A Role for the Tyrosine Kinase Pyk2 in Depolarization-induced Contraction of Vascular Smooth Muscle*♦

Background: Depolarization-induced tonic contraction of vascular smooth muscle involves tyrosine phosphorylation. Results: Depolarization activates the Ca2+-dependent tyrosine kinase Pyk2, leading to activation of the RhoA/Rho-associated kinase pathway. Conclusion: Activation of Pyk2 is required for the sustained phase of depolarization-induced contraction. Significance: Knowledge of the mechanisms responsible for sustained contraction is crucial for identification of defects leading to disease associated with vascular contractile dysfunction. Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC20). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca2+-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K+-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K+, Pyk2 autophosphorylation, and LC20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca2+, elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca2+ channel blocker nifedipine inhibited peak and sustained K+-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K+-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca2+ activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.

Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC 20 ). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca 2؉ -dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K ؉ -induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC 20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K ؉ , Pyk2 autophosphorylation, and LC 20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca 2؉ , elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca 2؉ channel blocker nifedipine inhibited peak and sustained K ؉ -induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K ؉ -induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca 2؉ activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC 20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.
Membrane depolarization evoked by neurotransmitter release is a key mechanism of activation of vascular smooth muscle contraction. Depolarization leads to activation of voltage-gated Ca 2ϩ channels, Ca 2ϩ entry from the extracellular space, myosin regulatory light chain (LC 20 ) 4 phosphorylation by Ca 2ϩ /calmodulin-dependent myosin light chain kinase (MLCK), and contraction (1,2). Although this mechanism accounts for the rapid (phasic) contraction elicited by membrane depolarization, this is followed by a sustained (tonic) contraction, which requires activation of the RhoA/Rho-associated kinase (ROK) pathway, leading to inhibition of myosin light chain phosphatase (MLCP) via phosphorylation of MYPT1, the myosin targeting subunit of MLCP (3)(4)(5).
It remains unclear, however, how membrane depolarization and Ca 2ϩ entry lead to the activation of RhoA. We demonstrated recently that genistein-sensitive tyrosine phosphorylation lies upstream of RhoA activation in response to membrane depolarization, and hypothesized that the Ca 2ϩ -dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) may be involved (6). We have addressed this hypothesis in de-endothelialized rat caudal arterial smooth muscle strips using a variety of Pyk2 inhibitors in conjunction with measurements of Pyk2 autophosphorylation (activation), RhoA translocation, and MYPT1 and LC 20 phosphorylation, and we conclude that Pyk2 is activated in response to depolarization-induced Ca 2ϩ entry and lies upstream of RhoA and ROK, leading to MLCP inhibition and sustained contraction.

EXPERIMENTAL PROCEDURES
Materials-Sodium salicylate was purchased from Wako Pure Chemical Industries (Osaka, Japan). PF-431396, PF-573228, and ionomycin were purchased from Sigma. NVP-TAE226 and PF-562271 were purchased from Selleck Chemicals. ML-9, sodium orthovanadate, and calyculin A were purchased from Calbiochem, and FAK inhibitor sc-203950 was purchased from Santa Cruz Biotechnology.
Force Measurements in Isolated Muscle Strips-Caudal arteries were removed from male Sprague-Dawley rats (300 -400 g) after sacrifice using protocols consistent with the standards of the Canadian Council on Animal Care and approved by the Institutional Ethics Committee for Animal Research at Meiji Pharmaceutical University and the University of Calgary Animal Care and Use Committee. De-endothelialized caudal arterial smooth muscle strips were prepared for force measurement as described previously (3,7). Experiments were carried out with endothelium-denuded vessels to eliminate interference from endothelial cell-derived vasoactive mediators. Tissues were stimulated with 60 or 87 mM KCl by replacing NaCl in Hepes-Tyrode's solution with equimolar KCl. These concentrations of KCl induce close-to-maximal contractile responses: 85.5% at 60 mM and 90.3% at 87 mM KCl.
Western Blotting-Tissues were harvested at the times indicated in the figure legends for protein extraction, SDS-PAGE (10% or 7.5% acrylamide), or Phos-tag SDS-PAGE (Wako Pure Chemical Industries) and Western blotting with enhanced FIGURE 1. Effect of pre-treatment with sodium salicylate on K ؉ -induced contraction. A and B, upper panels show the time courses of K ϩ -induced contraction without (E) or with (•) sodium salicylate (SS) pre-incubation (3 mM in A and 10 mM in B). Lower panels show the effects of sodium salicylate on the phasic (open bars) and tonic (filled bars; 30 min after K ϩ addition) components of K ϩ -induced contraction. Force is expressed as a percentage of the maximal force of the phasic contraction induced by K ϩ without sodium salicylate. Values represent the mean Ϯ S.E. (n ϭ 8). *, p Ͻ 0.001, significantly different from the value of the force without sodium salicylate. chemiluminescence detection, as described previously (6 -8). Interestingly, we found that transfer of Pyk2 to nitrocellulose membranes required SDS in the transfer buffer, whereas transfer of FAK was optimal in the absence of SDS. The levels of phosphorylation of Pyk2 at Tyr-402 and FAK at Tyr-397 were calculated from the ratio of signal intensities for pTyr-402-Pyk2 or pTyr-397-FAK:total Pyk2/FAK and/or actin. Anti-Pyk2 rabbit polyclonal antibody (Sigma, catalogue number SAB4500837) was used at 1:1500 dilution, and anti-pTyr-402-Pyk2 rabbit polyclonal antibody (Invitrogen, catalogue number 44-618G) was used at 1:1500 dilution. Anti-pTyr-397-FAK (Cell Signaling, catalogue number 3283) and anti-FAK (Cell   Signaling, catalogue number 3285) were used at 1:1000 dilution. The level of phosphorylation of MYPT1 at Thr-697 and Thr-855 was calculated from the ratio of signal intensities for pThr-697-MYPT1 or pThr-855-MYPT1:total MYPT1. Anti-pThr-697-MYPT1 rabbit polyclonal antibody (Millipore, catalogue number ABS45) was used at 1:3500 dilution, anti-pThr-855-MYPT1 rabbit polyclonal antibody (Millipore, catalogue number 36-003) was used at 1:3000 dilution, and anti-MYPT1 rabbit polyclonal antibody (Santa Cruz Biotechnology, catalogue number sc-25618) was used at 1:250 dilution. Anti-pTyr monoclonal antibody, clone 4G10, was purchased from Millipore (catalogue number 05-321), and monoclonal antibody P-Tyr-100 was purchased from Cell Signaling (catalogue num-ber 9411). For phosphotyrosine Western blots, transfer to nitrocellulose membranes was carried out with SDS in the transfer buffer; if SDS was eliminated, there was little transfer of phosphotyrosine-containing proteins. In the case of Phos-tag SDS-PAGE (which separates phosphorylated and unphosphorylated forms of LC 20 ), anti-LC 20 rabbit polyclonal antibody (Santa Cruz Biotechnology, catalogue number sc-15370) was used at 1:500 dilution.
RhoA Translocation-Separation of particulate and cytosolic fractions was achieved by the method of Gong et al. (9) as described in detail by Mita et al. (6).
Statistical Analysis-Data represent the mean Ϯ S.E. Values of n indicate the numbers of muscle strips utilized. Student's t test was used for statistical comparisons. One-way analysis of variance followed by Tukey-Kramer multiple-comparisons test was used to compare three or more groups. p values Ͻ 0.05 were considered to be statistically significant. These analyses were performed using JMP-5J (SAS Institute) or SigmaPlot.

RESULTS
Sodium salicylate has been identified as an inhibitor of Pyk2 (11), and its unique ability among non-steroidal anti-inflammatory drugs to induce vasodilation has been attributed to this action rather than its cyclooxygenase inhibitory activity (12). Incremental addition of sodium salicylate after steady-state force was attained in response to K ϩ -induced membrane depolarization of rat caudal arterial smooth muscle strips, and resulted in concentration-dependent relaxation with an IC 50 of FIGURE 6. Pyk2 is the major tyrosine-phosphorylated protein in K ؉ -stimulated rat caudal arterial smooth muscle. Rat caudal arterial smooth muscle strips were pre-incubated with PF-431396 (10 M) or vehicle for 30 min prior to stimulation with K ϩ for 10 min in the absence or presence of inhibitor. A and B, tissues were quick-frozen for analysis of protein tyrosine phosphorylation (A) and Pyk2 autophosphorylation (B) by Western blotting with anti-pTyr and anti-pTyr-402-Pyk2 (pY402-Pyk2), respectively. Actin was used as the loading control. C, control tissues and tissues treated for 10 min with K ϩ were subjected to SDS-PAGE and Western blotting with antibodies to Pyk2, pTyr, or FAK as indicated in the upper panel. The position of the 130-kDa marker is indicated. Blots were reprobed (lower panel) with anti-pTyr, anti-FAK, and anti-pTyr, respectively. Blots are representative of Ն3 replicates. APRIL 3, 2015 • VOLUME 290 • NUMBER 14 2.9 Ϯ 0.5 mM (n ϭ 13). Furthermore, pre-treatment with sodium salicylate (3 and 10 mM) reduced the tonic component of K ϩ -induced contraction without affecting the phasic component (Fig. 1). This inhibitory effect of sodium salicylate was very similar to that evoked by the ROK inhibitor Y-27632 (3), suggesting that Pyk2 is involved in activation of the RhoA/ ROK pathway responsible for force maintenance, but not the phasic contractile response to Ca 2ϩ entry leading to MLCK activation. In support of this conclusion, sodium salicylate had no effect on the rapid increase in LC 20 phosphorylation elicited by membrane depolarization, but abolished the maintenance of LC 20 phosphorylation levels at longer times corresponding to the sustained phase of the contractile response to K ϩ (Fig. 2).

Pyk2 in Depolarization-induced Vascular Smooth Muscle Contraction
The involvement of Pyk2 in the K ϩ -induced contractile response was supported by the inhibitory effect of PF-431396, a Pyk2/FAK inhibitor (13). Following sustained K ϩ -induced con-traction, PF-431396 induced a concentration-dependent relaxation with an IC 50 of 0.3 Ϯ 0.1 M (Fig. 3, A and B). Pre-treatment with PF-431396 not only inhibited the tonic component of the K ϩ -induced contractile response (IC 50 ϭ 0.49 Ϯ 0.07 M) but also the phasic contraction (Fig. 3C) with an IC 50 of 2.4 Ϯ 0.16 M (Fig. 3, D and E, and Table 1) (14 -19).
To determine whether membrane depolarization evokes Pyk2 activation, we investigated the effect of K ϩ stimulation on the autophosphorylation of Pyk2 at Tyr-402. K ϩ induced a time-dependent increase in phosphorylation of Pyk2 at Tyr-402 (Fig. 4A, left panel, and Fig. 4B). The time course of Pyk2 activation is consistent with its involvement in the tonic component of the contractile response to membrane depolarization (compare Figs. 1 and 4). The t1 ⁄ 2 of the phasic K ϩ -induced contraction was 12.1 Ϯ 0.5 s, and the time to maximal force was 46.0 Ϯ 2.0 s (n ϭ 76). PF-431396 pre-treatment inhibited basal (see below, Fig. 11B) and K ϩ -induced Pyk2 autophosphoryla-

Pyk2 in Depolarization-induced Vascular Smooth Muscle Contraction
tion at Tyr-402 (Fig. 4A, right panel, and Fig. 4B). Although PF-431396 inhibited K ϩ -induced contraction, it had no effect on the contractile response to the membrane-permeant phosphatase inhibitor calyculin-A (Fig. 5), indicating that the Pyk2/FAK inhibitor does not exert an off-target effect downstream of LC 20 phosphorylation, e.g. at the level of cross-bridge cycling.
Pyk2 co-migrates on SDS-PAGE with the major tyrosinephosphorylated protein in response to K ϩ treatment (Fig. 6). PF-431396 pre-treatment blocked tyrosine phosphorylation of this band (Fig. 6A) and, as shown earlier, abolished the auto-phosphorylation of Pyk2 at Tyr-402 (Fig. 6B). However, PF-431396 also inhibits the related tyrosine kinase FAK (13), and we observed that FAK autophosphorylation at Tyr-397 (equivalent to Tyr-402 of Pyk2) is increased upon K ϩ stimulation of rat caudal arterial smooth muscle in a time-dependent manner (Fig. 7). We were able to separate Pyk2 and FAK on 7.5% acrylamide SDS gels, and the results showed that Pyk2, but not FAK, co-migrated with the major tyrosine-phosphorylated band (Fig. 6C, upper blots). Reprobing these anti-Pyk2, -pTyr, and -FAK blots with anti-pTyr, -FAK, and -pTyr, respectively, clearly indicated that Pyk2 and not FAK is the major tyrosine-  (n ϭ 6). *, p Ͻ 0.001, **, p Ͻ 0.002, significantly different from the control K ϩ -induced sustained contractile response. B, tissues were quick-frozen 10 min after the addition of K ϩ for analysis of Pyk2 autophosphorylation by Western blotting with anti-pTyr-402-Pyk2 (pY402-Pyk2) and FAK autophosphorylation with anti-pTyr-397-FAK (pY397-FAK). Actin was used as the loading control, and anti-Pyk2 and anti-FAK were used to verify the total levels of the two proteins. C and D, relationship between Pyk2 (C) and FAK (D) autophosphorylation and the magnitude of the tonic K ϩ -induced contractile response. Values represent the mean Ϯ S.E. (n ϭ 4 (C) and n ϭ 3 (D)). phosphorylated protein in K ϩ -stimulated tissue (Fig. 6C, lower  blots).
To distinguish functionally between Pyk2 and FAK, the effects on K ϩ -induced contraction (Fig. 8A) and Pyk2 activation (Fig. 8B) of several inhibitors with similar and different inhibitory potencies toward these tyrosine kinases were examined. A close correlation (r 2 ϭ 0.87) was found between the ability of these inhibitors to inhibit Pyk2 autophosphorylation and K ϩ -induced contraction ( Fig. 8C and Table 1). This was not the case for FAK, however (r 2 ϭ 0.44) (Fig. 8D and Table 1), suggesting that Pyk2 rather than FAK plays an important role in the contractile response to membrane depolarization. The Pyk2/FAK inhibitors also affected sustained K ϩ -induced LC 20 phosphorylation with relative potencies that matched their effects on Pyk2 rather than FAK ( Fig. 9 and Table 1).
Sodium orthovanadate, a tyrosine phosphatase inhibitor, has long been known to induce vascular smooth muscle contraction (20). We investigated the possibility that vanadate may act by unmasking the basal activity of Pyk2. In support of this hypothesis, vanadate evoked a slow, sustained contraction (Fig.  10A, left panel) that was abolished by pre-incubation with PF-431396 (Fig. 10A, right panel) with an IC 50 of 66 Ϯ 17 nM (Fig. 10, C and D). Pyk2 autophosphorylation increased with a similar time course to contraction (Fig. 10B, left panel), and vanadate-induced Pyk2 autophosphorylation was abolished by pre-treatment with PF-431396 (Fig. 10B, right panel).
To determine whether Pyk2 activation is indeed downstream of Ca 2ϩ influx, the Ca 2ϩ ionophore ionomycin was used to increase [Ca 2ϩ ] i , and Pyk2 activation was assessed by measurements of Tyr-402 phosphorylation. In the presence of extracellular Ca 2ϩ , ionomycin induced a slow, sustained contraction (Fig. 11A, left panel) that was inhibited by pre-incubation with PF-431396 (Fig. 11A, right panel) with an IC 50 of 2.6 Ϯ 0.7 M (n ϭ 4). Ionomycin induced an increase in Pyk2 autophosphorylation in a Ca 2ϩ -dependent manner, and this was also inhibited by pre-treatment with the Pyk2 inhibitor (Fig. 11, C and D). We also examined the effect of blockade of voltage-gated Ca 2ϩ channels by nifedipine on K ϩ -induced contraction and Pyk2 autophosphorylation. Nifedipine inhibited peak (Fig. 12A) and sustained K ϩ -induced force (Fig. 12B), as well as Pyk2 phosphorylation at Tyr-402 (Fig. 12, C and D), in a concentration-dependent manner. We conclude from these ionomycin and nifedip- ine experiments that the influx of extracellular Ca 2ϩ is responsible for the activation of Pyk2. The MLCK inhibitor ML-9 inhibited K ϩ -induced contraction (Fig. 13, A and B) and LC 20 phosphorylation (data not shown) without affecting the K ϩ -in-duced increase in Pyk2 autophosphorylation (Fig. 13C), confirming that Pyk2 lies upstream of MLCK.
A connection from Pyk2 activation to RhoA activation and Ca 2ϩ sensitization was made with the observation that pre-  APRIL 3, 2015 • VOLUME 290 • NUMBER 14 treatment with sodium salicylate prevented (i) the K ϩ -induced translocation of RhoA from the cytosol to the membrane (Fig.  14) and (ii) the increase in phosphorylation of MYPT1 at Thr-855 and Thr-697 (Fig. 15). Finally, it was important to demonstrate that sodium salicylate does not have an off-target effect on ROK. An in vitro kinase assay indicated that purified ROK was unaffected by sodium salicylate; the rates of phosphorylation of MYPT1 peptide by ROK were 70.5 Ϯ 1.9 mol P i /min/mg of ROK in the absence of sodium salicylate, 66.5 Ϯ 2.9 mol P i /min/mg of ROK in the presence of 3 mM sodium salicylate, and 65.4 Ϯ 3.4 mol P i /min/mg of ROK in the pres-ence of 10 mM sodium salicylate (values indicate the mean Ϯ S.E., n ϭ 3 in each case).

DISCUSSION
Pyk2 (also known as FAK2, CAK␤, and RAFTK) is a nonreceptor, Ca 2ϩ -dependent protein-tyrosine kinase (21). Activation of Pyk2 involves autophosphorylation at Tyr-402, which enables the binding of Src via its SH2 domain and phosphorylation at Tyr-579 and Tyr-580 of Pyk2 within the kinase domain activation loop to generate maximal kinase activity. Kohno et al. (22) have proposed that the FERM domain of Pyk2 regu-

Pyk2 in Depolarization-induced Vascular Smooth Muscle Contraction
lates its activity by mediating Ca 2ϩ /calmodulin-dependent Pyk2 homodimer formation and transphosphorylation.
Our previous studies indicated that depolarization-induced Ca 2ϩ entry elicits Ca 2ϩ sensitization of vascular smooth muscle contraction via activation of the RhoA/ROK pathway (3) and implicated a genistein-sensitive tyrosine kinase upstream of RhoA activation (6). We considered Pyk2 as a strong candidate given its Ca 2ϩ dependence. This possibility was supported by the observation that sodium salicylate (a known inhibitor of Pyk2) relaxed rat caudal arterial smooth muscle pre-contracted by membrane depolarization and that the tonic, but not the phasic component of K ϩ -induced contraction was abolished by pre-treatment with salicylate (Fig. 1). This effect of salicylate was very similar to that of the ROK inhibitor Y-27632 (3). Consistent with these effects on force, sodium salicylate pre-treatment had no effect on the rapid increase in LC 20 phosphorylation induced by membrane depolarization, but abolished the sustained elevation of LC 20 phosphorylation (Fig. 2). The Pyk2/ FAK inhibitor PF-431396, on the other hand, inhibited both phasic and tonic components of K ϩ -induced force (Fig. 3) and LC 20 phosphorylation (Fig. 9), suggesting an off-target effect of this compound during the phasic contraction. A likely target is MLCK itself because skeletal muscle MLCK has been shown to be partially inhibited by PF-431396 (13).
One of the most important findings of this study was that Pyk2 autophosphorylation at Tyr-402, which correlates with activation of the enzyme (21), increased in response to membrane depolarization (Fig. 4) with a time course corresponding to that of the tonic component of the contractile response (Fig.  1). Furthermore, Pyk2 was identified as the major tyrosinephosphorylated protein in response to membrane depolarization (Fig. 6). As expected, PF-431396 inhibited both basal (Fig. 10B) and K ϩ -induced Pyk2 autophosphorylation (Fig.  4). Importantly, PF-431396 had no effect on contraction induced by the membrane-permeant Ser/Thr phosphatase inhibitor calyculin A (Fig. 5). Calyculin A induces a slow, sustained contraction of vascular smooth muscle through inhibition of MLCP, which unmasks the basal activities of integrin-linked kinase and zipper-interacting protein kinase, leading to phosphorylation of LC 20 at Thr-18 and Ser-19 and contraction (7). The fact that PF-431396 had no effect on calyculin A-induced contraction indicates the absence of an off-target effect of the compound downstream of LC 20 phosphorylation.    ϭ 6 -7). *, p Ͻ 0.05, significantly different from the value under resting conditions without sodium salicylate; #, p Ͻ 0.05, significantly different from the value following K ϩ stimulation for 5 or 15 min.
The related tyrosine kinase FAK was also found to be autophosphorylated at Tyr-397 (which corresponds to Tyr-402 of Pyk2) in response to K ϩ stimulation, with a time course similar to that of Pyk2 autophosphorylation (Fig. 7). However, quantitatively, Pyk2 tyrosine phosphorylation was found to be considerably greater than that of FAK (Fig. 6), and the relationship between autophosphorylation and contraction in the presence of various Pyk2/FAK inhibitors was linear for Pyk2 ( Fig. 8C; r 2 ϭ 0.87) but not for FAK ( Fig. 8D; r 2 ϭ 0.44). We conclude, therefore, that Pyk2 plays a major role in sustained K ϩ -induced contraction.
Use of the tyrosine phosphatase inhibitor sodium orthovanadate further supported a role for Pyk2 in sustained contraction of vascular smooth muscle. Thus the slow, sustained contraction induced by vanadate was abolished by pre-treatment with PF-431396, and this correlated with attenuation of vanadateinduced Pyk2 autophosphorylation (Fig. 10).
Treatment of rat caudal arterial smooth muscle strips with the Ca 2ϩ ionophore ionomycin induced Pyk2 autophosphorylation at Tyr-402 and a slow, sustained contractile response, both of which were blocked by pre-incubation with PF-431396 (Fig. 11), indicating that Ca 2ϩ influx activates Pyk2 and contraction. The IC 50 for inhibition of ionomycin-induced contraction was determined to be 2.6 Ϯ 0.7 M, i.e. similar to the IC 50 (2.4 Ϯ 0.16 M) for PF-431396-mediated inhibition of the phasic component of the K ϩ -induced contraction (see above).
These observations are consistent with (i) a slow ionomycininduced increase in [Ca 2ϩ ] i leading to slow activation of MLCK and sustained contraction due to the maintenance of elevated [Ca 2ϩ ] i and (ii) activation of Pyk2 by Ca 2ϩ , which is not required for ionomycin-induced contraction. That is, Pyk2mediated inhibition of MLCP is not required for the sustained ionomycin-induced contraction because the increase in [Ca 2ϩ ] i is presumably sufficient to activate MLCK to a level that overcomes that of MLCP. The conclusion that Ca 2ϩ entry is responsible for Pyk2 activation was supported by the observation that the Ca 2ϩ channel blocker nifedipine inhibited both K ϩ -induced force and Pyk2 autophosphorylation (Fig. 12).
Finally, a connection between Pyk2 activation and the RhoA/ ROK pathway leading to MLCP inhibition was established by the demonstration that sodium salicylate prevented both (i) the K ϩ -induced translocation of RhoA from the cytosolic to the particulate fraction (Fig. 14) and (ii) the K ϩ -induced phosphorylation of MYPT1 at the two inhibitory phosphorylation sites (23)(24)(25), Thr-697 and Thr-855 (Fig. 15). We also demonstrated that salicylate had no effect on the activity of purified recombinant ROK.
The mechanism whereby Pyk2 activates RhoA remains to be elucidated. Transfection experiments with primary aortic vascular smooth muscle cells in culture have suggested that the guanine nucleotide exchange factor PDZ-RhoGEF may connect activated Pyk2 to RhoA activation via phosphorylation and activation of its guanine nucleotide exchange factor (GEF) activity (26). This remains to be demonstrated in freshly isolated vascular smooth muscle tissue.
In conclusion, our results support a role for Pyk2 in activation of the RhoA/ROK pathway and Ca 2ϩ sensitization in the tonic contractile response of vascular smooth muscle to membrane depolarization. They also emphasize the importance of the development of specific Pyk2 inhibitors as therapeutic agents for the treatment of cardiovascular diseases associated with hypercontractility because up-regulation of the RhoA/ ROK pathway has been implicated in the etiology of hypertension (27,28). This work also extends the regulatory roles of Pyk2 in vascular smooth muscle; Pyk2 was recently implicated in stretch-stimulated growth in the rat portal vein (29).