). In

mammalian cells, there are two tyrosine phosphatases containing

SH2 domains, SHP-1 and SHP-2. However, unlike SHP-1, SHP-2

is ubiquitously expressed. The SH2 domains of SHP-2 target this

enzyme to the phosphotyrosine residues of a variety of growth factor

receptors and other signaling molecules (Neel et al., 2003). Here,

we have delineated the unique role of SHP-2 phosphatase, recruited

by D2DR in the dopamine-regulated dephosphorylation of VEGFR-

2 and this regulation is a time-dependent phenomenon. With

immunoprecipitation experiments, we showed increased association

of VEGFR-2 with D2DR upon dopamine pretreatment, but we could

not detect any change in the association of SHP-2 with D2DR and

VEGFR-2 in the presence or absence of dopamine by this method.

With confocal microscopy, it was evident that dopamine

pretreatment followed by VEGF induction significantly enhanced

the translocation of SHP-2 from the cytoplasm to the cell surface,

and its association with D2DR on the surface. However, because

immunoprecipitation was performed with whole-cell lysates and

because confocal microscopy only highlights changes at the cell

surface, it is not possible to draw a direct correlation between the

immunoprecipitation and confocal data.

Cell-surface biotinylation also confirms increased recruitment of

SHP-2 to the cell surface, and decreased VEGFR-2 internalization

upon dopamine pretreatment. From sucrose density gradient

experiments, we showed that VEGFR-2 and SHP-2 are in the same

lipid-enriched membrane fraction and we found that both VEGFR-

2 and SHP-2 were enriched in this fraction upon dopamine

pretreatment. Further studies will define the recruitment mechanism

of SHP-2 into the D2DR complex and the proper

compartmentalization that leads to its phosphatase function.

We propose that after being engaged by D2DR, SHP-2 becomes

phosphorylated and activated. The active SHP-2 then targets the

specific tyrosine residues on VEGFR-2, which renders the

dissociation of VEGFR-2 from D2DR; however, SHP-2 is not

responsible for the dopamine-mediated dephosphorylation of

VEGFR-2 at Y1175. Therefore, we speculate that other

phosphatases are involved in this dopamine-mediated differential

regulation of VEGFR-2 phosphorylation. We have previously

reported that dopamine regulates VEGFR-2 phosphorylation, which

in turn controls Src kinase activity (Bhattacharya et al., 2008b). It

has also been reported that phosphorylation of VEGFR-2 at Y951

allows for the binding and tyrosine phosphorylation of T-cell-

specific adapter (TSAd) (Matsumoto et al., 2005). TSAd then binds

to the cytoplasmic tyrosine kinase Src and regulates the migration

of endothelial cells in response to VEGF (Matsumoto et al., 2005).

After knockdown of SHP-2, dopamine could not inhibit the

VEGFR-2 phosphorylation at Y951, downstream Src

phosphorylation or Src kinase activity: thus HUVEC migration is

permitted. From this data, we infer that SHP-2 has a crucial role

in the dopamine-modulated inhibition of VEGF-induced endothelial

cell migration.

Journal of Cell Science 122 (18)

Fig. 7. Model for dopamine-mediatedregulation of VEGFR-2 phosphorylationby recruiting SHP-2. (A) In untreatedHUVECs, D2DR and VEGFR-2 remainassociated with each other. (B) UponVEGF induction, SHP-2 translocatesfrom the cytosol to the cell surface andbecomes associated with both VEGFR-2and D2DR. However, VEGF treatmentpromotes the dissociation of VEGFR-2from D2DR to subsequently induceVEGFR-2 phosphorylation anddownstream signaling. (C) Dopaminepretreatment, followed by VEGFstimulation for 10 minutes, leads to anincrease in the association of D2DR withVEGFR-2. Dopamine treatment alsoinduces an increased association betweenSHP-2 and D2DR at the cell surface andstimulates the phosphorylation of SHP-2and its phosphatase activity. Active SHP-2 then inhibits the phosphorylation ofVEGFR-2 at Y951, Y996 and Y1059,but not at Y1175. Decreasedphosphorylation of VEGFR-2 at Y951leads to a subsequent decrease in Srcphosphorylation at Y418 and its kinaseactivity, effectively blocking VEGF-induced migration.

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3391SHP-2 in D2DR and VEGFR-2 crosstalk

We were also interested in determining how SHP-2 activity is

regulated after dopamine challenge. Although previous reports

suggest that Fyn kinase is upstream of SHP-2 (Samayawardhena

et al., 2006; Tang et al., 1999), dopamine pretreatment did not

alter Fyn phosphorylation, and the knockdown of Fyn by siRNA

in HUVECs did not show any effect on the phosphorylation of

SHP-2 at Y542. However, inhibition of Src kinase activity by PP2

or SU6656 caused a significant reduction in the phosphorylation

of SHP-2 at Y542, with or without dopamine pretreatment. We

also found inhibition of SHP-2 phosphorylation at Y542 with or

without VEGF or dopamine treatment using siRNA against Src.

Therefore, Src kinase is probably responsible for the control of

SHP-2 phosphorylation in a feedback mechanism after both

VEGF and dopamine treatment. Additional in-depth studies are

ongoing to elucidate the feedback loop between SHP-2 and Src

in this context.

Our current hypothesis derived from our data is presented in

Fig. 7. We propose that D2DR and VEGFR-2 remain associated

with each other at the cell surface. Upon VEGF induction, SHP-

2 translocates from the cytosol to the cell surface and becomes

associated with both VEGFR-2 and D2DR. VEGF treatment also

promotes the dissociation of VEGFR-2 from D2DR and induces

VEGFR-2 phosphorylation and downstream signaling. Dopamine

pretreatment prevents VEGFR-2 internalization, resulting in an

increase association between D2DR with VEGFR-2. Dopamine

treatment also induces an increased association between SHP-2

and D2DR at the cell surface and stimulates the phosphorylation

of SHP-2 and its phosphatase activity. Active SHP-2 then inhibits

the phosphorylation of VEGFR-2 at Y951, Y996 and Y1059, but

not at Y1175. Decreased phosphorylation of VEGFR-2 at Y951

leads to subsequent decrease in the phosphorylation of Src at Y418

and its kinase activity, and that in turn, blocks VEGF-induced

migration.

Overall, our results provide a novel mechanistic insight into the

regulatory role of SHP-2 as a key mediator in the dopamine-

regulated D2DR and VEGFR-2 crosstalk and ultimately,

angiogenesis.

Materials and MethodsReagentsVEGF-A was obtained from R&D Systems, Minneapolis, MN. The antibodies to

VEGFR-2 (sc-504), SHP-2 (sc-280), D2DR (sc-7522), Src (sc-5266), VEGFR-2(996)-

P (sc-16629) and VEGFR-2(951)-P (sc-16628) were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA); VEGFR-2(1059)-P, Tyr-P 4G10 and Src antibodies

were purchased from Upstate. Antibodies against VEGFR-2(1175)-P (cat. no. 2478),

SHP-2(542)-P (3751), Fyn (4023) and Src(527)-P (2105) were purchased from Cell

Signaling. The Src(Tyr418-P) antibody was from BioSource International.Antibody

SHP-2 and β-actin were from BD Biosciences. siRNAs against SHP-2 (sc-36488)

and Fyn (sc-29321) were from Santa Cruz Biotechnology. siRNA against Src was

from Dharmacon (Lafayette, CO). PP2, PP3 and SU6656 were from EMD Biosciences

(San Diego, CA). Dopamine hydrochloride, quinpirole and all other chemicals not

listed were from Sigma.

Immunoprecipitation and western blot analysisSerum-starved (0.1% serum, for 24 hours) HUVECs were pretreated with 5 μM PP2

or PP3 or 10 μM SU6656 for 1 hour and then treated either with 10 μM dopamine

or quinpirole for 15 minutes before treatment with VEGF (10 ng/ml) for 5 or 10

minutes. Whole-cell lysates in RIPA buffer supplemented with protease inhibitor

cocktail and with or without phosphatase inhibitor were prepared from HUVECs.

The lysates were collected after centrifugation at 14,000 g for 10 minutes at 4°C.

250 μg protein was incubated with 2 μg respective antibody for 1 hour and 50 μl of

proteinA/G-conjugated agarose beads overnight at 4°C. Beads were washed with RIPA

buffer three times and immunoprecipitates were resuspended in SDS sample buffer

and electrophoresis was performed. All signals were detected using the

chemiluminescent detection method according to the manufacturer’s protocol

(Amersham, UK) and quantified using the NIH Image densitometry software.

Experiments were repeated at least three times.

Cell fractionationThe separation of light and heavy membrane fractions from HUVECs was performed

as described (Song et al., 1996). Briefly, the serum-starved HUVECs were pretreated

with 10 μM dopamine for 15 minutes before treatment with VEGF (10 ng/ml) for

10 minutes, and then cells were suspended in 2 ml of 0.5 M sodium carbonate buffer

(pH, 11.0) containing phosphatase and protease inhibitor. The cell suspension was

homogenized, adjusted to 45% sucrose by the addition of 2 ml of 90% sucrose prepared

in MBS buffer, and placed into ultracentrifuge tubes. A 5-45% discontinuous sucrose

gradient was then formed on top by the addition of 35% and 5% sucrose in MBS

buffer (4 ml each). The samples were centrifuged at 40,000 r.p.m. for 18 hours in a

SW41-Ti rotor (Beckman Instruments; Palo Alto, CA). The fractions were collected

from the top in 1 ml amounts. Equal volumes of each fraction from each sample

were then separated by SDS-PAGE, and transferred to nitrocellulose membranes.

The membranes were immunoblotted with the desired antibodies. All signals were

detected by the chemiluminescent detection method according to the manufacturer’s

protocol (Amersham).

Cell-surface biotinylationSerum-starved HUVECs were pretreated with 10 μM dopamine for 15 minutes before

treatment with VEGF (10 ng/ml) for 10 minutes. The biotinylation experiment was

performed without permeabilization at 4°C, following the manufacturer’s protocol

(Pierce, Rockford, IL). After VEGF or dopamine treatment, the cells were lysed with

a mild detergent and then cell surface biotinylated proteins were isolated with

immobilized neutravidin gel. The bound proteins released by incubating the resin

with SDS-PAGE sample buffer containing 50 mM DTT.

siRNA transfection1�105 HUVECs were seeded in 60 mm plates and cultured for 24 hours in EGM.

The next day, cells were washed with OPTI-MEM reduced-serum medium and

transfected with 50 nM SHP-2 or Fyn siRNA and 100 nM Src siRNA using

Oligofectamine (Invitrogen). After 4 hours, antibiotic-free EGM was added and cell

lysates were prepared 48 or 24 hours after transfection.

ImmunofluorescenceThe anti-VEGFR-2 monoclonal antibody used for immunofluorescence was from

Sigma and the anti-SHP-2 antibody was from BD Biosciences (San Jose, CA). Alexa

Fluor 488 anti-goat (green) or Alexa Fluor 647 anti-mouse (red) secondary antibodies

were purchased from Molecular Probes (Eugene, OR). 2�104 HUVECs were seeded

on collagen-coated Lab-Tek chamber slides. After 24 hours, cells were serum starved.

The next day, cells were pretreated with 10 μM of dopamine before induction with

VEGF (10 ng/ml). Further steps were carried out at 4°C. Slides were washed in PBS

and fixed in 4% paraformaldehyde (PFA) without permeabilization. Slides were

washed in PBS, blocked in 10% goat serum, and stained with the respective primary

antibody in 1% goat serum for 2 hours. Slides were then washed in PBS and incubated

for 1 hour with the 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-scanning microscope

with a C-Apochromat 100� NA 1.4 oil-immersion lens. Absence of signal crossover

was established using single-labeled samples and quantification of colocalization was

done using the Axio KS400 software (�15 cells/group).

SHP-2 phosphatase assayThe phosphatase assay was performed according to the manufacturer’s protocol

(Stratagene, Signal Scout Phosphatase Profiling System). Briefly, starved HUVECs

were pretreated for 15 minutes with or without dopamine and then with or without

10 ng/ml VEGF for 10 minutes. Cells were lysed (100 mM NaCl, 10 mM Tris-HCl,

pH 8.0, 1 mM EDTA, 1% Triton X-100, 5 mM DTT) and the resulting lysates were

precleared; protein concentration was determined using the Bradford assay. An equal

amount of protein from each sample was then immunoprecipitated with an SHP-2

antibody. One part was then subjected to western blot and another part was treated

as follows: 80 μl 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 μl pNPP

substrate (20 mM) was then added to each sample and absorbance at 405 nm was

recorded on a Tecan SpectraFluor Plus after a 30 minute incubation at 30°C. Data

presented were normalized with respect to the negative control. Three independent

experiments were performed.

Migration assay5�104 SHP-2 siRNA-treated serum-starved HUVEC cells were seeded into collagen-

coated Transwell chambers with a diameter of 6.5 mm and a pore size of 8 μm

(Corning CoStar Corporation, Cambridge, MA), which were then inserted into 24-

well plates containing serum-starved EGM. After incubation at 37°C for 1 hour, 10

μM dopamine was added to the cells in the Transwells, which were then incubated

for a further 15 minutes. VEGF was then added to a final concentration of 10 ng/ml

in the lower chamber. Following incubation for 4 hours at 37°C in a CO2 incubator,

cells that remained in the upper chamber were gently removed with a cotton swab.

Cells that had invaded through the filter were fixed in 4% PFA and stained with 0.2%

crystal violet dissolved in 2% ethanol. Migration was quantified by counting the

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number of cells on the filter using bright-field optics with a Nikon Diaphotmicroscope equipped with a 16-square reticule (1 mm2). Four separate fields werecounted for each filter. An average of three separate experiments was recorded.

Statistical analysisAll values are expressed as means ± s.d. Statistical significance was determined usingthe two-sided Student’s t-test and a value of P�0.05 was considered significant. Errorbars are given on the basis of calculated s.d. values.

We would like to thank Jim Tarara for help with the confocalmicroscopy experiments and Julie Lau for discussions. This work ispartly supported by NIH grants HL70567, HL72178 and CA78383,and also a grant from American Cancer Society to D.M. Deposited inPMC for release after 12 months.

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