The calcium-dependent activity of large-conductance, calcium-activated K+ channels is enhanced by Pyk2- and Hck-induced tyrosine phosphorylation

Shizhang Ling, Jian-Zhong Sheng, and Andrew P. Braun

Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Submitted 20 January 2004 ; accepted in final form 28 April 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent results showing that large-conductance, calcium-activated K+ (BKCa) channels undergo direct tyrosine phosphorylation in the presence of c-Src tyrosine kinase have suggested the involvement of these channels in Src-mediated signaling pathways. Given the important role for c-Src in integrin-mediated signal transduction, we have examined the potential regulation of BKCa channels by proline-rich tyrosine kinase 2 (Pyk2), a calcium-sensitive tyrosine kinase activated upon integrin stimulation. Transient coexpression of murine BKCa channels with either wild-type Pyk2 or hematopoietic cell kinase (Hck), a Src-family kinase, led to an enhancement of BKCa channel activity over the range of 1–10 µM free calcium, whereas coexpression with catalytically inactive forms of either kinase did not significantly alter BKCa gating compared with channels expressed alone. In the presence of either wild-type Pyk2 or Hck, BKCa {alpha}-subunits were found to undergo tyrosine phosphorylation, as determined by immunoprecipitation and Western blotting strategies. However, tyrosine phosphorylation of the BKCa {alpha}-subunit was not detected for channels expressed alone or together with inactive forms of either Pyk2 or Hck. Interestingly, wild-type, but not inactive, Pyk2 was also present in BKCa channel immunoprecipitates, suggesting that Pyk2 may coassociate with the BKCa channel complex after phosphorylation. Collectively, the observed modulation and phosphorylation of BKCa channels by Pyk2 and a Src-family kinase may reflect a general cellular mechanism by which G protein-coupled receptor and/or integrin activation leads to the regulation of membrane ion channels.

BK channels; tyrosine kinase; calcium; immunoprecipitation


THE MODULATION OF ION CHANNEL ACTIVITIES by phosphorylation/dephosphorylation events, protein-protein interactions, or membrane insertion/retrieval in response to extracellular stimuli plays an important role in the regulation of diverse physiological processes, including muscle contraction and neurotransmission (9, 23, 43). Cell surface molecules, such as G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and integrins, initiate signal transduction cascades involving serine/threonine and/or tyrosine phosphorylation events (3, 29, 39), and recent studies have demonstrated a role for protein tyrosine kinases (PTKs) in the control of membrane excitability via the regulation of voltage- and ligand-gated ion channels (9).

PTKs, such as focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), and Src-family kinases, are important components of integrin signaling complexes in most, if not all, cell types (13, 45). Initially described as cell adhesion receptors, integrins are composed of {alpha}- and {beta}-subunits linked in a noncovalent manner to form transmembrane heterodimers. Ligand binding or clustering of integrins by components of the extracellular matrix (ECM) leads to "outside-in" signal transduction and the activation of various protein kinases, including FAK, c-Src, Pyk2, and MAP kinases (20). Integrins present in the vasculature play an important role in the mechanotransduction pathways regulating angiogenesis (21), vascular tone, and myogenic responsiveness (7, 8). A number of studies have further reported that many of the functional consequences of integrin activation involve the modulation of voltage-dependent Ca2+ and K+ channels in either vascular endothelium or smooth muscle (for review, see Ref. 10).

Previously, our laboratory (26) and other investigators (1) reported that the activity of large-conductance, calcium-activated K+ (BKCa) channels may be modulated via phosphorylation by the prototypical tyrosine kinase c-Src. This observation, together with the important role of c-Src in integrin signaling pathways, the observed integrin-dependent modulation of ion channels, and the impact of BKCa channel activity on smooth muscle contractility, suggested the possibility that BKCa channels may undergo functional modulation by integrin-associated protein kinases. In addition to c-Src, several other major protein kinases are known to associate with integrin complexes, including integrin-linked kinase (ILK), FAK, and Pyk2. ILK is a recently identified serine kinase (16) that plays a critical role in the integrin-dependent activation of protein kinase B/Akt and downstream effectors, such endothelial nitric oxide synthase (44, 47). FAK and Pyk2 are closely related members of the same tyrosine kinase family (15, 38) and are distinct from Src-family kinases. FAK appears to be expressed ubiquitously and undergoes activation/autophosphorylation in response to integrin stimulation. Pyk2 displays more limited tissue distribution (hematopoietic cells, vascular smooth muscle and endothelium, spleen, and kidney, but enriched in the central nervous system) (2, 22). Protein kinase C appears to mediate the activation of Pyk2 by various calcium-mobilizing stimuli (14); however, the underlying molecular mechanism remains unclear. Recent observations have further suggested that Pyk2 may serve as a common link between integrin and GPCR signaling pathways, because Pyk2 becomes translocated to focal adhesions in response to either GPCR activation or cell adhesion to ECM proteins (27).

The regulation of ion channel activities by integrin- and Pyk2-dependent signaling pathways has been described in both neurons (12, 17, 19) and vascular smooth muscle (10), cell types in which BKCa channels have functionally important roles (32, 36, 37, 48). Collectively, these observations raised the possibility that BKCa channel activity may also undergo modulation by integrin-associated protein kinases, such as Pyk2 and the Src-family kinase Hck (hematopoietic cell kinase). To directly examine this question, we transiently expressed murine BKCa channels in HEK-293 cells alone or together with either wild-type or catalytically inactive forms of Pyk2 or the Src-family kinase Hck. We observed that Pyk2 or Hck coexpression enhanced the calcium-dependent gating of BKCa channels and that this enhancement was associated with tyrosine phosphorylation of the BKCa {alpha}-subunit, as detected by direct immunoprecipitation and Western blotting. Inactive forms of Pyk2 or Hck did not induce channel phosphorylation or alter BKCa currents compared with channels expressed alone. These findings thus describe a novel mechanism by which tyrosine kinases associated with GPCR and integrin signaling pathways contribute to the regulation of mammalian BKCa channels.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and chemicals. Lipofectamine and high-glucose-containing Dulbecco's modified Eagle's medium cell culture medium were purchased from Invitrogen. The anti-phosphotyrosine mouse monoclonal antibodies 4G10 and PY20 and the anti-hemagglutinin (HA) tag monoclonal antibody 12CA5 were purchased from Upstate Biotechnology (Lake Placid, NY) and Transduction Labs (Lexington, KY), respectively. The anti-Hck monoclonal antibody was purchased from BD Biosciences (Mississauga, ON, Canada). The anti-mSlo rabbit polyclonal antibody and horseradish peroxidase-linked goat anti-mouse and goat anti-rabbit secondary antibodies were obtained from Chemicon International (Temecula, CA). The SuperSignal chemiluminescence detection reagent was purchased from Pierce Chemical (Rockford, IL). Chemicals used in the preparation of solutions for electrophysiological recordings and Ca2+ ionophore A-23187 were from Sigma-Aldrich (Oakville, ON, Canada). A Lowry-style protein assay kit (detergent compatible), nitrocellulose membrane (0.2-µm pore diameter), and SDS-PAGE reagents were purchased from Bio-Rad Laboratories (Hercules, CA).

Construction and transfection of cDNA plasmids. Expression vectors containing cDNAs encoding the murine mSlo {alpha}-subunit (46) and wild-type green fluorescent protein (GFP) (5) were recently described (4, 26). Wild-type and catalytically inactive forms of Pyk2, in the pRK5 expression vector, containing a COOH-terminal HA epitope tag, were generously provided by Dr. Sima Lev (Department of Neurobiology, The Weizmann Institute, Rehovot, Israel). Wild-type and catalytically inactive forms of p59Hck in the LNCX expression vector were obtained from Dr. Stephen Robbins (University of Calgary) (35).

Transient transfection of HEK-293 cells was carried out as previously described (4, 26). After cells were replated onto sterile glass coverslips, electrophysiological recordings were typically performed on days 2–4 following transfection (day 1). For biochemical studies, cells were detached from 35-mm dishes on day 2 and then replated onto 100-mm culture dishes to prevent overgrowth. These cells were then harvested on days 3 and 4 following transfection.

Electrophysiology. Macroscopic BKCa channel currents were recorded at 35 ± 0.5°C from excised inside-out membrane patches of HEK-293 cells with the use of an Axopatch 200B patch-clamp amplifier and pCLAMP 7 software, as recently described (25). Briefly, membrane currents were activated by voltage-clamp pulses delivered from a holding potential of either –60 or –120 mV to membrane potentials ranging from –180 to 240 mV; tail currents were recorded at +50, –80, or –120 mV. Micropipettes were filled with a solution containing (in mM) 5 KCl, 125 KOH, 1 MgCl2, 1 CaCl2, and 10 HEPES, with pH adjusted to 7.3 with methanesulfonic acid, and had tip resistances of 2–4 M{Omega}. The bath solution contained (in mM) 5 KCl, 125 KOH, 1 MgCl2, 2 EGTA or HEDTA, and 10 HEPES, with pH adjusted to 7.2 with methanesulfonic acid; variable amounts of a 0.1 M CaCl2 solution were added to give the desired free calcium concentrations. The level of free calcium in each solution was confirmed using a calcium electrode (Orion model 93-20) with calibration standards (WPI, Sarasota, FL) ranging from pCa 8 to 2. The recording chamber (~0.3-ml volume) was perfused at a constant rate of 1–1.5 ml/min, using a set of manually controlled solenoid valves to switch between various solutions. Individual cells expressing BKCa channels were then identified visually by coexpression of the marker protein GFP under epifluorescence with the use of 480-nm excitation and 510-nm emission filters.

Western blotting. Transfected cells were detached on day 3 by brief incubation with sterile PBS containing 0.05% trypsin-0.5 mM EDTA, centrifuged in 15-ml culture tubes at ~100 g for 5 min, and stored at –80°C as intact cell pellets. These pellets were suspended in 0.5–1 ml of ice-cold lysis buffer [20 mM Tris·HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, 1% (vol/vol) Triton X-100, 1 mM EGTA, 2 mM EDTA, 1 mM DTT, 1 mM benzamidine, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 5 µg/ml each of leupeptin, aprotinin, and pepstatin A], followed by incubation on ice for 20–30 min. Crude lysates were centrifuged at 16,000 g in a microcentrifuge at 4°C for 20 min, and the protein concentration of the resulting supernatants was measured using a modified Lowry procedure (28). Supernatant fractions and immunoprecipitates (see Immunoprecipitation of BKCa channels or phosphotyrosine-containing proteins) were mixed with Laemmli sample buffer containing 0.5% (vol/vol) {beta}-mercaptoethanol and incubated for 20–30 min at 70°C, and the proteins were then separated by denaturing SDS-PAGE. The resolved proteins were electrotransferred to nitrocellulose membrane at 4°C overnight in a buffer containing 25 mM Tris, 192 mM glycine, 0.1% (wt/vol) SDS and 20% (vol/vol) methanol. Membranes were briefly rinsed in a buffer containing 20 mM Tris·HCl, pH 7.4, 150 mM NaCl, and 0.1% (vol/vol) Tween 20 (TTBS), incubated at room temperature for 20–30 min in TTBS containing 5–10% (wt/vol) skim milk powder to block nonspecific binding of antibodies, and then rinsed three times for 5 min each in TTBS. Incubation of membranes with primary antibodies was carried out in TTBS containing 1% (wt/vol) skim milk powder for 1–2 h at room temperature, followed by three to five washes for 10 min each with TTBS alone. Membranes were then incubated for ~1 h with the appropriate secondary antibody, also diluted in TTBS containing 1% (wt/vol) skim milk powder, followed by three to five washes for 5 min each with TTBS. After the final wash, blots were immediately developed by applying the SuperSignal chemiluminescence reagent for 3–5 min and were then exposed to X-ray film (Hyperfilm; Amersham).

Immunoprecipitation of BKCa channels or phosphotyrosine-containing proteins. BKCa channels and phosphotyrosine-containing proteins were immunoprecipitated as follows. Supernatants prepared from cell lysates were diluted to 0.4–0.6 mg protein/ml, and a 1.4-ml aliquot of the diluted material was transferred to a microcentrifuge tube. Bovine serum albumin (BSA) was added to a final concentration of 1 mg/ml, and the samples were then precleared by addition of 40 µl of a 50% slurry (vol/vol) of rehydrated protein A-Sepharose beads (Amersham Pharmacia), followed by rotation at 4°C for 2 h. Samples were centrifuged for 5 min at ~8,000 g to pellet the beads, and the soluble material was transferred to a clean microcentrifuge tube. Precleared supernatants were then incubated for 4–16 h at 4°C with either ~1.5 µg of anti-BKCa channel antibody or 3–5 µg of anti-phosphotyrosine antibody (4G10 + PY20), followed by further incubation for 2 h with 30 µl of protein A-Sepharose beads (50% slurry). The beads were pelleted by centrifugation at ~2,000 g for 5 min at 4°C and then washed three times by resuspension in 1 ml of wash buffer containing 20 mM Tris·HCl, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM DTT, 1 mM EDTA, 0.2 mM EGTA, 1 mM benzamidine, 0.5 mM PMSF, 0.1% (vol/vol) Triton X-100, and 2 µg/ml each of aprotinin, leupeptin, and pepstatin A. The beads were then resuspended in 30–40 µl of Laemmli sample buffer, heated to 70°C for 20–30 min, and centrifuged at ~2,000 g for 5 min. The soluble proteins were then resolved by SDS-PAGE and analyzed by Western blotting.

Statistical analysis. Values of the membrane voltage producing half-maximal activation (V1/2) for BKCa channels expressed alone or together with either wild-type or catalytically inactive Pyk2 or Hck (refer to Fig. 4) were examined statistically using a one-way analysis of variance. Pairwise comparisons were made using an unpaired Student's t-test, and differences between values were considered to be statistically significant at a level of P < 0.05.



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Fig. 4. Plot of half-maximal voltages of activation (V1/2) for coexpressed BKCa channels vs. the concentration of free cytosolic calcium. BKCa channels were expressed alone or together with WT Pyk2, dead Pyk2, WT Hck, or dead Hck. V1/2 values were derived from single Boltzmann functions fit to the normalized conductance-voltage curves, as described in Fig. 3. Plotted values are expressed as means ± SE of 3–8 individual measurements under each of the conditions described. For the purpose of clarity, symbols representing BKCa channels coexpressed with either active or dead Pyk2 or Hck have been horizontally offset from the symbol for BKCa channel alone. The experimental free calcium concentration for each cluster of V1/2 values is as follows: ~5 nM, 1 µM, 4 µM, 10 µM, and 100 µM. V1/2 values for BKCa channels coexpressed with WT Pyk2 or Hck are significantly different from those for BKCa channels expressed alone (*P < 0.05); no statistical differences were noted between V1/2 values for BKCa channels expressed in either the absence or presence of dead Pyk2 or Hck.

 

    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The potential modulation of BKCa channel activity by integrin-associated signaling molecules (i.e., Pyk2 and a Src-family tyrosine kinase) was investigated in a cultured cell system by transient coexpression of murine BKCa channels with either wild-type or catalytically inactive ("dead") forms of human Pyk2 or human Hck in HEK-293 cells. The protein expression profiles of transfected cells were then confirmed by Western blot analysis of total cellular lysates (Fig. 1). The pore-forming BKCa {alpha}-subunit was detected as an ~120-kDa immunoreactive band, and the level of protein expression was not noticeably altered by cotransfection with cDNAs encoding either Pyk2 or Hck (Fig. 1A). Previously, our laboratory (26, 42) had been unable to detect BKCa channel immunoreactivity in untransfected HEK-293 cells. With the use of kinase-specific antibodies, immunoreactive bands of ~130 and ~60 kDa were detected in cells cotransfected with cDNAs encoding the wild-type and inactive forms of Pyk2 and Hck, respectively (Fig. 1, B and C). Hck immunoreactivity was not detected in untransfected HEK-293 cells, because this kinase is typically expressed in cells of hematopoietic origin (33, 49). Similarly, Pyk2 expression was observed only in transfected cells, because the anti-HA tag antibody specific for the recombinant kinase would not be expected to detect any endogenous Pyk2 that might be present in HEK-293 cells.



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Fig. 1. Expression of large-conductance, calcium-activated K+ (BKCa) channels in HEK-293 cells in the absence or presence of coexpressed wild-type (WT) or catalytically inactive ("dead") forms of Pyk2 or Hck. Whole cell lysates were prepared from transiently transfected HEK-293 cells (see top for transfection conditions), and equivalent amounts of protein were analyzed by Western blotting for the expression of BKCa channels ({alpha}-BK; A), WT and dead Pyk2 ({alpha}-Pyk2; B), and Hck ({alpha}-Hck; C). To demonstrate the expected enzymatic activity of coexpressed Pyk2 or Hck, or lack thereof, cellular lysates were probed with an anti-phosphotyrosine antibody ({alpha}-pTyr; D). Under 2 conditions, cotransfected cells were briefly stimulated by the calcium ionophore A-23187 (1 µM for 5 min) before lysis. The relative expression of the ~120-kDa BKCa channel {alpha}-subunit under the various transfection conditions, as quantified by scanning densitometry, is as follows (values expressed as means ± SD; n = 4): BK alone, 1.0; BK + Pyk2, 1.21 ± 0.22; BK + dead Pyk2, 1.48 ± 0.64; BK + Pyk2 + A-23187, 1.28 ± 0.29; BK + dead Pyk2 + A-23187, 1.37 ± 0.40; BK + Hck, 0.97 ± 0.30; BK + dead Hck, 1.27 ± 0.36. The electrophoretic mobilities of molecular mass markers (in kDa) are indicated at right.

 
To demonstrate that both Pyk2 and Hck exhibited enzymatic activity after coexpression with BKCa channels, we probed a Western blot of the same samples shown in Fig. 1A with a monoclonal antibody (4G10) recognizing phosphotyrosine-containing proteins (Fig. 1D). Cells expressing either wild-type Pyk2 or Hck displayed robust anti-phosphotyrosine immunoreactivity compared with cells transfected with BKCa channels alone. Importantly, cells coexpressing a dead form of either Pyk2 or Hck displayed only modest levels of tyrosine-phosphorylated proteins, similar to that observed in cells expressing only BKCa channels. In cells cotransfected with Pyk2, a calcium-sensitive tyrosine kinase (22), elevation of intracellular calcium by exposure to the ionophore A-23187 (1 µM, 5-min exposure) did not noticeably increase the overall level of phosphotyrosine-containing proteins. Collectively, the results shown in Fig. 1 confirm the coexpression of BKCa channels with either Pyk2 or Hck in HEK-293 cells, along with the expected enzymatic activity of both wild-type and dead kinase isoforms.

Coexpression of BKCa channels with either Pyk2 or Hck enhances steady-state gating. To examine the functional consequence of either coexpressed Pyk2 or Hck catalytic activity on BKCa channel gating, we recorded macroscopic currents in excised inside-out membrane patches from transfected HEK-293 cells. As shown in Fig. 2, coexpression of BKCa channels with either wild-type Pyk2 (B) or Hck tyrosine kinase (D) led to an enhancement of channel activity in the presence of 4 µM cytosolic free calcium. However, BKCa channels coexpressed with catalytically inactive, or dead, forms of either tyrosine kinase (Fig. 2, C and E) did not display enhanced gating compared with BKCa channels expressed alone. Figure 2 further shows that only very low levels of membrane current were recorded in HEK-293 cells expressing wild-type Pyk2 alone (F), indicating that Pyk2 activity did not dramatically increase an endogenous background current in transfected cells. Similar results also were observed for cells transfected with wild-type Hck alone (data not shown).



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Fig. 2. Coexpression of BKCa channels with either Pyk2 or Hck tyrosine kinase enhances steady-state channel gating. A–E: representative current traces of BKCa channel activity recorded from different cells transfected using the indicated conditions. F: membrane currents recorded from a cell transfected with WT Pyk2 alone. Macroscopic BKCa currents were recorded from excised inside-out membrane patches in the presence of 4 µM free cytosolic calcium. Voltage-clamp steps were delivered over the range of –90 to +180 mV, in 10-mV increments, from a holding potential of –60 mV; tail currents were recorded at –80 mV (refer to inset for voltage-clamp protocol). Vertical and horizontal scale bars apply to all current traces shown in A–F.

 
After this initial examination, the functional consequences of Pyk2 or Hck coexpression on BKCa channel activity were further characterized over a broader range of membrane voltage and cytosolic free calcium concentrations. Figure 3 shows normalized conductance-voltage (G-V) relationships for BKCa channels expressed in the presence or absence of either wild-type or dead Pyk2 or Hck and recorded over free calcium concentrations ranging from <10 nM to 100 µM. As evidenced by the degree of leftward shift in the steady-state G-V relationships, wild-type Pyk2 and Hck produced significant enhancements of macroscopic currents recorded under the conditions of 1–10 µM free calcium; however, little effect by either kinase was observed in the presence of 100 µM free calcium. Under the same recording conditions, BKCa channels coexpressed with dead Pyk2 or Hck displayed no change in steady-state gating compared with channels expressed alone (see Fig. 3). These kinase-induced shifts in BKCa channel gating are further quantified in a plot of the membrane voltages producing half-maximal activation (V1/2 values) of BKCa current vs. the concentrations of cytosolic free calcium (Fig. 4). As shown, V1/2 values for BKCa channels coexpressed with dead Pyk2 or Hck did not differ from those for channels expressed alone; in contrast, V1/2 values for BKCa channels coexpressed with wild-type Pyk2 or Hck were significantly more negative over the free calcium concentration range of 1–10 µM. Thus, in the presence of 1–10 µM cytosolic calcium, the open probability of BKCa channels is greater at any given voltage in the presence of Pyk2 or Hck, compared with BKCa channels coexpressed with dead Pyk2 or Hck or BKCa channels expressed alone. V1/2 values at both nominally free cytosolic calcium (2 mM EGTA only) and 100 µM free calcium were not statistically different among the various groups.



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Fig. 3. Effects of coexpressed WT or dead Pyk2 (A) or Hck (B) on the voltage- and calcium-dependent gating of BKCa channel activity. Open and filled symbols represent the WT and inactive forms of the coexpressed kinase, respectively. Macroscopic BKCa currents were recorded from inside-out membrane patches over the indicated membrane voltages in response to a wide range of free calcium concentrations [<10 nM (2 mM EGTA), 1 µM, 4 µM, 10 µM, and 100 µM]. Normalized conductance (G/Gmax)-voltage relationships were calculated from tail current amplitudes measured 0.25 ms after voltage-clamp step to a fixed tail potential (e.g., +50, –80, or –120 mV). Data points were fit with a single Boltzmann function (solid line) according to the equation

where Vm is the experimental test potential (in volts), V1/2 is the half-maximal voltage of activation (in volts), defined as the membrane potential at which 50% of the channels are open, and slope is defined as RT/zF, where z is the equivalent gating charge, and RT/F = 26.54 mV at 35°C. Each data point represents the mean ± SE of current measurements recorded from 3–8 excised membrane patches under the indicated voltage and free calcium conditions.

 
BKCa channels undergo direct tyrosine phosphorylation in the presence of Pyk2 or Hck. The observation that BKCa channel gating remained enhanced in excised membrane patch recordings after coexpression with either Pyk2 or Hck strongly suggested that the channel complex had undergone a long-lasting or covalent modification, such as protein phosphorylation. To examine this possibility, we utilized the anti-phosphotyrosine antibodies PY20 + 4G10 to "pull down" or isolate phosphotyrosine-containing proteins from lysates of HEK-293 cells expressing BKCa channels either alone or together with either wild-type or dead Pyk2 or Hck. Immunoprecipitates were resolved by SDS-PAGE and then examined by Western blot analyses. In cells coexpressing BKCa channels with wild-type Pyk2 or Hck, we detected significantly higher levels of phosphotyrosine-containing proteins compared with cells expressing dead kinase or BKCa channels alone (Fig. 5A). The major immunoreactive band visible at ~130 kDa likely corresponds to the expressed form of Pyk2. Importantly, when these same immunoprecipitates were reprobed with an anti-BKCa channel antibody, immunoreactive bands of ~120 kDa were readily detected in samples from cells coexpressing BKCa channels and wild-type Pyk2 or Hck but not in samples from cells expressing inactive kinase (Fig. 5B). Interestingly, prior stimulation of intact cells coexpressing BKCa channels and Pyk2 with the calcium ionophore A-23187 (1 µM for 5 min) led to a modest increase in the intensity of the anti-BKCa immunoreactive band. The reported calcium sensitivity of Pyk2 (22) could explain this observed enhancement of Pyk2-induced phosphorylation. As expected, wild-type Pyk2 and Hck tyrosine kinases both were readily detected as phosphotyrosine-containing proteins (Fig. 5, C and D), most likely due to autophosphorylation of their catalytic sites. In contrast, inactive forms of either Pyk2 or Hck were not detected as phosphoproteins.



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Fig. 5. BKCa channels undergo immunoprecipitation by anti-phosphotyrosine antibodies. A: a Western blot of detergent-soluble proteins immunoprecipitated (IP) from transfected cells by anti-phosphotyrosine antibodies and immunoblotted (IB) with an anti-phosphotyrosine antibody ({alpha}-pTyr). Immunoprecipitates from HEK-293 cells cotransfected with BKCa channels and WT Pyk2 or Hck displayed overall higher levels of protein tyrosine phosphorylation compared with cells transfected with either BKCa channels alone or BKCa channels coexpressed with catalytically inactive Pyk2 or Hck. BKCa channel {alpha}-subunits are clearly detectable in anti-phosphotyrosine immunoprecipitates from cells coexpressing WT Pyk2 or Hck but are absent under all other conditions ({alpha}-BK; B). C and D: antibodies specific for Pyk2 and Hck ({alpha}-Pyk2 and {alpha}-Hck) readily detect the WT but not catalytically inactive forms of these kinases in the corresponding anti-phosphotyrosine immunoprecipitates. The electrophoretic positions of molecular mass markers (in kDa), along with the monomeric antibody heavy chain (IgG), are indicated at right. Results shown are representative of 1 additional experiment that yielded very similar data.

 
Although the above findings are consistent with the tyrosine phosphorylation of BKCa channels by either Pyk2 or Hck, we also considered the possibility that BKCa channels may be complexed with phosphotyrosine-containing accessory proteins, thus leading to indirect coimmunoprecipitation. To address this question, we directly immunoprecipitated BKCa {alpha}-subunits from HEK-293 cells cotransfected with BKCa channels and either wild-type or dead forms of Pyk2 or Hck, as described above. Under all transfection conditions, we detected similar amounts of immunoreactive BKCa channel {alpha}-subunit in each immunoprecipitate (Fig. 6A). Consistent with the above observations, when these BKCa channel immunoprecipitates were further probed with an anti-phosphotyrosine antibody, immunoreactive bands of ~120 kDa were detected in immunoprecipitates from cells coexpressing BKCa channels and wild-type Pyk2 or Hck but not in immunopreciptates from cells expressing inactive forms of these kinases (Fig. 6B). This finding thus provides direct evidence of BKCa channel phosphorylation and is consistent with the data presented in Fig. 5B. Interestingly, probing these same samples with a monoclonal antibody recognizing the expressed form of Pyk2 revealed that wild-type Pyk2, but not the dead isoform, remained coassociated with BKCa channel complexes after immunoprecipitation (Fig. 6C). In contrast, Hck immunoreactivity was not detected in these BKCa channel immunoprecipitates (Fig. 6D); this latter result is thus similar to our earlier data showing that coexpressed c-Src was not present in BKCa channel immunoprecipitates (26). The identity of the ~65-kDa phosphotyrosine-containing protein in the BKCa channel immunoprecipitate from cells coexpressing wild-type Hck is currently unknown. Taken together, the results presented in Figs. 5 and 6 support our initial hypothesis that the BKCa channel {alpha}-subunit undergoes direct tyrosine phosphorylation in the presence of Pyk2 or Hck tyrosine kinase. This phosphorylation may thus account for the enhancement of BKCa channel activity observed in excised membrane patches in the presence of micromolar concentrations of free calcium.



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Fig. 6. BKCa channels undergo tyrosine phosphorylation in the presence of WT but not catalytically inactive Pyk2 or Hck. After lysis and detergent solubilization, BKCa channels were immunoprecipitated from transfected HEK-293 cells (see MATERIALS AND METHODS), and immunoprecipitates were probed by immunoblotting for the BKCa channel {alpha}-subunit ({alpha}-BK; A). To examine potential kinase-dependent modifications, we further probed BKCa channel immunoprecipitates with an anti-phosphotyrosine antibody ({alpha}-pTyr; B). Additional probing of these same immunoprecipitates for interacting proteins revealed the presence of WT but not inactive Pyk2 in BKCa channel immunoprecipitates ({alpha}-Pyk2; C). No Hck immunoreactivity was detected in these same immunoprecipitate samples ({alpha}-Hck; D). IgG denotes the position of the monomeric antibody heavy chain carried over from the immunoprecipitation procedure. The electrophoretic mobilities of molecular mass markers (in kDa) are indicated at left. Results shown are representative of 1 additional experiment that yielded very similar data.

 
In an earlier study from our laboratory (26), Tyr766 in the BKCa {alpha}-subunit was identified as a residue directly phosphorylated by c-Src in situ. To determine whether this same site was also involved in the Pyk2-induced phosphorylation of the BKCa channel observed in situ, we immunoprecipitated wild-type and mutant BKCa channels expressed in either the absence or presence of Pyk2 and performed Western blot analyses. Similar to results shown in Fig. 6B, direct tyrosine phosphorylation of the BKCa {alpha}-subunit was observed for wild-type BKCa channels coexpressed with Pyk2, but no anti-phosphotyrosine immunoreactivity was detected for channels expressed either alone or in the presence of catalytically inactive Pyk2 (Fig. 7A). Interestingly, Pyk2-induced tyrosine phosphorylation of mutant BKCa channels containing single Tyr-to-Phe substitutions at positions 766, 935, or 1027 did not appear to be dramatically decreased compared with wild-type channel phosphorylation. Reprobing these immunoprecipitates with an anti-BKCa channel antibody further demonstrated that similar amounts of BKCa {alpha}-subunit were also present in the channel immunoprecipitates (Fig. 7B).



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Fig. 7. BKCa channels containing Tyr-to-Phe substitutions still undergo Pyk2-dependent tyrosine phosphorylation in situ. WT BKCa channels or mutants containing a single Tyr-to-Phe substitution at position 766, 935, or 1027 (Y766F, Y935F, or Y1027F, respectively) were expressed in HEK-293 cells in either the absence or presence of Pyk2 (as indicated at top) and then directly immunoprecipitated. BKCa channel immunoprecipitates were first probed with an anti-phosphotyrosine antibody ({alpha}-pTyr; A), followed by an antibody recognizing the BKCa {alpha}-subunit ({alpha}-BK; B). The electrophoretic positions of molecular mass markers (in kDa) are indicated at left. Arrowheads at right denote the positions of the tyrosine phosphorylated BKCa {alpha}-subunit (pY-BK), the immunoprecipitated BKCa {alpha}-subunit (BK), and the IgG heavy chain (IgGH).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recently, our laboratory (26) and other investigators (1) reported that BKCa channel activity may be regulated by the prototypic tyrosine kinase c-Src via direct phosphorylation of the BKCa {alpha}-subunit. In the present study, we observed that 1) the BKCa channel {alpha}-subunit undergoes direct tyrosine phosphorylation in situ in the presence of coexpressed wild-type, but not catalytically inactive, forms of either Pyk2 or Hck, and 2) tyrosine phosphorylation of the BKCa {alpha}-subunit is associated with enhancement of the calcium-dependent gating of BKCa current. These new data thus suggest that BKCa channels may be regulated by multiple tyrosine kinases in addition to cyclic nucleotide-dependent serine/threonine kinases (40).

An interesting aspect of our study is the finding that Pyk2 and Hck influence primarily the calcium-dependent gating of BKCa current, with little effect on gating by voltage alone (see Figs. 3 and 4). This result may be explained by recent data indicating that membrane voltage and intracellular calcium appear to act via independent and parallel mechanisms to promote BKCa channel gating (18). On the basis of such a model, it is readily apparent how the observed tyrosine phosphorylation of the BKCa {alpha}-subunit is able to selectively enhance calcium-dependent channel opening without significantly altering voltage-dependent processes. Mechanistically, such phosphorylation events may produce their effect by influencing the kinetics of channel gating. We recently reported (25) that interaction of BKCa channels with the SNARE protein syntaxin 1A positively influences the time constants of channel activation and deactivation, leading to enhancement of channel activity. Similarly, macroscopic current recordings of BKCa channels coexpressed with wild-type Pyk2 or Hck (see Fig. 2) appear to be consistent with somewhat faster current activation and slowed deactivation compared with BKCa channels expressed alone.

An important feature of our data is that BKCa channels in situ undergo tyrosine phosphorylation in the presence of wild-type, but not inactive, forms of either Pyk2 or Hck (see Figs. 5 and 6). The fact that Hck produced functional effects similar to those observed earlier with c-Src (26) suggests that Src-family kinases in general may be capable of BKCa channel modulation. Furthermore, because the Pyk2-induced phosphorylation of BKCa channels in situ did not appear to be dramatically reduced by a Tyr-to-Phe substitution at a residue (Tyr766) previously shown to be modified by c-Src in situ (26), it is likely that Pyk2 and Src family kinases phosphorylate distinct sites in the BKCa {alpha}-subunit (Fig. 7). Direct tyrosine phosphorylation of the BKCa {alpha}-subunit by either Pyk2 or Hck thus represents a plausible mechanism to explain the observed enhancement of channel activity in the presence of either kinase. Tyrosine phosphorylation of the BKCa channel complex or Pyk2 itself, or both, may further underlie the observed coassociation between BKCa channels and Pyk2, because no interaction was detected in the presence of the dead Pyk2 isoform (see Fig. 5). Our data showing that this coassociation of Pyk2 with BKCa channels, as well as the level of Pyk2-dependent tyrosine phosphorylation of BKCa {alpha}-subunits, was increased by elevated intracellular calcium are further consistent with the reported calcium-dependent actions of Pyk2 (22). Earlier observations have suggested that a protein kinase C isoform may contribute to the calcium-sensitive effects of Pyk2 (14, 41).

While the simplest interpretation of these data is that Pyk2 and Hck are directly responsible for the observed BKCa channel phosphorylation and enhancement of gating, additional tyrosine kinase molecules also may play a role. For example, Pyk2 is reported to interact with Src-family kinases (11), as well as with FAK (14, 24). Recently, Rezzonico et al. (34) reported that FAK is able to associate with native BKCa channels (hSlo) in human osteoblasts, suggesting the presence of BKCa channels in focal adhesion complexes. In this context, both K+ and Ca2+ channels are reported to undergo modulation by integrin-dependent signaling pathways typically associated with focal adhesions (10). Together, these observations suggest that ion channels may colocalize with integrins at focal adhesion complexes to form a spatially compact signaling domain.

How would Pyk2-dependent modulation of BKCa channel activity contribute to the functional state of an intact blood vessel? Vascular smooth muscle is known to express several types of integrin complexes, including {alpha}4{beta}1, {alpha}5{beta}1, or {alpha}V{beta}3 (31). Biological events affecting the integrity of the ECM (i.e., mechanical forces, denaturation, enzymatic degradation by collagenases, matrix metalloproteinases) expose or generate soluble/insoluble integrin ligands from ECM substrates (e.g., fibronectin, vitronectin, and collagens), which often contain a RGD binding motif (6, 30). The binding of such ligands to integrins promotes the ligation or clustering of integrin molecules, leading to the activation of intracellular protein kinases (e.g., FAK and Pyk2) (13). Such a process could thus lead to the phosphorylation and enhancement of BKCa channel activity, as we have described. Physiologically, such enhancement may contribute to integrin-mediated mechanotransduction events that underlie myogenic tone (30). Under conditions of tissue injury, integrin-induced enhancement of smooth muscle BKCa channels may lead to changes in local blood flow that could promote swelling and infiltration of leukocytes as part of the cellular processes underlying wound repair. Modulation of BKCa channel activity by Pyk2, Src-family kinases, and, possibly, FAK thus may contribute to integrin-mediated regulation of blood flow, under the context of myogenic tone, as well as to injury-related changes in flow, as a result of degradation/denaturation of the ECM.


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This work was supported by research funding from the Heart and Stroke Foundation of Alberta, Northwest Territories and Nunavut (to A. P. Braun).


    ACKNOWLEDGMENTS
 
We thank Drs. S. Lev and S. Robbins for kindly providing the Pyk2 and Hck constructs, respectively. S. Ling was partially supported by a graduate student scholarship from the University of Calgary. A Senior Scholar Award to A. P. Braun from the Alberta Heritage Foundation for Medical Research is gratefully acknowledged.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. P. Braun, Dept. of Pharmacology and Therapeutics, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Drive, N.W., Calgary, Alberta, Canada T2N 4N1 (E-mail: abraun{at}ucalgary.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Alioua A, Mahajan A, Nishimaru K, Zarei MM, Stefani E, and Toro L. Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction. Proc Natl Acad Sci USA 99: 14560–14565, 2002.[Abstract/Free Full Text]

2. Avraham H, Park SY, Schinkmann K, and Avraham S. RAFTK/Pyk2-mediated cellular signalling. Cell Signal 12: 123–133, 2000.[CrossRef][ISI][Medline]

3. Boudreau NJ and Jones PL. Extracellular matrix and integrin signalling: the shape of things to come. Biochem J 339: 481–488, 1999.[CrossRef][ISI][Medline]

4. Braun AP, Heist EK, and Schulman H. Inhibition of a mammalian large conductance, calcium-sensitive K+ channel by calmodulin-binding peptides. J Physiol 527: 479–492, 2000.[Abstract/Free Full Text]

5. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC. Green fluorescent protein as a marker for gene expression. Science 263: 802–805, 1994.[ISI][Medline]

6. Davis GE, Bayless KJ, Davis MJ, and Meininger GA. Regulation of tissue injury responses by the exposure of matricryptic sites within extracellular matrix molecules. Am J Pathol 156: 1489–1498, 2000.[Abstract/Free Full Text]

7. Davis MJ and Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev 79: 387–423, 1999.[Abstract/Free Full Text]

8. Davis MJ, Wu X, Nurkiewicz TR, Kawasaki J, Davis GE, Hill MA, and Meininger GA. Integrins and mechanotransduction of the vascular myogenic response. Am J Physiol Heart Circ Physiol 280: H1427–H1433, 2001.[Abstract/Free Full Text]

9. Davis MJ, Wu X, Nurkiewicz TR, Kawasaki J, Gui P, Hill MA, and Wilson E. Regulation of ion channels by protein tyrosine phosphorylation. Am J Physiol Heart Circ Physiol 281: H1835–H1862, 2001.[Abstract/Free Full Text]

10. Davis MJ, Wu X, Nurkiewicz TR, Kawasaki J, Gui P, Hill MA, and Wilson E. Regulation of ion channels by integrins. Cell Biochem Biophys 36: 41–66, 2002.[ISI][Medline]

11. Dikic I, Tokiwa G, Lev S, Courtneidge SA, and Schlessinger J. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature 383: 547–550, 1996.[CrossRef][ISI][Medline]

12. Felsch JS, Cachero TG, and Peralta E. Activation of protein tyrosine kinase PYK2 by the m1 muscarinic acetylcholine receptor. Proc Natl Acad Sci USA 95: 5051–5056, 1998.[Abstract/Free Full Text]

13. Giancotti FG and Ruoslahti E. Integrin signaling. Science 285: 1028–1032, 1999.[Abstract/Free Full Text]

14. Girault JA, Costa A, Derkinderen P, Studler JM, and Toutant M. FAK and PYK2/CAKb in the nervous system: a link between neuronal activity, plasticity and survival? Trends Neurosci 22: 257–263, 1999.[CrossRef][ISI][Medline]

15. Hanks SK and Polte TR. Signaling through focal adhesion kinase. Bioessays 19: 137–145, 1997.[ISI][Medline]

16. Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, Bell JC, and Dedhar S. Regulation of cell adhesion and anchorage-dependent growth by a new {beta}1-linked protein kinase. Nature 379: 91–96, 1996.[CrossRef][ISI][Medline]

17. Heidinger V, Manzerra P, Wang XQ, Strasser U, Yu SP, Choi DW, and Behrens MM. Metabotropic glutamate receptor 1-induced upregulation of NMDA receptor current: mediation through the Pyk2/Src-family kinase pathway in cortical neurons. J Neurosci 22: 5452–5461, 2001.[ISI]

18. Horrigan FT and Aldrich RW. Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. J Gen Physiol 120: 267–305, 2002.[Abstract/Free Full Text]

19. Huang YQ, Lu WY, Ali DW, Pelkey KA, Pitcher GM, Lu YM, Aoto H, Roder JC, Sasaki T, Salter MW, and MacDonald JF. CAK{beta}/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus. Neuron 29: 485–496, 2001.[ISI][Medline]

20. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687, 2002.[ISI][Medline]

21. Ingber DE. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ Res 91: 877–887, 2002.[Abstract/Free Full Text]

22. Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, and Schlessinger J. Protein tyrosine kinase Pyk2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions. Nature 376: 737–745, 1995.[CrossRef][ISI][Medline]

23. Levitan IB. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu Rev Physiol 56: 193–212, 1994.[CrossRef][ISI][Medline]

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

25. Ling S, Sheng JZ, Braun JEA, and Braun AP. Co-association of syntaxin 1A with native rat brain and cloned large conductance, calcium-activated K+ channels in situ. J Physiol 553: 65–81, 2003.[Abstract/Free Full Text]

26. Ling S, Woronuk G, Sy L, Lev S, and Braun AP. Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase. J Biol Chem 275: 30683–30689, 2000.[Abstract/Free Full Text]

27. Litvak V, Tian D, Shaul YD, and Lev S. Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades. J Biol Chem 275: 32736–32746, 2000.[Abstract/Free Full Text]

28. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]

29. Luttrell LM, Daaka Y, and Lefkowitz RJ. Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11: 177–183, 1999.[CrossRef][ISI][Medline]

30. Martinez-Lemus LA, Wu X, Wilson E, Hill MA, Davis GE, Davis MJ, and Meininger GA. Integrins as unique receptors for vascular control. J Vasc Res 40: 211–233, 2003.[CrossRef][ISI][Medline]

31. Moiseeva EP. Adhesion receptors of vascular smooth muscle cells and their functions. Cardiovasc Res 52: 372–386, 2001.[CrossRef][ISI][Medline]

32. Nelson MT and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799–C822, 1995.[Abstract/Free Full Text]

33. Quintrell NA, Lebo R, Varmus H, Bishop JM, Pettenati MJ, Le Beau MM, Diaz MO, and Rowley JD. Identification of a human gene (Hck) that encodes a protein-tyrosine kinase and is expressed in hemopoietic cells. Mol Cell Biol 7: 2267–2275, 1987.[ISI][Medline]

34. Rezzonico R, Cayatte C, Bourget-Ponzio I, Romey G, Belhacene N, Loubat A, Rocchi S, van Obberghen E, Girault JA, Rossi B, and Schmid-Antomarchi H. Focal adhesion kinase pp125FAK interacts with the large conductance calcium-activated hSlo potassium channel in human osteoblasts: potential role in mechanotransduction. J Bone Miner Res 18: 1863–1871, 2003.[ISI][Medline]

35. Robbins SM, Quintrell NA, and Bishop JM. Mercuric chloride activates the Src-family protein kinase, Hck in myelomonocytic cells. Eur J Biochem 267: 7201–7208, 2000.[Abstract/Free Full Text]

36. Roberts WM, Jacobs RA, and Hudspeth AJ. Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J Neurosci 10: 3664–3684, 1990.[Abstract]

37. Robitaille R, Garcia ML, Kaczorowski GJ, and Charlton MP. Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. Neuron 11: 645–655, 1993.[ISI][Medline]

38. Schlaepfer DD, Hauck CR, and Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol 71: 435–478, 1999.[CrossRef][ISI][Medline]

39. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 103: 211–225, 2000.[ISI][Medline]

40. Schubert R and Nelson MT. Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 22: 505–512, 2001.[CrossRef][ISI][Medline]

41. Siciliano JC, Toutant M, Derkinderen P, Sasaki T, and Girault JA. Differential regulation of proline-rich tyrosine kinase 2/cell adhesion kinase {beta} (PYK2/CAK{beta}) and pp125FAK by glutamate and depolarization in rat hippocampus. J Biol Chem 271: 28942–28946, 1996.[Abstract/Free Full Text]

42. Swayze RD and Braun AP. A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP. J Biol Chem 276: 19729–19737, 2001.[Abstract/Free Full Text]

43. Swope SL, Moss SJ, Blackstone CD, and Huganir RL. Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. FASEB J 6: 2514–2523, 1992.[Abstract/Free Full Text]

44. Troussard AA, Mawji N, Ong C, Mui A, St-Arnaud R, and Dedhar S. Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation. J Biol Chem 278: 22374–22378, 2003.[Abstract/Free Full Text]

45. Vuori K. Integrin signaling: tyrosine phosphorylation events in focal adhesions. J Membr Biol 165: 191–199, 1998.[CrossRef][ISI][Medline]

46. White RE, Darkow DJ, and Falvo Lang JL. Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP-dependent mechanism. Circ Res 77: 936–942, 1995.[Abstract/Free Full Text]

47. Wu C and Dedhar S. Integrin-linked kinase (ILK) and its interactions: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. J Cell Biol 155: 505–510, 2001.[Abstract/Free Full Text]

48. Yazejian B, DiGregorio DA, Vergara J, Poage RE, Meriney SD, and Grinnell AD. Direct measurements of presynaptic calcium and calcium-activated potassium currents regulating neurotransmitter release at cultured Xenopus nerve-muscle synapses. J Neurosci 17: 2990–3001, 1997.[Abstract/Free Full Text]

49. Ziegler SF, Marth JD, Lewis DB, and Perlmutter RM. Novel protein-tyrosine kinase gene (hck) preferentially expressed in cells of hematopoietic origin. Mol Cell Biol 7: 2276–2285, 1987.[ISI][Medline]