G13α-mediated PYK2 Activation

G12α/G13α transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G12α/G13α signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G12α or G13α but not by RhoA. The GTPase-deficient form of G13α triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G13α, although not G12α, readily associates with PYK2. Thus, G-protein-coupled receptors via G13α activation can use PYK2 to link to SRE-dependent gene expression.

Growth factors rapidly induce the c-fos gene, a response that depends upon a critical cis regulatory site located in the c-fos promoter termed the serum response element (SRE). 1 Two transcriptional factors bind the SRE, the 67-kDa serum response factor (SRF), which binds as a dimer to the CarG box of the SRE, and the ternary complex factor (TCF), which binds SRF along with a purine-rich sequence at the 5Ј-end of the SRE (1). TCF, which is encoded by several ets-related genes including Elk-1 and SRF accessory proteins 1 and 2, can only bind the SRE in conjunction with SRF. TCF phosphorylation enhances ternary complex formation and its transcriptional activity. At least three major mitogen-activated kinases including ERK1/2, p38, and stress-activated protein kinase (SAPK, also referred to as Jun kinase or JNK) phosphorylate TCF (2)(3)(4). A mutant SRE that lacks the TCF binding site no longer responds to activators of these kinase pathways but remains responsive to stimuli that activate SRF (5,6).
Agonists of some GPCRs also activate the tyrosine kinase PYK2 (11)(12)(13)(14). GPCRs that couple to G q ␣ subfamily members stimulate phospholipase C-␤, which causes the release of intracellular calcium and protein kinase C activation and a subsequent enhancement of PYK2 kinase activity (13,15). Downstream of PYK2 signaling lie the p38, SAPK, and Erk pathways, each of which can lead to TCF phosphorylation (11, 16 -18). In addition, PYK2 triggers c-src activation (11), which can stimulate SRF-dependent SRE activation (10). Thus, in cell types that express PYK2, GPCR signaling by triggering PYK2 activation could enhance SRE-mediated transcription through TCF-dependent and -independent pathways. To examine this possibility, we used HeLa cells, a cell line that contains significant endogenous levels of PYK2. Surprisingly we found a requirement for PYK2 in SRF activation triggered by engagement of the M1R or by expression of GTPase-deficient G 12 ␣ and G 13 ␣. In the course of these studies, we observed that GTP-G 13 ␣ potently stimulates PYK2 kinase activity and that it associates with PYK2 in vivo.
Transfections and Reporter Gene Assays-HeLa and COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and transfected using Su-perFect (Qiagen Inc., Valencia, CA). The collected cells were lysed in 200 l of reporter lysis buffer (Promega, Madison, WI) for 20 min on ice. After centrifugation, 20 l of the supernatant was tested for ␤-galactosidase activity using galactan chemiluminescent substrate (Tropix, Bedford, MA) or luciferase activity using a luciferase substrate (Promega). For the CAT assays, the transfected HeLa cells were lysed by three cycles of freeze-thaw, and CAT activity was determined using a previously described liquid scintillation assay (20). Data from all the transfection assays were normalized using the activity of a control reporter gene.
Immunoblotting and Immunoprecipitations-HeLa cell lysates were prepared using a solution containing 150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA, and 1% Triton X-100 along with a mixture of protease inhibitors for 20 min on ice. The detergent-insoluble material was removed by microcentrifugation for 10 min at 4°C. Equal amounts of protein from each sample were fractionated by SDS-PAGE and transferred to pure nitrocellulose. Membranes were blocked with 10% milk in TTBS (Tween 20, Tris, base, salt) for 1 h and then incubated with an appropriate dilution of the primary antibody in 5% milk and 0.05% sodium azide in TTBS overnight. The blots were washed twice with TTBS before the addition of a biotinylated goat anti-rabbit immunoglobulin (DAKO, Carpinteria, CA) diluted 1:1500 in TTBS containing 5% fetal calf serum. Following a 1-h incubation, the blot was washed twice with TTBS and then incubated with streptavidin conjugated to horseradish peroxidase (DAKO). The signal was detected by enhanced chemiluminescence (ECL) following the recommendations of the manufacturer (Amersham Pharmacia Biotech).
The co-immunoprecipitations were performed using lysates (20 mM Tris, pH 8.0, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate plus protease inhibitors) prepared from HeLa cells coexpressing HA-G 12 ␣QL or HA-G 13 ␣QL in conjunction with Myc-PYK2. Anti-Myc or -HA monoclonal antibodies were added, and the immunoprecipitates were collected with the appropriate secondary antibodycoupled magnetic beads (Dynal Corp., Lake Success, NY). They were washed three times in lysis buffer, twice in lysis buffer with 0.5 M NaCl, fractionated by SDS-PAGE, and analyzed by immunoblotting with the appropriate antibody. Alternatively, HeLa cells were treated for 20 min with AlF 4 Ϫ or not and washed twice using phosphate-buffered saline prior to the addition of lysis buffer. PYK2 immunoprecipitates were collected and washed as above prior to fractionation on SDS-PAGE and immunoblotting using the G 13 ␣-specific antiserum.
RhoA Translocation-The RhoA translocation experiment was done by a modification of a previously published procedure (10). Briefly, COS-7 cells transfected with PYK2 or PYK2 KD expression vector were incubated in a hypotonic buffer containing 20 mM Tris and protease inhibitors for 30 min on ice, lysed by several cycles of freezing and thawing, and sonicated for five 10-s intervals. To make the membrane preparations, the samples were centrifuged at 3000 ϫ g for 5 min, and the supernatant was recentrifuged at 52,000 rpm for 30 min at 4°C. The membrane pellets were washed three times with the hypotonic buffer and dissolved in sample buffer. The proteins in the supernatant were precipitated with 6% (m/v) trichloroacetic acid and 0.0125% (m/v) deoxycholate. Equal amount of proteins from the membrane and the supernatant were loaded onto SDS-PAGE, and the proteins were detected by the PYK2-and RhoA-specific antibodies, respectively.
Rho Binding Assay-Bacterially expressed GST-Rhotekin Rho binding domain protein (GST-RBD) was purified from isopropyl-1-thio-␤-D-galactopyranoside (1 mM)-induced BL21 cells previously transformed with the appropriate plasmid (21). The transfected HeLa cells were washed with ice-cold Tris-buffered saline, and lysis buffer was added (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl 2 , 10 g/ml of leupeptin and aprotinin, and 1 mM phenylmethlylsulfonyl fluoride). The lysate was spun at 13,000 ϫ g at 4°C for 2 min, and the cleared lysate was incubated with GST-RBD beads (25 g) on ice for 90 min. The beads were washed four times with Tris buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM MgCl 2 , 10 g each of leupeptin and aprotinin, and 0.1 mM phenylmethlylsulfonyl fluoride. The wash buffer was removed, and SDS sample buffer was added. The samples were fractionated on 13% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting for Rho.

RESULTS
GTPase-deficient G 12 ␣ and G 13 ␣, M1R Signaling, and PYK2 Stimulated an SRE Reporter Gene-To determine which activated G-proteins enhance SRE-dependent gene expression in HeLa cells, we transfected constructs that directed the expression of GTPase-deficient forms of G␣ or ␤1 and ␥2 along with a reporter gene that contains five copies of the SRE and a minimal promoter fused to luciferase. Similar to the results reported with NIH 3T3 cells (9), GTPase-deficient G 12 ␣ and G 13 ␣ potently induced SRE-luciferase activity (Fig. 1A). G 12 ␣QL and G 13 ␣QL both increased the reporter gene activity more than 200-fold compared with the basal activity. In contrast, G q ␣QL induced only a 10-fold increase, and the other stimuli had minimal effects, yet G q ␣QL and G s ␣QL substantially elevated the intracellular levels of inositol phosphates and cAMP, respectively, and G i2 ␣QL reduced intracellular levels of cAMP (data not shown).
PYK2 expression also activated the SRE-luciferase reporter construct. While not as efficient as the GTPase deficient forms of G 12 ␣ or G 13 ␣, PYK2 induced a 20 -25-fold increase in SREluciferase activity (Fig. 1B). Signaling to the SRF by activators of heterotrimeric G-proteins requires RhoA, while c-src triggers SRF activation through a RhoA-independent pathway (10). To determine whether PYK2 induces SRE reporter gene activation by activating RhoA, we simultaneously expressed the botulinum C3 exoenzyme. The toxin ADP-ribosylates RhoA at asparagine 41, thereby deactivating it and preventing the activation of downstream effectors (5). Surprisingly, the co-expression of the C3 toxin markedly impaired the ability of PYK2 to activate the SRE reporter construct, indicating a requirement for Rho activation (Fig. 1B).
This result suggested that PYK2 activates the SRE through an SRF-dependent mechanism, a possibility we tested using a TCF-insensitive SRE-CAT construct (5). TCF can not bind because of a mutation in the TCF binding site (pSREmutL). PYK2 readily activated this construct (Fig. 1C). Furthermore, C3 toxin and a specific RhoA inhibitor, which inhibits the dissociation of GDP from RhoA (RhoGDI) (19), both inhibited the activation. As expected, both C3 toxin and RhoGDI blocked activation of the reporter gene by p115 RhoGEF, a Rho exchange factor whose expression activates endogenous Rho (Fig.  1D). Thus, in HeLa cells, PYK2 expression activates the SRE reporter gene at least in part through an SRF-and RhoA-dependent pathway.
PYK2 Involvement in SRE-mediated Transcription Induced by G 12 ␣, G 13 ␣, and M1R Signaling-If PYK2 participates in G 12 ␣and G 13 ␣-induced SRE transcription in HeLa cells, a kinase inactive form of PYK2 (PYK2 KD) should inhibit the response. The same construct attenuates SAPK and p38 activation by stress signals (17,18) and ERK activation by angiotensin II signaling (22), thereby implicating PYK2 in these signaling pathways. We found that the co-expression of PYK2 KD significantly impaired G 12 ␣QLand G 13 ␣QL-induced activation of the SRE-reporter ( Fig. 2A). In both instances, the PYK2 KD construct inhibited SRE activation nearly as well as did the C3 toxin. A similar analysis of M1R signaling to the SRE revealed a similar inhibition (Fig. 2B). To verify that M1R signaling triggered the SRE reporter through a G 12 /G 13 -linked signaling pathway in HeLa cells, we repeated the M1R signaling experiments in the presence of the RGS domain of p115 RhoGEF (p115 RGS), a GTPase-activating protein for G 12 ␣ and G 13 ␣ (7). The expression of p115 RGS nicely blocked the activation of the SRE reporter construct induced by signaling through the M1R (Fig. 2C). These data indicate that GPCRs, which couple to G 12 ␣ and G 13 ␣, can activate SRF-dependent transcription through a PYK2-dependent pathway. G 13 ␣QL and to a Lesser Degree G 12 ␣QL Activate PYK2-While M1R signaling leads to PYK2 tyrosine phosphorylation and enhanced PYK2 kinase activity (13), the effects of G 12 /G 13 on PYK2 have not been previously reported. To examine their effects, we transfected the PYK2 expression vector in the presence or absence of constructs that directed the expression of G 12 ␣QL or G 13 ␣QL and subjected immunoprecipitated PYK2 to an in vitro kinase using an exogenous substrate, poly(Glu, Tyr) (4:1). Both G 12 ␣QL and G 13 ␣QL enhanced PYK2 kinase activity toward the exogenous substrate; however, G 13 ␣QL induced a higher level (Fig. 3A). We also noted a phosphorylated band that migrated at a rate identical to that of PYK2 in the lysates from stimulated cells. To directly determine if the stimuli triggered PYK2 tyrosine phosphorylation, we analyzed the PYK2 immunoprecipitates by immunoblotting with an anti-phosphotyrosine antibody. While G 13 ␣ and M1R signaling enhanced PYK2 tyrosine phosphorylation, G 12 ␣ had a very modest effect. The expression of G 13 ␣ or G 12 ␣ did not alter PYK2 expression levels. By immunoblotting with PYK2 phosphotyrosine-specific antibodies (Fig. 3B), we discovered that G 13 ␣QL led to the phosphorylation of PYK2 on residues Tyr 402 , a major autophosphorylation site and site of interaction with Src (11); Tyr 579 and Tyr 580 in the kinase domain; and Tyr 881 in the proline-rich region. In contrast, G 12 ␣ failed to induce high levels of reactivity with the antibodies. These results suggest that while both G 13 ␣ and G 12 ␣ activate PYK2, G 13 ␣ is more efficient.
In HeLa Cells, PYK2 Acts Predominantly Upstream of RhoA and Downstream of G 12 /G 13 -The inhibition of M1R, G 12 ␣QL, and G 13 ␣QL triggered SRE reporter gene activity by PYK2 KD did not delineate where in the signaling pathway PYK2 functions. However, the dependence of PYK2-induced SRE reporter gene activity upon RhoA activation implied a role for PYK2 upstream of RhoA. To test that prediction, we examined whether the PYK2 KD construct inhibited RhoA-induced SRE reporter gene activity. We found that the same concentration of PYK2 KD that inhibited G 13 ␣QL-induced SRE reporter gene activity by 75% only reduced RhoA-induced reporter gene activity by 10 -20% (Fig. 4A). Furthermore, G 13 ␣QL enhanced PYK2 kinase activity independent of RhoA activation, because C3 toxin co-expression had no effect on the PYK2 kinase activity (data not shown).
Since a tyrosine phosphorylation event precedes the translocation of RhoA to the plasma membrane (23), PYK2 expression could alter the intracellular location of RhoA (Fig. 4B). Indeed, the expression of PYK2 but not PYK2 KD in COS-7 cells enhanced the amount of endogenous RhoA found in a crude membrane fraction. In contrast, neither the wild type PYK2 nor the kinase defective form altered the amount of G i ␣ in the membrane preparations. This led us to examine whether PYK2 expression increased the amount of activated Rho in HeLa cells. To do so, we used the RBD of Rhotekin to affinityprecipitate GTP-Rho (21) from HeLa cells lysates expressing RhoA and PYK2 (Fig. 4C). As controls, we performed similar experiments with PYK2 KD or p115 RhoGEF. We found that PYK2 KD did not alter the basal amount of affinity-precipitated GTP-Rho; however, both wild type PYK2 and the positive control, p115 RhoGEF, increased the amount. Thus, the ex- Finally, to verify the specificity of p115 RGS and to analyze the effect of RhoA on PYK2 activation, we expressed GTPasedeficient forms of G 12 ␣, G 13 ␣, G q ␣, RhoA, and M1R in the presence of PYK2 and p115 RGS or not. By binding to GTP-G 12 ␣ and GTP-G 13 ␣, p115 RGS should inhibit their ability to stimulate downstream effectors (7). We found that co-expression of p115 RGS blocked PYK2 activation by GTPase-deficient forms of G 13 ␣ and G 12 ␣ but not by GTPase-deficient G q ␣ (Fig.  5). Suggesting that in HeLa cells G 12 /G 13 activation was important for M1R signaling to PYK2, p115 RGS markedly inhibited PYK2 activation. Consistent with PYK2 acting predomi-nantly upstream of RhoA, activated RhoA had a minimal effect on PYK2 kinase activity. G 13 ␣ and PYK2 Associate in Vivo-The activation of G 13 ␣ could either directly or indirectly lead to PYK2 activation. If G 13 ␣ directly stimulated PYK2, we should be able to detect an interaction between the two proteins. To test whether the two proteins interact, we co-transfected HeLa cells with G 13 ␣QL and PYK2. G 13 ␣QL was epitope-tagged with HA, which was useful for immunoprecipitating the protein but not for immunoblotting. We analyzed either HA or FLAG (control) antibody immunoprecipitates for the presence of PYK2 using a Myc tag-specific antibody, which recognized Myc-tagged PYK2 (Fig.  6A). We found that the HA-G 13 ␣QL immunoprecipitates contained readily detectable PYK2, while the control FLAG immunoprecipitates did not. In similar experiments using HA-tagged G 12 ␣QL and PYK-2, the two proteins did not co-immunoprecipitate. We confirmed the presence of G 12 ␣QL and G 13 ␣QL in the immunoprecipitates by immunoblotting with G-protein specific antibodies. Conversely, we examined Myc-Pyk2 immunoprecipitates for the presence of G 12 ␣QL or G 13 ␣QL via immunoblotting with G-protein-specific antibodies (Fig. 6B). Again, G 13 ␣QL readily co-immunoprecipitated with PYK2, while G 12 ␣QL did not.
These results indicate that GTP-G 13 ␣ can associate in vivo with PYK2. To provide better evidence that this is the case, we determined whether G-protein activation leads to a detectable FIG. 2. PYK2 KD inhibits G 12 ␣QL-, G 13 ␣QL-, and carbacholinduced SRE reporter gene activity. A, PYK2 KD inhibited G 12 ␣QLand G 13 ␣QL-induced SRE reporter gene activity. HeLa cells transfected with constructs directing expression of G 12 ␣QL (0.05 g) or G 13 ␣QL (0.05 g) in the presence or absence of PYK2 KD (0.5 or 1 g) or C3 toxin (0.25 g) were assessed for SRE-luciferase activity. The levels of the expressed proteins were detected by immunoblotting. Data are from three experiments. B, PYK2 KD inhibited carbachol signaling through the M1R to the SRE reporter. SRE-luciferase activity was detected in HeLa cells expressing M1R (0.5 g), PYK2 KD (0.5 or 1 g), and C3 (0.25 g) as indicated. The cells were exposed to carbachol (100 M) for the last 5 h of the culture. Data are from two experiments. C, p115 RGS blocks carbachol signaling through the M1R to the SRE reporter. SRE-luciferase activity was detected in HeLa cells expressing M1R (0.2 g) and p115 RGS (0.5, 1.0, or 1.5 g) or not. The cells were exposed to carbachol (100 M) for the last 5 h of the culture. Data are representative of two experiments performed. G 13 ␣-PYK2 complex in nontransfected HeLa cells. We used a nonspecific activator of heterotrimeric G-proteins, treatment with aluminum fluoride, to stimulate endogenous heterotrimeric G-proteins and then examined PYK2 immunoprecipitates for the presence of G 13 ␣ by immunoblotting (Fig. 6C). We found a clear band reactive with the G 13 ␣ antisera, which migrated with the appropriate molecular mass when we used cells that had been treated with aluminum fluoride for 30 min prior to lysis. Control immunoprecipitates failed to contain any such band. Thus, the activation of G 13 ␣ probably leads to its transient association with PYK2 in PYK2-expressing cells.

DISCUSSION
Three sets of observations support our conclusion that G 13 ␣ can use PYK2 to link to downstream signaling pathways. First, in HeLa cells, overexpression of either PYK2 or G 13 ␣QL activates SRF through a RhoA-dependent mechanism, and a kinase-inactive form of PYK2 blocks G 13 ␣QL from inducing SREdependent transcription. This inhibition appears specific, since the kinase-inactive PYK2 does not block the triggering of SREdependent transcription by RhoA. Second, expression of GT-Pase-deficient G 13 ␣ activates PYK2 as assessed by an in vitro kinase assay using a known PYK2 substrate and by immuno-blotting with phosphorylation state-specific antibodies. The latter verifies that G 13 ␣ triggers tyrosine phosphorylation of the major PYK2 autophosphorylation site. Furthermore, the RGS domain of p115 RhoGEF impairs PYK2 activation by either M1R or G 13 ␣QL. Third, GTP-G 13 ␣ and PYK2 interact. In cells expressing PYK2 and G 13 ␣QL, the two proteins readily co-immunoprecipitate. In contrast, a similar interaction did not occur when we substituted G 12 ␣QL for G 13 ␣QL. Confirming that a physiologically relevant G 13 ␣/PYK2 interaction occurs, the activation of endogenous heterotrimeric G-proteins leads to a rapid association between G 13 ␣ and PYK2. While the easy detection of G 13 ␣ with PYK2 indicates that PYK2 could be a direct G 13 ␣ effector, the activation of PYK2 may be secondary to other signaling pathways that G 13 ␣ stimulates. We are currently mapping the regions in PYK2 necessary to detect the interaction with G 13 ␣.
We also find that G 12 ␣QL expression activates PYK2 and that PYK2 KD interferes with the activity of an SRE reporter following G 12 ␣QL expression in HeLa cells. G 12 ␣QL induces a very high level of SRE reporter gene activity even greater than that we observe with G 13 ␣QL. However, an interaction between G 12 ␣QL and PYK2 sufficient to allow the proteins to co-immunoprecipitate does not occur, suggesting a fundamental difference in how G 12 ␣ and G 13 ␣ activate PYK2. Further studies will be needed to illuminate how G 12 ␣ and G 13 ␣ activate PYK2.
A recent study implicates Tec/Bmx nonreceptor tyrosine kinases in the regulation of Rho and serum response factor by G 12 ␣/G 13 ␣ (10). These results parallel our work with PYK2 in HeLa cells. Several explanations could reconcile the two studies. Since a kinase defective Bmx more readily impairs SRF activation by G 12 ␣QL (10), while kinase-deficient PYK2 better inhibits SRF activation by G 13 ␣QL (our data), G 12 ␣ may preferentially use Tec kinases to activate the SRF, while G 13 ␣ uses PYK2 for a similar purpose. Because different cell types ex- press varying amounts of the Tec kinases and PYK2, in some cell types G 13 ␣ may largely use PYK2 and in others it may use Tec kinases to activate SRF, whereas in some cells it may use both kinases. Arguing that both kinases may contribute to SRF activation, Tec kinase or PYK2 overexpression enhances SRE reporter activity significantly less than does GTPase-deficient G 12 ␣/G 13 ␣. To examine the potential for Tec kinases to activate the SRF in HeLa cells, we have tested whether Bruton's tyrosine kinase (BTK) enhances transcription of the SRE-luciferase reporter. Arguing that expression of BTK should be functionally equivalent to the expression of other Tec kinases, Bmx, Txk, Tec, and Itk all reconstitute BTK signaling in cell lines deficient for BTK (24). We find that a constitutively active form of BTK induces only a 2-3-fold increase in reporter gene activ-ity, while wild type PYK2 induces a 20-fold enhancement; however, the combination of PYK2 and BTK leads to a 60-fold increase. Indicating that is not downstream of BTK, the expression of PYK2 KD does not block SRE activation by BTK. 2 How do Tec kinases and PYK2 lead to Rho activation and hence the SRF? The basal activity of Tec kinase and PYK2 in NIH 3T3 and HeLa cells leads to SRF activation in the respective cell type (Ref. 10 and our data). In both instances, the activation of SRF requires functional kinase domains and Rho activation, implying the need for the phosphorylation of downstream effectors and for GTP-Rho. Both Tec kinase and PYK2 enhance the translocation of Rho to COS-7 cell membranes, results consistent with the inhibition of Rho translocation by tryosine kinase inhibitors (23). Tec overexpression induces cytoskeletal reorganization in NIH 3T3 cells (10), and PYK2 overexpression increases the amount of GTP-Rho in HeLa cells. Thus, both Tec kinase and PYK2 can cause Rho activation. RhoGEFs, RhoGDIs, and RhoGAPs control Rho activity (GTPversus GDP-bound state) by triggering the exchange of GTP for GDP, inhibiting the release of GDP from RhoA, or by enhancing the rate that GTP-Rho hydrolyzes GTP back to GDP, respectively (reviewed in Ref. 25). G 13 ␣ itself can stimulate Rho by activating RhoGEFs. Tec kinases and PYK2 or one of their targets could regulate Rho activity by potentiating G 13 ␣'s activation of the exchange factors. While the Tec TH domain binds to VAV, a hematopoietic specific exchange factor for RhoA, Rac, and Cdc42 (25), no evidence currently links PYK2 to RhoGEFs. Further studies will determine whether PYK2 enhances their activity. Alternatively, PYK2 or Tec kinase could either directly or indirectly deactivate RhoGDIs or RhoGAPs, which would enhance the likelihood of RhoA being GTP-bound and able to activate downstream effectors.
Besides involvement in SRF activation by G 13 ␣ in some cell types, PYK2 could also participate in G 13 ␣'s induction of SAPKs. G 12 ␣ and G 13 ␣ activate SAPK via different mechanisms depending upon the cell type. In COS-7 cells, they activate SAPK through Cdc42, while in NIH 3T3 cells (26) and COS-1 cells Ras is critical (27). In HEK 293 cells, a dominant negative form of RhoA and C3 exoenzyme blocks G 12 ␣-mediated SAPK activation as does C-terminal Src kinase (Csk), suggesting the additional involvement of a Src kinase (28). In NIH 3T3 cells, a dominant negative Ras inhibits G 12 ␣-mediated SAPK activation, but it fails to do so in HEK 293 cells, where a dominant negative form of Rac does so (29). These differences probably arise because of the differing availability of downstream mediators of G 12 ␣ and G 13 ␣ signaling. In those cells that express PYK2, we would expect that G 13 ␣ activation should lead to PYK2 and Src kinase activation, both of which can contribute to SAPK activation.
Activation of certain heterotrimeric G-proteins causes neurite retraction and cell rounding through a RhoA-dependent pathway (30). In PC12 cells, the induction of neurite retraction and cell rounding by GTPase-deficient G 12 ␣, G 13 ␣, or G q ␣ also depends upon RhoA (31). The tyrosine kinase inhibitor tyrphostin A25 blocks the morphological changes triggered by GTPasedeficient G 13 ␣ or G q ␣ but not by G 12 ␣, while the inhibition of protein kinase C or elimination of extracellular calcium inhibits the morphological changes induced by GTPase-deficient G q ␣ but not by G 12 ␣ or G 13 ␣ (32). Furthermore, lysophosphatidic acid induces neurite retraction through a G 12 /G 13 -initiated pathway that requires tyrosine kinase activity (33). Since PC12 and neuronal cells express high levels of PYK2 (32), its activation by G q ␣ via the release of intracellular calcium or by G 13 ␣ through a yet unknown mechanism could account for these 2 C-S. Shi, unpublished observations. FIG. 6. PYK2 interacts with G 13 ␣. A, PYK2 immunoprecipitated with G 13 ␣QL. HeLa cells were co-transfected with constructs directing the expression of Myc-PYK2 (1 g) and HA-G 12 ␣QL (1 g) or HA-G 13 ␣QL (1 g). HA (G-protein) or FLAG (control) immunoprecipitates were immunoblotted for Myc (PYK2), G 12 ␣ or G 13 ␣. PYK2 and Gprotein expression is shown in the cell lysates. B, G 13 ␣QL immunoprecipitated with PYK2. Similar experiment as shown in A except that Myc (PYK2) or FLAG (control) immunoprecipitates were immunoblotted for Myc (PYK2), G 12 ␣, or G 13 ␣. C, endogenous PYK2 and G 13 ␣ co-immunoprecipitated following G-protein activation. HeLa cells were treated with AlF 4 Ϫ or not prior to lysis. PYK2 or control immunoprecipitates were immunoblotted for PYK2 (left panel) or G 13 ␣ (right panel). PYK2 levels are shown in the cell lysate. W.B., Western blot; I.P., immunoprecipitation; Ab., antibody.
results. Finally, in PC12 cells transfected with the ␣-1A adrenergic receptor, norepinephrine activates mitogen-activated protein kinase pathways and triggers PYK2 tyrosine phosphorylation independent of calcium release or protein kinase C activation (34). These results suggest that either GTP-G q can stimulate PYK2 through a pathway not dependent upon calcium release or protein kinase C or that the ␣-1A adrenergic receptor uses G 12 ␣/G 13 ␣ to activate PYK2.
Poised to receive activation signals from GPCRs both via the activation of G q ␣ and G 13 ␣ and located at a critical juncture in many signaling pathways, PYK2 processes upstream information to coordinate the activation of downstream signaling pathways, which alter the activity of transcription factors such as SRF and TCF. Interestingly, also subject to many of the same input signals including G q ␣ and G 12 ␣/G 13 ␣, the Tec kinases link to many of the same downstream signaling pathways that PYK2 links to. Whether PYK2 and Tec kinases cross-regulate each other is an interesting question for future studies.