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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2548.)
© 2005 American Heart Association, Inc.

Vascular Biology

Role of Focal Adhesion Kinase in Flow-Induced Dilation of Coronary Arterioles

Ryoji Koshida; Petra Rocic; Shuichi Saito; Takahiko Kiyooka; Cuihua Zhang; William M. Chilian

From the Department of Physiology, Louisiana State University Health Sciences Center, New Orleans.

Correspondence to William M. Chilian, PhD, Louisiana State University, 1201 Perdido St, New Orleans, LA 70112. E-mail chilian{at}lsuhsc.edu

	Abstract

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Backgound— Flow-induced regulation of endothelial NO synthase (eNOS) depends on integrin signaling and tyrosine kinase activation. Integrins cluster in focal adhesion complexes, where the extracellular matrix is connected to the cytoskeleton and where focal adhesion kinase (FAK) is located. FAK plays a central role in integrin signaling and Src activation. Accordingly, we hypothesized that FAK plays an important role in flow-induced dilation (FID).

Methods and Results— To inactivate FAK-dependent signaling, anti-FAK, phosphospecific (Tyr³⁹⁷) antibody (FAKab), which binds against the FAK autophosphorylation site, was incorporated into endothelium of rat coronary arterioles using liposomal transfection. The responses to flow, acetylcholine (Ach), or the NO donor MAHAMANONOate (NOC-9) were observed before and after FAKab. In control and vehicles (denatured antibody or transfecting reagent alone), flow produced progressive dilation to a maximal value of 35% increase in diameter, which was inhibited by N-nitro-L-arginine methyl ester (L-NAME). However, FAKab prevented FID (P<0.01 versus control). Combined treatment with FAKab and L-NAME did not produce inhibition greater than FAKab alone. FAKab did not blunt Ach- or NOC-9–induced dilation. Western analysis demonstrated that FAKab prevented flow-induced phosphorylation of FAK (pY397-FAK), Akt (pS473-Akt), and eNOS (pS1179-eNOS).

Conclusion— Our study demonstrates the pivotal role of FAK in NO-mediated FID. Inhibition of FAK signaling with FAKab impaired FID and phosphorylation of Akt and eNOS. Our data suggest that the activation of FAK is central to the mechanotransduction of FID via regulation of activation of Akt and eNOS.

Although NO-mediated flow-induced dilation (FID) has been observed by many groups, the signal transduction pathway is still not totally resolved. We sought to determine the role of focal adhesion kinase in FID by loading endothelial cells of intact, isolated coronary resistance vessels with an anti-FAK that prevented its downstream signaling. Anti-FAK blocked FID but not dilation to endothelium-dependent and -independent agonists. Anti-FAK also prevented the flow-dependent phosphorylation of Akt and eNOS, 2 enzymes essential for FID. We conclude that FAK plays a pivotal role in NO-mediated FID.

Key Words: coronary microcirculation • resistance vessels • nitric oxide • Akt

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fluid shear stress is one of the main physiological stimuli that regulates function of vascular endothelial cells (ECs).¹ The effect of shear stress on vascular caliber is also termed flow-induced dilation (FID) or shear stress–induced dilation.^2,3 In the absence of endothelial dysfunction or cardiovascular disease, FID in coronary arteries and arterioles is mediated by the production of NO from endothelial NO synthase (eNOS).^4,5 Integrin signaling plays an important role in flow-induced regulation of eNOS.⁶ Previous work from our laboratory demonstrated that inhibition of integrin signaling using ß₃ integrin antibody or an antagonist to the matrix–integrin binding site (RGD peptide) attenuated FID and tyrosine phosphorylation of ECs in coronary arterioles.⁶ Src activation is also important in flow-induced eNOS regulation.^7,8 In cultured ECs, pharmacological inactivation of Src reduced the phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, and eNOS.^8,9 In addition, we reported that inhibition of tyrosine kinases compromises FID.^6,10 Also critical to this integrin-activated pathway is the cytoskeleton to which the integrins are coupled, and along this line, disruption of the cytoskeleton was found to impair flow-dependent dilation.¹¹

Although the roles for integrins and the downstream kinase cascade (SrcPI3KAkteNOS) activated by shear stress are fairly well established,^12–17 the connection between integrin signaling and the downstream kinases has not been established in FID. Focal adhesion kinase (FAK) plays a central role in integrin signaling and Src activation.^18–20 FAK is a 125-kDa cytosolic protein nonreceptor tyrosine kinase localized in the focal adhesion complexes.²¹ The focal adhesion complexes also are points where integrins bind externally to extracellular matrix proteins and internally to cytoplasmic proteins that are bound to the cytoskeleton. Shear stress also activates and clusters integrins at focal adhesions, which results in rapid phosphorylation of FAK at Tyr³⁹⁷.^21–23 Tyr³⁹⁷ is the autophosphorylation site of FAK, and this phosphorylation event is essential for downstream signaling.^9,18,24–29 FAK activation generates a high-affinity binding site to the SH2 domain of Src family tyrosine kinases and causes recruitment and activation of Src.^18,19,30 FAK activation also leads to activation of PI3K, which binds to SH2 domain of FAK.^9,27,30–33 Accordingly, we hypothesized that FAK plays a critical role in FID and eNOS activation via the PI3K–Akt pathway. In the present study, we demonstrated that inhibition of FAK signaling prevents FID and phosphorylation of Akt and eNOS.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
General Preparations
Male Wister rats (125 to 175 g; Harlan; Indianapolis, Ind) were anesthetized (sodium pentobarbital; 100 mg/kg IP). The chest was opened, and the heart was removed and placed in chilled (4°C) physiological saline solution (PSS).³³

Isolation of Coronary Arterioles
Coronary arterioles (75 to 128 µm) were dissected from the interventricular septum. Arterioles were cannulated with glass micropipettes (tip diameter 4565 µm), which are connected to hydrostatic reservoirs filled with PSS with 1 g/100 mL of BSA. Arterioles were pressurized at 60 mm Hg and incubated with PSS to develop spontaneous tone. Changes in diameter of arterioles were measured by videomicroscopy.^34–38

Incorporation of Antibody Into Endothelium
To inactivate FAK, we incorporated FAKab into endothelium of coronary arterioles³⁸ using polycationic liposome transfection reagent Lipofectamine 2000 (Lipo2000; Invitrogen). The FAKab/Lipo2000 complex was applied intraluminally.

Measurement of Vasodilatation
After development of spontaneous tone, responses to flow (generated by producing a pressure drop across the arterioles by raising and lowering reservoirs connected to the inflow and outflow pipettes in equal but opposite directions [P=4, 10, 20, 40, or 60 cm H₂O]), to acetylcholine (Ach; 1x10^–8 to 1x10^–5 mol/L), and to MAHAMANONOate (NOC-9; 1x10^–8 to 1x10^–5 mol/L) were measured before and after N-nitro-L-arginine methyl ester (L-NAME), denatured antibody (DN-ab), and Lipo2000 without FAKab or FAKab. Percent dilation was calculated as the percentage increase in diameter from basal. In this regard, because the vessels developed spontaneous tone of 40% on average (40% reduction in diameter from passive state), dilation of 40% would be maximal.

Shear stress () is calculated from the equation where =viscosity, v=velocity of flow, and r=radius of the arteriole: =4v/r.

In this paradigm, velocity of flow is derived from the P, which is linearly related to flow.

Western Blotting
Five groups were analyzed: control, flow, flow+FAKab, pressure, and pressure+FAKab. The duration of flow was 1 minute (generated from 60 cm H₂O of P). In pressurized groups, arterioles were pressurized at 60 mm Hg for 30 minutes after treatments. Six coronary arterioles were pooled to obtain sufficient amounts of protein. Western blotting was performed to assess the expressions of total FAK and Akt and phosphorylation of Tyr³⁹⁷-FAK, Ser⁴⁷³-Akt, and Ser¹¹⁷⁹-eNOS.^40,41 Densitometric analysis (band sizexintensity of the signal) were performed on the signals using a Bio-Rad Versadoc imaging system.

Statistical Analysis
Differences within and between groups were determined using 2-way ANOVA for repeated measures with Fisher’s post hoc test. Results are presented as mean±SD, and statistical significance was deemed to have been achieved when P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vasodilatory Responses
Coronary arterioles developed tone spontaneously in response to intraluminal pressure at 60 mm Hg (69±2% of the passive diameter). Administration of FAKab/Lipo2000 complex, Lipo2000 without antibody, or denatured FAKab/Lipo2000 had no effects on the baseline tone (69±3, 67±4, and 65±2%; P=0.96, 0.71, and 0.36 versus control). Calculated shear stress was 2.23±0.2, 4.64±0.41, 8.65±0.76, 16.68±1.47, and 24.71±2.17 dyne/cm² at P of 4, 10, 20, 40, and 60 cm H₂O, respectively.

Figure 1 shows the effect of NOS inhibition on FID. FID was significantly attenuated in the presence of L-NAME. This result shows that FID is largely mediated by NO production in rat coronary arterioles.

View larger version (22K): [in this window] [in a new window]   Figure 1. FID of rat coronary arterioles in the presence or absence of L-NAME (50 µmol/L; n=7). *P<0.01, control responses vs after NOS inhibition with L-NAME. L-NAME significantly attenuated FID of rat coronary arterioles. The baseline diameter (without flow) was not changed by L-NAME: 69±15 µm (control) vs 73±14 µm (L-NAME); mean±SD.

Figure 2 shows the results of the control vehicle experiments. Administration of lipofectamine alone (Figure 2A and 2B) or a combination of lipofectamine with DN-Ab (Figure 2C and 2D) did not influence dilation to flow or to Ach. Thus, endothelium-dependent vasodilation to flow or Ach was not affected in a nonspecific manner by our maneuvers or the introduction of protein into ECs.

View larger version (30K): [in this window] [in a new window]   Figure 2. Responses to flow and Ach in vehicle experiments. A, FID before and after lipofectamine treatment without FAKab (Lipo2000; n=6). B, Responses to Ach before and after treatments with lipofectamine alone (n=6). C, FID before and after incorporation with denatured FAKab (DN-Ab; n=6). D, Responses to Ach before and after incorporation with denatured FAKab (DN-Ab; n=6). Lipo2000 had no effects on flow (vs control; P=0.37) or on Ach-induced dilation (vs control; P=0.97). DN-Ab incorporation did not affect either FID (vs control; P=0.60) or Ach-induced dilation (vs control; P=0.59).

Figure 3 shows the effect of blocking FAK-dependent signaling (by the use of the FAKab) on FID and Ach-induced dilation. In control preparations, FID was graded and dependent on the level of flow (Figure 3A; maximal response 38±5% increase in diameter). Treatment with FAKab significantly attenuated FID (Figure 3A; maximal response 15±4%; P<0.01 versus control; n=10). Addition of L-NAME to the FAKab treatment (n=4) did not further reduce the dilation (data not shown). The percentage differences in dilation at P of 4, 10, 20, 40, and 60 cm H₂O between the 2 interventions were 1.1±0.6%, 0.6±0.4%, 0.22±0.2%, 0.5±0.3%, and 0.0±0.2%, respectively (all NS). Thus, even with the combination of L-NAME and FAKab, a component of shear stress–dependent dilation remained. Figure 3B also shows that Ach-induced dilation was not affected by the FAKab (P=0.437; n=10).

View larger version (14K): [in this window] [in a new window]   Figure 3. Responses to flow and Ach before and after FAK inhibition. A, FID before and after FAK inhibition (n=10). *P<0.01, control responses vs after FAK inhibition. FID was attenuated after FAK inhibition. B, Ach-induced dilation before and after FAK inhibition (n=10). The responses to Ach were preserved despite FAK inhibition.

Figure 4 shows that FAK inhibition had no effects on vasodilation to the NO donor NOC-9 (P=0.625; n=4), indicating that the incorporation of FAKab did not affect the dilation to NO. These data demonstrate that inhibition of FAK signaling prevents NO-mediated FID but not agonist (endothelium-dependent or -independent)-induced dilation.

View larger version (17K): [in this window] [in a new window]   Figure 4. Responses to NOC-9–induced dilation before and after FAK inhibition (n=4). FAK inhibition had no effects on NOC-9–induced dilation.

Phosphorylation of FAK, Akt, and eNOS
Western blotting for FAK and Akt protein and phosphorylated forms of FAK, Akt, and eNOS is shown in Figure 5. The signals for total Akt and FAK protein were virtually identical in all groups. However, flow increased pY397-FAK, pS473-Akt, and pS1179-eNOS above that in all other groups (Figure 5). Inhibition of FAK signaling by FAKab incorporation prevented flow-induced phosphorylation of FAK, Akt, and eNOS.

View larger version (68K): [in this window] [in a new window]   Figure 5. Western blot of rat coronary arterioles under nonstimulated, pressurized, and flow-stimulated conditions with or without FAK inhibition. Flow increased phosphorylation of FAK (pY397-FAK), of Akt (pS473-Akt), and eNOS (pS1179-eNOS). FAK inhibition (FAKab) markedly reduced flow-induced phosphorylation of FAK, Akt, and eNOS. The loading for total FAK and Akt were virtually identical in the different groups. The Table summarizes densitometric analysis of the signals for the phosphorylated proteins. The loading for total FAK and Akt were virtually identical in the different groups and are not summarized in the Table.

View this table: [in this window] [in a new window]   Densitometric Analysis of Protein Phosphorylation

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The new finding of the present study is that FAK plays a critical role in FID of rat coronary arterioles. We demonstrated that FID is impaired by inhibition of FAK signaling through the incorporation of an antibody directed against the autophosphorylation site of FAK into ECs. FAK inhibition also reduced flow-induced phosphorylation of Akt at Ser⁴⁷³ and eNOS at Ser¹¹⁷⁹, which is consistent with preventing flow-induced activation of Akt and eNOS. These results indicate that FAK is a key molecule involved in the mechanotransduction of shear stress that culminates in NO production.

In various organs and species, flow- or shear stress–induced vasodilation is an ubiquitous phenomenon in vessels ranging from tiny precapillary arterioles to large arteries.^1,35–39 In the absence of vascular disease and compromised endothelial function, NO is considered one of the most important effectors of FID.^4,5 Fluid shear stress plays an important role in the maintenance of the function and structure of the vessel.¹ Despite this importance, the mechanism of shear stress sensing, transduction, and signaling is not completely understood.

Integrin signaling and tyrosine kinase activation play important roles in flow-induced regulation of eNOS.^6,10,42–44 However, the link between these signaling events in FID has not been established, despite the observation that FAK plays a central role in integrin signaling and Src activation.^{8,18,19,23,45} Autophosphorylation of FAK at Tyr³⁹⁷ is involved in FAK activation, and this site is critical for the docking of Src family tyrosine kinases and its activation.^{18,19,23,29,33,45} Shear stress rapidly activates several tyrosine kinases, including FAK, the Src family kinases, proline-rich tyrosine kinase (Pyk2) and vascular endothelial growth factor receptor 2 in ECs.^{8,18,19,45,46} In addition, pharmacological inactivation of Src prevents the phosphorylation of PI3K, Akt, and eNOS.^8,9 A previous report from our laboratory demonstrates that inhibition of integrin activation prevents FID and tyrosine phosphorylation in ECs of porcine coronary arterioles.⁶ In the present study, we demonstrate that inhibition of FAK autophosphorylation significantly prevents FID. Our present findings suggest that tyrosine autophosphorylation of FAK is essential for flow-induced NO-mediated dilation, and that FAK is likely involved in the integrin-mediated mechanotransduction of shear stress.

We also observed that the antibody that targets autophosphorylated FAK reduced its autophosphorylation. Our explanation is that the antibody is developed against a sequence of amino acids that flank Tyr³⁹⁷ and the phosphorylated tyrosine; thus, there is a likelihood that the antibody can bind to the autophosphorylation site, even without the tyrosine being phosphorylated (but with lower affinity than when the residue is phosphorylated).

Recent reports demonstrated that inhibition of PI3K reduced NO-dependent FID. PI3K is activated on Src-induced phosphorylation of p130Cas, which allows the p85 and p115 subunits of PI3K to dock and become activated.^{9,27,31–33,47} PI3K activates Akt by phosphorylating at Ser473, and Akt phosphorylates eNOS, which then induces NO production.^12–16 Our results connect well with this scheme; specifically, FAK-initiated signaling, critical for Src activation, is a prerequisite for FID. We also demonstrated that the flow-induced phosphorylation of Akt and eNOS is blocked by antagonism of FAK-dependent signaling. Our data suggest that shear-induced autophosphorylation of FAK initiates a signaling pathway involving activation of Akt and eNOS in coronary arterioles.

Another result that bears on conclusion about the role of FAK in shear stress–dependent NO-mediated dilation was our finding that the inhibition of FID by the combination of L-NAME and FAKab had no further effect than the antibody alone. This implies that the inhibition is along a series of biochemical steps, as we have proposed, rather than parallel pathways. We say this because if the pathways were parallel, we would have expected an additive effect, but this was not observed. Furthermore, this remaining component of FID appears to be independent of FAK signaling and NO production; thus, there may be alternative mechanisms responsible for the production of other endothelium-dependent dilators, endothelium-derived hyperpolarizing factor, or prostacyclin by shear. Although this component of dilation was small, under conditions such as chronic inhibition or loss of NO, these alternative endothelial dilators may assume a primary role.^48,49

A point we would like to mention is that we used 6 arterioles in each group for the Western analyses. This number of vessels was essential to provide 20 µg of protein for loading to allow reprobing of the membrane with the various antibodies. Although a disadvantage of pooling the vessels is that we essentially have a sample size of 1 for each treatment in the Western analyses, the pooling of vessels allows us to evaluate Akt, FAK, pY397-FAK, pS473-Akt, and pS1179-eNOS in the same pooled samples. Although we could not probe for total eNOS protein, we believe that the total eNOS protein was similar among the different treatment groups because total FAK and Akt and the total amount of protein loaded per lane were equivalent. We perfused arterioles with antibody/liposome reagent complex intraluminally to confine the effects of the FAKab to the arteriolar endothelium. Therefore, we opine that the change in phosphorylation of FAK and Akt represent signals from ECs. Moreover, pressurization (which would reflect primarily smooth muscle) did not affect the signals, but phosphorylation was affected by flow. These observations further strengthen our argument that the effects of FAKab were localized to the endothelium.

It is known that FAK plays central roles in integrin signaling and Src activation.^{18–20,25,30,45} Activation of FAK is enhanced by various stimuli such as shear stress, cyclic stretching, vascular endothelial growth factor, angiopoietin-I, _vß₃, or ß₁ integrins.^{12,24–26,28,47,50,51} The question of how FAK selects and regulates the signaling between multiple stimuli and multiple downstream outcomes is key¹⁹ but remains unanswered. However, we can state with conviction that in the present study, we demonstrated a critical role for FAK in flow-induced signaling mechanisms leading to vasodilation.

In summary, inhibition of FAK signaling by interfering with the autophosphorylation at Tyr³⁹⁷ impairs flow- or shear stress–dependent dilation. Also, inhibition of FAK signaling prevents flow-induced phosphorylation of Akt at Ser⁴⁷³ and eNOS at Ser¹¹⁷⁹. Therefore, we conclude that activation of FAK is central to the mechanotransduction of shear stress and to FID via the regulation of Akt and eNOS.

	Acknowledgments

 
This work was supported by National Heart, Lung, and Blood Institute grants HL32788 and HL65203 to W.M.C. and American Heart Association (0455435B), Atorvastatin Research Award (2004-37), and COBRE P20 RR18766 to C.Z.

Received April 22, 2005; accepted September 7, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995; 75: 519–560.[Abstract/Free Full Text]

2. Holtz J, Giesler M, Bassenge E. Two dilatory mechanisms of anti-anginal drugs on epicardial coronary arteries in vivo: indirect, flow-dependent, endothelium-mediated dilation and direct smooth muscle relaxation. Z Kardiol. 1983; 72 (suppl 3): 98–106.

3. Lamping KG, Dole WP. Flow-mediated dilation attenuates constriction of large coronary arteries to serotonin. Am J Physiol. 1988; 255: H1317–H1324.

4. Amezcua JL, Palmer RM, de Souza BM, Moncada S. Nitric oxide synthesized from L-arginine regulates vascular tone in the coronary circulation of the rabbit. Br J Pharmacol. 1989; 97: 1119–1124.[Medline] [Order article via Infotrieve]

5. Lamontagne D, Pohl U, Busse R. Mechanical deformation of vessel wall and shear stress determine the basal release of endothelium-derived relaxing factor in the intact rabbit coronary vascular bed. Circ Res. 1992; 70: 123–130.[Abstract/Free Full Text]

6. Muller JM, Chilian WM, Davis MJ. Integrin signaling transduces shear stress–dependent vasodilation of coronary arterioles. Circ Res. 1997; 80: 320–326.[Abstract/Free Full Text]

7. Davis ME, Cai H, Drummond GR, Harrison DG. Shear stress regulates endothelial nitric oxide synthase expression through c-Src by divergent signaling pathways. Circ Res. 2001; 89: 1073–1080.[Abstract/Free Full Text]

8. Jin ZG, Ueba H, Tanimoto T, Lungu AO, Frame MD, Berk BC. Ligand-independent activation of vascular endothelial growth factor receptor 2 by fluid shear stress regulates activation of endothelial nitric oxide synthase. Circ Res. 2003; 93: 354–363.[Abstract/Free Full Text]

9. Kumar P, Amin MA, Harlow LA, Polverini PJ, Koch AE. Src and phosphatidylinositol 3-kinase mediate soluble E-selectin-induced angiogenesis. Blood. 2003; 101: 3960–3968.[Abstract/Free Full Text]

10. Muller JM, Davis MJ, Chilian WM. Coronary arteriolar flow-induced vasodilation signals through tyrosine kinase. Am J Physiol. 1996; 270: H1878–H1884.

11. Sun D, Huang A, Sharma S, Koller A, Kaley G. Endothelial microtubule disruption blocks flow-dependent dilation of arterioles. Am J Physiol Heart Circ Physiol. 2001; 280: H2087–H2093.[Abstract/Free Full Text]

12. Berk BC, Corson MA, Peterson TE, Tseng H. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow. J Biomech. 1995; 28: 1439–1450.[CrossRef][Medline] [Order article via Infotrieve]

13. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999; 399: 597–601.[CrossRef][Medline] [Order article via Infotrieve]

14. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605.[CrossRef][Medline] [Order article via Infotrieve]

15. Fisslthaler B, Dimmeler S, Hermann C, Busse R, Fleming I. Phosphorylation and activation of the endothelial nitric oxide synthase by fluid shear stress. Acta Physiol Scand. 2000; 168: 81–88.[CrossRef][Medline] [Order article via Infotrieve]

16. Luo Z, Fujio Y, Kureishi Y, Rudic RD, Daumerie G, Fulton D, Sessa WC, Walsh K. Acute modulation of endothelial Akt/PKB activity alters nitric oxide-dependent vasomotor activity in vivo. J Clin Invest. 2000; 106: 493–499.[Medline] [Order article via Infotrieve]

17. Scotland RS, Morales-Ruiz M, Chen Y, Yu J, Rudic RD, Fulton D, Gratton JP, Sessa WC. Functional reconstitution of endothelial nitric oxide synthase reveals the importance of serine 1179 in endothelium-dependent vasomotion. Circ Res. 2002; 90: 904–910.[Abstract/Free Full Text]

18. Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995; 15: 954–963.[Abstract]

19. Xing Z, Chen HC, Nowlen JK, Taylor SJ, Shalloway D, Guan JL. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. Mol Biol Cell. 1994; 5: 413–421.[Abstract]

20. Parsons JT. Focal adhesion kinase: the first ten years. J Cell Sci. 2003; 116: 1409–1416.[Abstract/Free Full Text]

21. Chan PY, Kanner SB, Whitney G, Aruffo A. A transmembrane-anchored chimeric focal adhesion kinase is constitutively activated and phosphorylated at tyrosine residues identical to pp125FAK. J Biol Chem. 1994; 269: 20567–20574.[Abstract/Free Full Text]

22. Li X, Dy RC, Cance WG, Graves LM, Earp HS. Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase. J Biol Chem. 1999; 274: 8917–8924.[Abstract/Free Full Text]

23. Toutant M, Studler JM, Burgaya F, Costa A, Ezan P, Gelman M, Girault JA. Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms. Biochem J. 2000; 348 Pt 1: 119–128.

24. Ishida T, Peterson TE, Kovach NL, Berk BC. MAP kinase activation by flow in endothelial cells. Role of beta 1 integrins and tyrosine kinases. Circ Res. 1996; 79: 310–316.[Abstract/Free Full Text]

25. Jalali S, Li YS, Sotoudeh M, Yuan S, Li S, Chien S, Shyy JY. Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells. Arterioscler Thromb Vasc Biol. 1998; 18: 227–234.[Abstract/Free Full Text]

26. Jalali S, del Pozo MA, Chen K, Miao H, Li Y, Schwartz MA, Shyy JY, Chien S. Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands. Proc Natl Acad Sci U S A. 2001; 98: 1042–1046.[Abstract/Free Full Text]

27. Chen HC, Appeddu PA, Isoda H, Guan JL. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J Biol Chem. 1996; 271: 26329–26334.[Abstract/Free Full Text]

28. Wang JG, Miyazu M, Matsushita E, Sokabe M, Naruse K. Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem Biophys Res Commun. 2001; 288: 356–361.[CrossRef][Medline] [Order article via Infotrieve]

29. Reiske HR, Zhao J, Han DC, Cooper LA, Guan JL. Analysis of FAK-associated signaling pathways in the regulation of cell cycle progression. FEBS Lett. 2000; 486: 275–280.[CrossRef][Medline] [Order article via Infotrieve]

30. Shen TL, Guan JL. Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts. FEBS Lett. 2001; 499: 176–181.[CrossRef][Medline] [Order article via Infotrieve]

31. Chen HC, Guan JL. Stimulation of phosphatidylinositol 3'-kinase association with foca adhesion kinase by platelet-derived growth factor. J Biol Chem. 1994; 269: 31229–31233.[Abstract/Free Full Text]

32. Chen HC, Guan JL. Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc Natl Acad Sci U S A. 1994; 91: 10148–10152.[Abstract/Free Full Text]

33. Reiske HR, Kao SC, Cary LA, Guan JL, Lai JF, Chen HC. Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration. J Biol Chem. 1999; 274: 12361–12366.[Abstract/Free Full Text]

34. Koshida R, Ou J, Matsunaga T, Chilian WM, Oldham KT, Ackerman AW, Pritchard KA Jr. Angiostatin: a negative regulator of endothelial-dependent vasodilation. Circulation. 2003; 107: 803–806.[Abstract/Free Full Text]

35. Kuo L, Chilian WM, Davis MJ. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am J Physiol. 1991; 261: H1706–H1715.

36. Kuo L, Davis MJ, Chilian WM. Endothelium-dependent, flow-induced dilation of isolated coronary arterioles. Am J Physiol. 1990; 259: H1063–H1070.

37. Kuo L, Davis MJ, Chilian WM. Longitudinal gradients for endothelium-dependent and -independent vascular responses in the coronary microcirculation. Circulation. 1995; 92: 518–525.[Abstract/Free Full Text]

38. Butler PJ, Weinbaum S, Chien S, Lemons DE. Endothelium-dependent, shear-induced vasodilation is rate-sensitive. Microcirculation. 2000; 7: 53–65.[CrossRef][Medline] [Order article via Infotrieve]

39. Vequaud P, Thorin E. Endothelial G-protein beta-subunits trigger nitric oxide-but not endothelium-derived hyperpolarizing factor-dependent dilation in rabbit resistance arteries. Circ Res. 2001; 89: 716–722.[Abstract/Free Full Text]

40. Rocic P, Govindarajan G, Sabri A, Lucchesi PA. A role for PYK2 in regulation of ERK1/2 MAP kinases and PI 3-kinase by Ang II in vascular smooth muscle. Am J Physiol Cell Physiol. 2001; 280: C90–C99.[Abstract/Free Full Text]

41. Rocic P, Griffin TM, McRae CN, Lucchesi PA. Altered PYK2 phosphorylation by Ang II in hypertensive vascular smooth muscle. Am J Physiol Heart Circ Physiol. 2002; 282: H457–H465.[Abstract/Free Full Text]

42. Corson MA, James NL, Latta SE, Nerem RM, Berk BC, Harrison DG. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res. 1996; 79: 984–991.[Abstract/Free Full Text]

43. Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ Res. 1996; 78: 750–758.[Abstract/Free Full Text]

44. Fleming I, Bauersachs J, Busse R. Calcium-dependent and calcium-independent activation of the endothelial NO synthase. J Vasc Res. 1997; 34: 165–174.[Medline] [Order article via Infotrieve]

45. Okuda M, Takahashi M, Suero J, Murry CE, Traub O, Kawakatsu H, Berk BC. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J Biol Chem. 1999; 274: 26803–26809.[Abstract/Free Full Text]

46. Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, Shyy JY. Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases. J Biol Chem. 1997; 272: 30455–30462.[Abstract/Free Full Text]

47. Li E, Stupack DG, Brown SL, Klemke R, Schlaepfer DD, Nemerow GR. Association of p130CAS with phosphatidylinositol-3-OH kinase mediates adenovirus cell entry. J Biol Chem. 2000; 275: 14729–14735.[Abstract/Free Full Text]

48. Huang A, Sun D, Carroll MA, Jiang H, Smith CJ, Connetta JA, Falck JR, Shesely EG, Koller A, Kaley G. EDHF mediates flow-induced dilation in skeletal muscle arterioles of female eNOS-KO mice. Am J Physiol Heart Circ Physiol. 2001; 280: H2462–H2469.[Abstract/Free Full Text]

49. Huang A, Wu Y, Sun D, Koller A, Kaley G. Effect of estrogen on flow-induced dilation in NO deficiency: role of prostaglandins and EDHF. J Appl Physiol. 2001; 91: 2561–2566.[Abstract/Free Full Text]

50. Kim I, Kim HG, Moon SO, Chae SW, So JN, Koh KN, Ahn BC, Koh GY. Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion. Circ Res. 2000; 86: 952–959.[Abstract/Free Full Text]

51. Tai LK, Okuda M, Abe J, Yan C, Berk BC. Fluid shear stress activates proline-rich tyrosine kinase via reactive oxygen species-dependent pathway. Arterioscler Thromb Vasc Biol. 2002; 22: 1790–1796.[Abstract/Free Full Text]

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