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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BK channels; tyrosine kinase; calcium; immunoprecipitation
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 - and
-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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction and transfection of cDNA plasmids.
Expression vectors containing cDNAs encoding the murine mSlo -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 24 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 24 M. 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 11.5 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 0.51 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 2030 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)
-mercaptoethanol and incubated for 2030 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 2030 min in TTBS containing 510% (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 12 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 35 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 0.40.6 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 416 h at 4°C with either
1.5 µg of anti-BKCa channel antibody or 35 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 3040 µl of Laemmli sample buffer, heated to 70°C for 2030 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.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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).
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 -subunit (Fig. 7). Direct tyrosine phosphorylation of the BKCa
-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
-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 4
1,
5
1, or
V
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.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Avraham H, Park SY, Schinkmann K, and Avraham S. RAFTK/Pyk2-mediated cellular signalling. Cell Signal 12: 123133, 2000.[CrossRef][ISI][Medline]
3. Boudreau NJ and Jones PL. Extracellular matrix and integrin signalling: the shape of things to come. Biochem J 339: 481488, 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: 479492, 2000.
5. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC. Green fluorescent protein as a marker for gene expression. Science 263: 802805, 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: 14891498, 2000.
7. Davis MJ and Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev 79: 387423, 1999.
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: H1427H1433, 2001.
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: H1835H1862, 2001.
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: 4166, 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: 547550, 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: 50515056, 1998.
13. Giancotti FG and Ruoslahti E. Integrin signaling. Science 285: 10281032, 1999.
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: 257263, 1999.[CrossRef][ISI][Medline]
15. Hanks SK and Polte TR. Signaling through focal adhesion kinase. Bioessays 19: 137145, 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 1-linked protein kinase. Nature 379: 9196, 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: 54525461, 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: 267305, 2002.
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/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus. Neuron 29: 485496, 2001.[ISI][Medline]
20. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 110: 673687, 2002.[ISI][Medline]
21. Ingber DE. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ Res 91: 877887, 2002.
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: 737745, 1995.[CrossRef][ISI][Medline]
23. Levitan IB. Modulation of ion channels by protein phosphorylation and dephosphorylation. Annu Rev Physiol 56: 193212, 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: 89178924, 1999.
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: 6581, 2003.
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: 3068330689, 2000.
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: 3273632746, 2000.
28. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265275, 1951.
29. Luttrell LM, Daaka Y, and Lefkowitz RJ. Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11: 177183, 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: 211233, 2003.[CrossRef][ISI][Medline]
31. Moiseeva EP. Adhesion receptors of vascular smooth muscle cells and their functions. Cardiovasc Res 52: 372386, 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: C799C822, 1995.
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: 22672275, 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: 18631871, 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: 72017208, 2000.
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: 36643684, 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: 645655, 1993.[ISI][Medline]
38. Schlaepfer DD, Hauck CR, and Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol 71: 435478, 1999.[CrossRef][ISI][Medline]
39. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 103: 211225, 2000.[ISI][Medline]
40. Schubert R and Nelson MT. Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 22: 505512, 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 (PYK2/CAK
) and pp125FAK by glutamate and depolarization in rat hippocampus. J Biol Chem 271: 2894228946, 1996.
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: 1972919737, 2001.
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: 25142523, 1992.
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: 2237422378, 2003.
45. Vuori K. Integrin signaling: tyrosine phosphorylation events in focal adhesions. J Membr Biol 165: 191199, 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: 936942, 1995.
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: 505510, 2001.
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: 29903001, 1997.
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: 22762285, 1987.[ISI][Medline]