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(Circulation Research. 1997;81:575-584.)
© 1997 American Heart Association, Inc.

Articles

A Role for Ca²⁺/Calmodulin-Dependent Protein Kinase II in the Mitogen-Activated Protein Kinase Signaling Cascade of Cultured Rat Aortic Vascular Smooth Muscle Cells

S. Thomas Abraham, Holly A. Benscoter, Charles M. Schworer, , Harold A. Singer

From the Weis Center for Research, Geisinger Clinic, Danville, Pa.

Correspondence to Harold A. Singer, PhD, Henry Hood MD Research Program, Weis Center for Research, PennState College of Medicine, 100 N Academy Ave, Danville, PA 17822-2612.

	Abstract

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Abstract Exposure of cultured rat aortic vascular smooth muscle (VSM) cells to the Ca²⁺ ionophore ionomycin produced an increase in extracellular signal–regulated kinase 1/2 (ERK1/2) activity that was maximal between 2 and 5 minutes but then declined to basal values within 20 minutes of stimulation. Elevation of [Ca²⁺]_i in VSM cells leads to an even more rapid activation of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II); thus, it was postulated that the Ca²⁺-dependent component of ERK1/2 activation was mediated by CaM kinase II. Transient ERK1/2 activation by ionomycin was almost completely abolished by pretreating cells with 30 µmol/L KN-93, a CaM kinase II inhibitor. Treatment of cells with KN-93 did not antagonize the ability of ionomycin to mobilize intracellular Ca²⁺ but prevented CaM kinase II and ERK1/2 activation with almost identical potencies. Consistent with a role for Ca²⁺ and calmodulin in intracellular Ca²⁺–induced activation of ERK, cells pretreated with calmodulin inhibitors (W-7 or calmidazolium) exhibited an attenuated ERK response to ionomycin. ERK1/2 activation in response to phorbol esters and platelet-derived growth factor were not significantly affected by KN-93, whereas the response to angiotensin II and thrombin were attenuated by 60% and 40%, respectively. Transient expression of wild-type ₂ CaM kinase II in COS-7 cells resulted in increased ERK2 activity, whereas coexpression of wild-type and a kinase-negative mutant resulted in a diminution of this response. These data suggest that regulation of cellular responses by Ca²⁺-dependent pathways in VSM cells may be mediated in part by CaM kinase II–dependent activation of ERK1/2.

Key Words: Ca²⁺/calmodulin-dependent protein kinase II • Ca²⁺ • mitogen-activated protein kinase • extracellular signal–regulated kinase • vascular smooth muscle

	Introduction

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The ERK1/2 or MAP kinase cascade is a well-described signaling event that regulates important cellular functions in many cells. Classical growth factors such as PDGF and epidermal growth factor initiate this cascade by binding to and activating a membrane-bound receptor, tyrosine kinase, which upon tyrosine phosphorylation recruits a complex of adapter proteins (shc, Grb2, and mSoS^{1 2 3} ). This association of growth factor receptors and adapter proteins causes the association of ras (a small G protein) with the complex, which becomes active after an exchange of bound GDP for GTP. This activated complex participates in the translocation of raf kinase to the plasma membrane to cause its activation.^{4 5} Active raf kinase phosphorylates MEK on two serine residues, which in turn phosphorylates ERK1/2 on Thr¹⁸³ and Tyr¹⁸⁵ (for rat ERK2^{6 7} ) to cause its full activation. The ERK pathway has been proposed to regulate diverse cellular functions, including cell proliferation,^{8 9} differentiation,¹⁰ gene expression,^{11 12 13} smooth muscle contraction,¹⁴ and learning and memory formation.¹⁵

A number of Ca²⁺-mobilizing agonists (angiotensin II, endothelin, phenylephrine, etc) have also been shown to activate ERK1/2 in certain cell types.^{16 17 18 19 20} Binding of these agonists to specific receptors results in the activation of membrane-associated heterotrimeric G proteins, leading to the activation of membrane-bound phospholipase C. Active phospholipase C hydrolyzes membrane phospholipid to inositol trisphosphate and diacylglycerol, which are thought to be responsible for the mobilization of intracellular Ca²⁺ from sarcoplasmic stores^{21 22} and activation of cellular PKC,²³ respectively. Activation of PKC leads to the rapid and robust activation of ERK1/2 in many cell types,^{17 24 25} probably by activation of raf,^{26 27} although some controversy exists involving the exact site of action of PKC.²⁸ Elevation of intracellular Ca²⁺ by receptor-independent means also leads to the activation of ERK1/2 in some cell types.^{29 30} The mechanism for this action of Ca²⁺ is not entirely clear, although neuronal cells may use a Ca²⁺-activated tyrosine kinase.³¹ In addition to PKC activation and elevation of intracellular Ca²⁺, some evidence exists for the activation of the ERK1/2 pathway by the ß subunits of G proteins.³

Earlier work in our laboratory indicated that VSM cells contain specific isoforms of CaM kinase II that are distinct from the most abundant forms found in the brain.³² In a more recent report, we described the activation of CaM kinase II in cultured VSM cells in response to Ca²⁺ ionophore and receptor agonists (angiotensin II, vasopressin, and PDGF³³ ). Cultured VSM cells contain relatively high amounts of this multifunctional serine/threonine kinase; however, its functions in these cells are largely unknown. In other cell types, CaM kinase II has been proposed to play a role in learning/memory,³⁴ gene expression,^{35 36} cell cycle regulation,^{37 38 39} and muscle contractility,^{40 41 42} functions that overlap with proposed MAP kinase–regulated processes.

The present study was undertaken to test the hypothesis that CaM kinase II mediates the Ca²⁺-induced activation of ERK1/2 in cultured rat aortic VSM cells. Since one multifunctional protein kinase (PKC) has been shown to stimulate the MAP kinase signaling cascade, we reasoned that another with a similar broad specificity (CaM kinase II) may regulate ERK1/2 activity in VSM cells. Using pharmacological inhibitors of CaM kinase II, we demonstrate that ERK1/2 activation in VSM cells, by Ca²⁺ ionophore, is dependent on the activation of CaM kinase II. MAP kinase activation by Ca²⁺-mobilizing receptor agonists also required CaM kinase II activation, whereas growth factors and phorbol esters were able to induce MAP kinase in a CaM kinase II–independent manner. In addition, transient transfection of ₂ CaM kinase II into COS-7 cells led to activation of ERK, which was antagonized by coexpression with the kinase-negative mutant of CaM kinase II. The results presented herein suggest that the multifunctional CaM kinase II mediates the activation of the MAP kinase pathway in VSM cells in response to elevated intracellular Ca²⁺. Portions of the present study have been previously published.⁴³

	Materials and Methods

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Cell Culture
VSM cells were dispersed from the medial layer of thoracic aortas of Sprague-Dawley rats weighing 150 to 200 g, as described earlier,⁴⁴ cultured in DMEM/F-12 medium containing 10% fetal calf serum (Hyclone) under standard conditions, and subcultured twice a week. Cells from passages 5 to 10 were used in the experiments after being grown to confluence and growth-arrested in 0.4% serum–containing medium for 16 to 20 hours. One to 2 hours before use, the cultures were incubated in fresh 0.4% serum–containing medium.

Cell Extract Preparation
VSM cells were pretreated with various inhibitors 30 minutes before exposure to stimulants for the times indicated. To extract MAP kinase from cells, the stimulant challenge was stopped by washing twice with 3 mL of ice-cold Ca²⁺/Mg²⁺-free HBSS (GIBCO-BRL) and scraping the cells into 250 to 400 µL of extraction buffer (20 mmol/L Tris-HCl [pH 7.5], 2 mmol/L EGTA, 1 mmol/L orthovanadate, 10 mmol/L ß-glycerophosphate, 1 mmol/L dithiothreitol, 0.1 mmol/L PMSF, and 100 U/mL aprotinin). The cells were disrupted with a Branson sonicator (three 1-second bursts), and the supernatant fraction was retained after centrifugation at 17 000g. The samples were stored at -20°C until kinase assays were performed. CaM kinase II was extracted from VSM cells in an equal mixture of Ca²⁺/Mg²⁺-free HBSS and a buffer containing 50 mmol/L MOPS (pH 8.6), 3 mmol/L EGTA, 250 mmol/L NaCl, 100 mmol/L NaF, 100 mmol/L sodium pyrophosphate, 1% NP-40, 2 mmol/L dithiothreitol, 0.2 mmol/L PMSF, and 0.4 U/mL aprotinin, as described previously,³³ and the samples were kept on ice until the kinase assay was performed.

In Vitro Kinase Assay
CaM kinase II assays were performed as indicated in Abraham et al³³ with the synthetic peptide autocamtide-2 (KKALRRQETVDAL) as the substrate for the enzyme. The relative specificity of this peptide as a substrate for CaM kinase II was demonstrated by >90% loss in autocamtide-2 kinase activity from cell extracts previously immunoprecipitated with an antibody against the carboxy terminus of CaM kinase II isoforms.³³ The "autonomous" fraction (or independent activity) of CaM kinase II (activity assayed in the absence of Ca²⁺ and calmodulin) was expressed as a percent of the total CaM kinase II activity (assayed in the presence of optimal Ca²⁺ and calmodulin).

In-Gel MAP Kinase Assay
The assay was performed according to the method described for the in-gel detection of specific kinase activities.^{18 45 46} Three to 10 µg of extract proteins was resolved by electrophoresis in 10% SDS-polyacrylamide gels (100x60x0.75 mm) containing 0.5 mg/mL of myelin basic protein copolymerized in the separating gel. The gels were washed with 20% 2-propanol in 50 mmol/L Tris-HCl (pH 8.0) for 1 hour with three changes, followed by 1 hour of incubation in buffer A (50 mmol/L Tris-HCl [pH 8.0] and 5 mmol/L 2-mercaptoethanol) containing 6 mol/L guanidine HCl. The separated proteins were renatured with four washes of buffer A containing 0.04% Tween 40 at 4°C over a 16- to 20-hour period. The gels were preincubated in 5 mL of buffer B (40 mmol/L HEPES [pH 8.0], 0.2 mmol/L dithiothreitol, 0.1 mmol/L EGTA, and 5 mmol/L magnesium acetate) for 30 minutes at room temperature. The kinase reaction was carried out for 1.5 hours at room temperature in 5 mL of buffer B containing 50 to 100 µCi of [-³²P]ATP and 20 µmol/L ATP. The reactions were stopped by six 20-minutes washes of the gels with 5% trichloroacetic acid/1% sodium pyrophosphate (200 mL per wash), and radioactivity incorporated into the substrate was detected on x-ray film and quantified on a PhosphorImager or Laser densitometer (Molecular Dynamics). Active recombinant human ERK2 obtained from New England BioLabs served as a positive control for the assay.

Immunodetection of Active ERK1/2
Protein extracts (7.5 to 30 µg) were separated by electrophoresis in 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked in TBS containing 5% nonfat dry milk and 0.2% NP-40 for 30 minutes and then incubated with either anti–phospho-MAP kinase (New England Biolabs) or anti–active MAP kinase antibody (Promega) in TBS containing 5% milk, 0.2% NP-40, and 0.1% SDS. The membranes were washed in fresh blocking buffer with three changes, followed by incubation with horseradish peroxidase–conjugated anti-rabbit antibody (1:1000 dilution, Amersham) in blocking buffer. Immunoreactive proteins on the membrane were visualized on x-ray film by enhanced chemiluminescence (Amersham), and the intensities of the visible bands were quantified by laser densitometer in the linear range of film sensitivity.

Intracellular Ca²⁺ Measurement
Measurement of free [Ca²⁺]_i was performed using fura 2-AM as described earlier.³³ Essentially, cells were loaded with fura 2-AM (2 µmol/L) while still attached to the culture dish (100-mm diameter), then dislodged by trypsin treatment, and washed in solutions containing soybean trypsin inhibitor. The cells were suspended in HBSS containing 1.8 mmol/L Ca²⁺ (Worthington), and [Ca²⁺]_i was determined using a SPEX DM3000 Fluorolog spectrometer (SPEX Industries) according to the method of Grynkiewicz et al.⁴⁷

Vector Constructs and Cell Transfection
The ₂ CaM kinase II insert DNA was obtained by reverse-transcriptase PCR of RNA from cultured rat aortic smooth muscle cells. The sequences for the upstream and downstream PCR primers were from a region 50 and 70 bases into the 5' and 3' untranslated regions of rat brain CaM kinase II, respectively. The PCR product was cloned into the EcoRV site of the plasmid vector pBluescript II KS+. The 1650-bp product of an Apa I digest was then cloned into the pRc/CMV expression vector (Invitrogen). The Transformer Site-Directed Mutagenesis Kit (Clonetech) was used to generate an Ala substitution for Lys⁴³ in the K43A ATP-binding defect mutant. The plasmid vectors (0.5 to 1.0 µg DNA) were transfected into COS-7 cells grown on 35- or 60-mm dishes using 8% lipofectamine (Life Technologies) in Opti-MEM I reduced-serum medium (GIBCO-BRL). Transfections were allowed to continue for 5 to 9 hours, at which time the medium was removed, and cells were maintained in regular growth medium for a further 24 hours. Six hours before the experiments, the growth medium was replaced with 0.4% serum–containing medium, and then cells were treated and extracts were made as for the VSM cells.

Materials
Angiotensin II was obtained from Bachem California; ionomycin, thapsigargin, Ro 31-8220, bisindolylmaleimide, and fura 2-AM were from Calbiochem. KN-93 was purchased from Seikagaku America or Calbiochem, and KN-92 was obtained from Calbiochem or RBI. Thrombin and myelin basic protein were from Sigma Chemical Co, and [-³²P]ATP was obtained from Amersham. Autocamtide-2 was synthesized with >95% purity in the Core Molecular Biology Laboratory at the Weis Center for Research.

	Results

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In VSM and other cell types (eg, fibroblasts, PC-12, and cardiac myocytes) elevation of [Ca²⁺]_i has been shown to result in MAP kinase activation, but the mechanism by which this occurs is not known. The present study was carried out to test the hypothesis that CaM kinase II is involved in the activation of MAP kinase by intracellular Ca²⁺ in cultured aortic VSM cells.

Exposure of VSM cells to ionomycin results in an increase in [Ca²⁺]_i, which is the primary mediator of CaM kinase II activation in VSM cells.³³ When extracts from ionomycin-treated cells were analyzed by the in-gel myelin basic protein kinase assay, both ERK1 and ERK2 activities were rapidly but transiently increased, with activities returning to basal values within 15 to 20 minutes (Fig 1A). The maximum induction by ionomycin at 2 to 5 minutes was 6- to 15-fold greater than ERK activities in untreated cells (n=4) and was equivalent to responses elicited by 40 ng/mL PDGF, 100 nmol/L PMA, or 300 nmol/L angiotensin II (see Fig 7 below). To test for a role of CaM kinase II in the ionophore-induced activation of ERK1/2, the cells were pretreated with 30 µmol/L of the CaM kinase II inhibitor KN-93.^{33 48} Analysis of these extracts revealed that KN-93 attenuated >90% of the ionomycin-induced ERK activity (Fig 1B).

View larger version (47K): [in this window] [in a new window]   Figure 1. Activation of ERK1 and ERK2 in VSM cells by ionomycin: effect of KN-93 pretreatment. A, VSM cells were exposed to 1 µmol/L ionomycin for up to 45 minutes before preparation of cytosolic extracts and kinase assay by an in-gel method (10 µg protein, see "Materials and Methods"). B, The cells were incubated with 30 µmol/L KN-93 for 30 minutes before treatment with ionomycin. Equal amounts of cytosolic protein (10 µg) were loaded in each lane, except on the far right lane in both panels, which had 5 ng of purified, recombinant, human ERK2. Data are representative of three separate experiments.

View larger version (48K): [in this window] [in a new window]   Figure 7. The effect of KN-93 on MAP kinase activation in VSM cells by various agents. A, Cells were exposed to 1 µmol/L ionomycin (IONO, 5 minutes), 40 ng/mL PDGF (10 minutes), or 100 nmol/L PMA (20 minutes), with or without 30 µmol/L KN-93 pretreatment, followed by in-gel assay. B, VSM cells were exposed to 300 nmol/L angiotensin II (AII, 5 minutes) or 300 nmol/L thrombin (5 minutes) in control or KN-93–treated cells before in-gel assay. Each of the sample lanes had 5 µg of sample protein loaded; lanes marked with ERK contained various amounts of purified active human ERK2.

Probing Western blots of cytosolic extracts from ionomycin-treated cells using the phospho-MAP kinase antibody revealed that both ERK1 and ERK2 became phosphorylated on a tyrosine residue (Tyr¹⁸⁵), in a manner parallel to the kinase activities detected by the in-gel assay (Fig 2A). Pretreatment of VSM cells with KN-93 caused a near total loss in tyrosine phosphorylation of MAP kinase induced by ionomycin (Fig 2B). Interestingly, the tyrosine phosphorylation of ERK1 (44 kD) was not well detected by the phospho-MAP kinase antibody, even though the in-gel assay clearly showed ERK1 activation. This result may be due to poor recognition of the phospho-ERK1 protein by the antibody. Alternatively, the amount of activated (phosphorylated) ERK1 in these extracts may be small relative to its enzymatic activity as detected by the in-gel assay. These data suggest that KN-93 has no direct inhibitory effect on ERK activity but interferes with an upstream component that prevents activation of MEK, resulting in a lack of ERK phosphorylation. A role for MEK in the ionomycin-induced ERK1/2 cascade was demonstrated by the ability of PD98059 (an inhibitor of MEK activation, 10 µmol/L) to prevent ERK activation (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 2. Tyrosine phosphorylation of ERK1/2 after ionomycin treatment of VSM cells, with or without KN-93 pretreatment. A, VSM cells were treated with ionomycin for up to 45 minutes before equal protein amounts (30 µg) of cell extract were separated by electrophoresis and transferred to nitrocellulose membranes. The blots were probed with the anti–phospho-MAP kinase antibody. B, VSM cells were pretreated with 30 µmol/L KN-93 before ionomycin exposure, and 30 µg of protein extract was resolved by electrophoresis and blotted for immunodetection using the anti–phospho-MAP kinase antibody. The extreme right lanes in both panels show the immunoreactivity of the anti–phospho-MAP kinase antibody to 12.5 ng of active, purified, human ERK2.

To define the specificity of KN-93, its effects on the ionomycin-induced mobilization of [Ca²⁺]_i was determined. Exposure of fura 2–loaded VSM cells to 1 µmol/L ionomycin resulted in a rapid increase in [Ca²⁺]_i that was maximal within 15 to 30 seconds (maximal change, 1537±145 nmol/L; n=4; Fig 3). Pretreatment of the cells with 30 µmol/L KN-93 for 30 minutes did not attenuate the maximal [Ca²⁺]_i or the rate of Ca²⁺-mobilization in response to ionomycin (Fig 3). Actually, KN-93 pretreatment appeared to increase the amount of [Ca²⁺]_i mobilized by ionomycin, particularly after 3 minutes of stimulation (not statistically significant). Inhibition of CaM kinase II does not appear to be responsible for this effect, since this response was more overt in cells treated with 30 µmol/L KN-92, the inactive analogue of KN-93 (P<.05, one-way ANOVA). Concentrations of KN-93 or KN-92 as low as 1 µmol/L showed qualitatively similar results, and the mechanism of this action is not presently clear.

View larger version (31K): [in this window] [in a new window]   Figure 3. Ionomycin (IONO)-induced Ca2+ mobilization in control and in KN-93– or KN-92–treated VSM cells. Fura 2–loaded VSM cells were stimulated with 1 µmol/L IONO for up to 5 minutes, and changes in intracellular Ca2+ were recorded. Each bar represents the mean±SEM [Ca2+]i of three or four separate experiments.

The specificity of KN-93 was further investigated by comparing its ability to inhibit two apparently distinct cellular events (CaM kinase II or ERK activation) in intact VSM cells by constructing KN-93 inhibition curves. A 15-second exposure of VSM cells to 1 µmol/L ionomycin resulted in the generation of Ca²⁺-independent or "autonomous" activity, which reached 37.3±3.1% (n=5) of the total CaM kinase II activity in the lysates (Fig 4A; resting autonomous activity was 5% to 10% of total activity). The generation of autonomous activity results from the autophosphorylation of CaM kinase II on a specific residue (Thr²⁸⁶ for -CaM kinase II³⁴ ) in the presence of Ca²⁺ and calmodulin to become partially independent of Ca²⁺/calmodulin for its activity. This autophosphorylation can be measured in vitro as the increase in the Ca²⁺/calmodulin-independent autocamtide-2 kinase activity and is a reflection of the activation of CaM kinase II in situ.³³ A 30-minute preincubation of cells with increasing concentrations of KN-93 (10 nmol/L to 100 µmol/L) antagonized the ionomycin-induced autonomous CaM kinase II activity, with an IC₅₀ of 14 µmol/L, as calculated by nonlinear regression analysis using GraphPad Prism software (n=5, Fig 4A). Pretreatment of the cells with the inactive analogue KN-92 did not result in significant inhibition of the ionomycin-induced autonomous activity (n=3; Fig 4A, inset) A 5-minute treatment of VSM cells with 1 µmol/L ionomycin resulted in 4-fold ERK1/2 activation as measured by the in-gel kinase assay and quantification by PhosphorImager (n=4, Fig 4B). Pretreatment of the cells with increasing concentrations of KN-93 resulted in successively greater inhibition of ERK1/2 induction by ionomycin (IC₅₀, 13 µmol/L; n=3 or 4; Fig 4B). As in the case of CaM kinase II, the inactive analogue KN-92 had no significant effect on the ability of ionomycin to elicit ERK1/2 activation (Fig 4B, inset). The similar potencies of KN-93 for prevention of either CaM kinase II or ERK induction by ionomycin argues for a common site of action for the antagonist in these two events, and this site is most likely CaM kinase II.

View larger version (29K): [in this window] [in a new window]   Figure 4. Concentration-dependent inhibition of CaM kinase II and ERK1/2 activation by KN-93. A, VSM cells were treated with 1 µmol/L ionomycin (IONO) for 15 seconds after 30-minute incubation of the cells with various concentrations of KN-93. The total and autonomous CaM kinase II activity was determined ("Materials and Methods"), and autonomous activity was expressed as a percentage of the total (n=5). The effect of the inactive analogue KN-92 on generation of autonomous kinase activity is shown in the inset (n=6). B, The cells were treated with increasing concentrations of KN-93 before a 5-minute exposure to 1 µmol/L IONO. ERK1/2 activity was determined by in-gel assay, and radioactivity in the gels was quantified by PhosphorImager (Molecular Dynamics) ("Materials and Methods") and expressed as a percent ERK activity elicited by IONO alone (n=3 or 4). The effect of KN-92 on ionomycin-induced ERK1/2 activation is demonstrated in the inset (n=3). The IC50 values for the inhibition curves were determined by nonlinear regression fit using GraphPad Prism software.

Because of the lack of other reliable CaM kinase II inhibitors, the calmodulin antagonists W-7 and calmidazolium were used to demonstrate a role for calmodulin in the ionomycin-induced activation of ERK1/2. Pretreatment of VSM cells with 30 µmol/L W-7 for 30 minutes resulted in an attenuation of the ionomycin-induced MAP kinase activation, with a >60% decrease at the 5-minute time point (Fig 5A). A structurally dissimilar calmodulin inhibitor, calmidazolium (60 µmol/L), was also able to antagonize ERK1/2 activation after 5 minutes of ionomycin treatment (67% of ionomycin control, Fig 5B). These data are consistent with a role for CaM kinase II in the Ca²⁺-induced activation of the ERK cascade, since CaM kinase II requires both Ca²⁺ and calmodulin for initiation of its activity.

View larger version (33K): [in this window] [in a new window]   Figure 5. Effect of the calmodulin inhibitors W-7 and calmidazolium on the ionomycin (IONO)-induced ERK1/2 activity. A, VSM cells were incubated with 30 µmol/L W-7 or left untreated before exposure to IONO for up to 45 minutes. ERK1/2 activity in each sample (5 µg protein per lane) was determined by in-gel assay. The lanes marked ERK2 indicate the activity of various amounts of active ERK2 as detected by the in-gel assay. These experiments were repeated twice, with similar results. B, VSM cells were treated with 60 µmol/L of calmidazolium before exposure to IONO for 5 minutes, in-gel kinase assay, and quantification by PhosphorImager (Molecular Dynamics) (data are the mean of two experiments).

Elevation of [Ca²⁺]_i also leads to the activation of Ca²⁺-dependent isoforms of PKC,^{23 49 50} and PKC activation has been shown to activate ERK1/2 in various cell types. To rule out PKC as a mediator of the Ca²⁺-induced activation of ERK1/2, exposure of cells to ionomycin was preceded by incubation with up to 1 µmol/L of the specific PKC inhibitor, Ro 31-8220. This treatment had no significant effect on ERK1/2 induction by ionomycin; however, the effects of the phorbol ester, PDB, were significantly inhibited by increasing concentrations of Ro 31-8220 (n=3, Fig 6A). A second PKC inhibitor, bisindolylmaleimide I, also selectively inhibited ERK1/2 activation in response to 1 µmol/L PDB but not ionomycin (n=3, Fig 6B). These results indicate that PKC does not play a significant role in the Ca²⁺-induced activation of ERK1/2 in VSM cells.

View larger version (19K): [in this window] [in a new window]   Figure 6. Effect of the PKC inhibitors Ro 31-8220 (Ro) and bisindolylmaleimide I (Bis) on ERK1/2 activation by ionomycin (IONO) and phorbol esters. A, Treatment of VSM cells with various concentrations of Ro was followed by exposure to 1 µmol/L IONO (for 5 minutes) or 1 µmol/L PDB (for 20 minutes), in-gel MAP kinase assay, and quantification by PhosphorImager (Molecular Dynamics). B, VSM cells were treated with different concentrations of Bis before exposure to PDB or IONO, as in panel A, and ERK activity assay. For each sample, 5 µg of protein was assayed, and activity units are expressed as a percentage of the PDB or IONO control response (mean±SEM of three or four independent experiments). *P<.05 vs PDB alone, by one-way ANOVA and Bonferroni's post hoc test.

To further investigate the scope of the KN-93 effect, its ability to inhibit ERK1/2 activation by various agents was studied. Pretreatment of VSM cells with KN-93 for 30 minutes prevented the development of ionomycin-induced ERK1/2 activity. However, ERK activity induced by PDGF or the phorbol ester PMA was unchanged after KN-93 pretreatment (Fig 7A). In contrast, the ERK response to the Ca²⁺-mobilizing agonists angiotensin II (300 nmol/L) and thrombin (300 nmol/L) were attenuated by 60% and 40%, respectively, after KN-93 treatment (Fig 7B). In addition, the inactive analogue KN-92 at this concentration did not significantly attenuate ERK1/2 activation in response to angiotensin II or thrombin. These results are consistent with a role for CaM kinase II in the activation of ERK by Ca²⁺-mobilizing agonists, since these agents have been shown to elicit CaM kinase II activation by increasing [Ca²⁺]_i.³³

In order to confirm the pharmacological data obtained in VSM cells, COS-7 cells were transfected with CaM kinase II to study the effect of this maneuver on ERK activity. Cultured VSM cells express the ₂ isoform of CaM kinase II most abundantly,³² so we transfected this form of the kinase into COS-7 cells. COS-7 cells were selected for these experiments because they have low endogenous amounts of total and autonomous CaM kinase II activity (3.33±0.64 and 0.21±0.06 nmol · min^-1 · mg protein^-1, respectively; n=5). Transient transfections with ₂ CaM kinase II cDNA resulted in 49.83±2.55 nmol · min^-1 · mg protein^-1 (autonomous activity, 0.61±0.09 nmol · min^-1 · mg protein^-1; n=5) of Ca²⁺/calmodulin-dependent kinase activity with transfection efficiencies in the range of 20% to 25% (by immunofluorescence microscopy, not shown). COS-7 cells were transfected with the CMV vector, vector containing ₂ CaM kinase II cDNA, or vector containing the K43A mutant or were cotransfected with ₂ CaM kinase II–containing and K43A-containing vectors. As shown in Fig 8, expression of CaM kinase II in COS-7 cells resulted in a 1.32-fold increase in basal ERK2 activity (n=5, not significantly different from CMV control at P<.05); however, in cells expressing ₂ CaM kinase II, ionomycin elicited a 2-fold increase in ERK2 activity (P<.05, relative to CMV control). ERK2 activation by ionomycin in CaM kinase II–expressing cells was also significantly greater than that in cells expressing CMV vector (Fig 8B, P<.05). Furthermore, the enhanced ERK2 activation (in response to ionophore treatment) in wild-type kinase–expressing cells was prevented by the coexpression of the K43A mutant kinase in these cells (n=2, Fig 8B).

View larger version (32K): [in this window] [in a new window]   Figure 8. Effect of CaM kinase II (CaMKII) overexpression on ERK activity in COS-7 cells as determined by in-gel activity. A, Cells were transiently transfected with control CMV vector or vector containing 2CaMKII cDNA or were cotransfected with both the wild-type 2 and K43A mutant cDNA. Basal ERK activities or ERK activity after ionomycin (IONO, 5 minutes) treatment was determined by in-gel method and quantified. Each lane contained 5 µg of extract protein, except the far right lane, which contained 5 ng of active ERK2. B, Summarized data are shown from five independent transfection experiments (mean±SEM), except for 2CaMKII+K43A and IONO+2CaMKII+K43A bars, which are the means of two experiments. *P<.05 vs CMV control; **P<.05 vs CMV control and CMV+IONO by one-way ANOVA (repeated measures), using Bonferroni's post hoc test. C, Activation of MAP kinase in COS-7 cells, transfected with the CMV, 2CaMKII, K43A, or 2CaMKII+K43A vectors, was detected using the anti–active MAP kinase antibody. Each lane in the blot contained 5 µg of lysate proteins and was treated in a manner similar to that indicated in panel A above.

In a separate experiment, the effects of CaM kinase II transfection on ERK activity were determined by probing Western blots of the various samples with the anti–active MAP kinase antibody. This antibody is made against a dually phosphorylated peptide that corresponds to the active form of ERKs. COS-7 cells were transfected, as indicated in "Materials and Methods," with different CaM kinase II constructs, and the amount of activated ERK in the respective extracts was analyzed. Expression of ₂ CaM kinase II in these cells resulted in a 3.5-fold increase in inactive ERK2 in unstimulated cells compared with CMV-transfected cells, whereas the expression of the inactive (K43A) mutant alone did not elevate the amount of basal ERK2 (Fig 8C). Coexpression of the wild-type and mutant proteins resulted in only a 1.9-fold increase in basal active ERK. In CMV-expressing cells, ionomycin elicited a 2.7-fold increase in active ERK2 over basal values, and this was enhanced by 140% in cells expressing wild-type ₂ CaM kinase II (3.7-fold over basal, Fig 8C and 8D). The data from these transfection experiments support the observations made in the VSM cells and intimate that CaM kinase II can influence the MAP kinase signaling pathway under certain conditions.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Receptor agonists that are coupled to activation of the G_q/11 type of G proteins cause the hydrolysis of membrane phospholipid, leading to intracellular Ca²⁺ mobilization and PKC activation. By an as-yet-unclear mechanism, these agents are able to elicit ERK activation in many cells. Direct activation of PKC by phorbol esters has been shown to activate the MAP kinase pathway, possibly by the phosphorylation of raf or another similar MEK kinase.^{26 27} However, there appears to be an additional mechanism by which Ca²⁺-mobilizing agonists elicit the activation of this cascade, since direct elevation of intracellular Ca²⁺ by pharmacological agents is able to produce robust ERK1/2 activation in some cell types. Additionally, experiments involving downregulation or inhibition of PKC have revealed that Ca²⁺-mobilizing agonists are, at most, only partially dependent on this enzyme for stimulating the ERK pathway.^{19 24 51} In VSM cells, where Ca²⁺-mobilizing agonists produce a marked stimulation of ERK1/2, we have shown that CaM kinase II is also rapidly activated in a Ca²⁺-dependent manner.³³ On the basis of the present results with the CaM kinase inhibitor and overexpression studies in COS-7 cells, we conclude that CaM kinase II mediates the Ca²⁺-induced activation of the MAP kinase cascade in VSM cells.

Exposure of VSM cells to ionomycin resulted in a rapid but transient activation of ERK1/2 as determined by the in-gel kinase assay (Fig 1A). Probing Western blots containing proteins from similarly treated cells (with the anti–phospho-MAP kinase antibody) showed that activation of ERK1/2 corresponded to the tyrosine phosphorylation state of the proteins (Fig 2A). Pretreating VSM cells with the CaM kinase II inhibitor KN-93 caused an almost complete loss in ERK1/2 activity in response to ionomycin (Fig 1B), whereas its inactive analogue (KN-92) had no significant effect (Fig 4B, inset). In parallel to the observed decrease in ERK activities, KN-93 treatment resulted in diminished tyrosine phosphorylation of ERK1/2, as indicated by immunoblotting (Fig 2B). These results indicate that KN-93 exerts its effects at a point proximal to ERK1/2 phosphorylation, which is normally catalyzed by MEK.

KN-93 does not appear to exert its effects by preventing Ca²⁺ ion mobilization by ionomycin, since [Ca²⁺]_i induced by the ionophore was not diminished by pretreatment with the inhibitor (Fig 3). Both KN-93 and the inactive analogue KN-92 were observed to enhance intracellular Ca²⁺ levels in response to ionophore treatment, particularly after 2 to 3 minutes of stimulation. The reason for this response is not clear, but it may be due to nonspecific actions of the KN compounds in a manner independent of CaM kinase II inhibition. KN-93 inhibited ERK1/2 or CaM kinase II activation in response to ionomycin with essentially identical potencies (IC₅₀s of 14 and 13 µmol/L, Fig 4), supporting the notion that the inhibitor produced its effects at a single site. Since CaM kinase II activation precedes ERK1/2 activation, it is most likely that CaM kinase II is the point of inhibition in the Ca²⁺-induced ERK activation.

As alternative pharmacological probes, the calmodulin inhibitors W-7 and calmidazolium were used to demonstrate a role for Ca²⁺ and calmodulin in the ionomycin-induced activation of ERK1/2 (and so support a role for CaM kinase II in this process). Both calmodulin inhibitors attenuated the ERK1/2 response to ionomycin by >60% (5-minute time point) in a manner consistent with a role for CaM kinase II in this process (Fig 5). Elevation of [Ca²⁺]_i is also thought to cause the activation of certain isoforms of PKC, which have been documented to activate the ERK pathway. To rule out the possibility that ionomycin activates PKC, which then induces ERK1/2, VSM cells were treated with the specific PKC inhibitors Ro 31-8220 or bisindolylmaleimide before exposure to the ionophore. Both PKC inhibitors showed remarkable selectivity toward phorbol ester–induced activation of ERK1/2, whereas ERK activation in response to ionomycin was essentially unaffected (Fig 6). These results indicate that Ca²⁺ and calmodulin, but not PKC, are involved in the induction of the ERK cascade by Ca²⁺ ionophore. These data are also similar to those reported by Eguchi et al,⁵² who demonstrated a role for Ca²⁺ and calmodulin (but not PKC) in the Ca²⁺-induced activation of MAP kinase in cultured VSM cells.

PDGF- and PMA-induced activation of MAP kinase was not affected by pretreatment with KN-93, whereas the thrombin and angiotensin II responses were inhibited by 40% and 60%, respectively (Fig 7). The results of these experiments indicate that the inhibitory effects of KN-93 are specific for Ca²⁺-mobilizing agents and not for MAP kinase induction by PKC or receptor tyrosine kinase activation. We have previously shown that Ca²⁺-mobilizing agonists, such as angiotensin II, are able to produce marked CaM kinase II activation in VSM cells and may use this pathway to activate the ERK cascade. The less than complete attenuation of the thrombin and angiotensin II response (compared with ionomycin, Fig 7B) suggests that these Ca²⁺-mobilizing agonists may use another MAP kinase–activating pathway(s) in addition to the one sensitive to KN-93.

The isoquinolinyl sulfonamide KN-62 and its more hydrophilic analogue KN-93 prevent the activation of CaM kinase II by interacting with the calmodulin-binding domain of the kinase.^{48 53} These inhibitors show a high degree of selectivity for CaM kinase II, with lesser effects on myosin light chain kinase, PKC, and PKA.^{48 53} Although these compounds appear to be specific with regard to CaM kinase II inhibition, other nonspecific effects on cellular function cannot be entirely ruled out. However, the lack of effect of KN-93 on MAP kinase induction by phorbol esters and PDGF demonstrates that this compound does not exert its effects by acting as a general protein kinase inhibitor. The data indicate that the inhibitory effects of KN-93 are restricted to MAP kinase activation by Ca²⁺-mobilizing agents and suggest that intracellular Ca²⁺ and growth factors/phorbol esters activate distinct signaling cascades, which impinge on ERK1/2 activation.

To obtain more direct evidence for an interaction between CaM kinase II and the ERK signaling pathway, overexpression studies of ₂ CaM kinase II in COS-7 cells were performed. COS-7 cells were chosen for this purpose because of the low amounts of endogenous CaM kinase II detectable by kinase assay (5- to 10-fold lower than in VSM cells) and for their ease of transfection. Expression of CaM kinase II in COS-7 cells resulted in elevation of both the resting and ionomycin-induced levels of ERK2 activity (Fig 8). The elevated basal values of ERK2 activity in unstimulated cells, expressing wild-type kinase, may be due to the fact that resting independent or autonomous activities in these cells is 3-fold higher than in the CMV-transfected cells. The specificity of the response to wild-type protein expression was demonstrated by the expression of the kinase-negative K43A mutant, which by itself did not enhance basal ERK2 activity nor did it inhibit the activation in response to ionomycin. This protein is unable to bind ATP because of a critical mutation at Lys⁴³ in the ATP-binding domain, which renders the protein inactive (C.M. Schworer, unpublished data, 1997). However, coexpression of the wild-type and inactive mutant resulted in lower basal ERK2 activities, relative to ₂ CaM kinase II expression alone, as well as a decrease in the ionomycin-stimulated activity. CaM kinase II, when expressed in COS-7 cells, forms large multimeric complexes composed of 10 to 12 individual subunits⁵⁴ and, when coexpressed with the K43A mutant, may form complexes that are partially inactive and are therefore unable to maintain sufficient autophosphorylation for the activation of substrates. Taken as a whole, the data from the transfection studies provide good supportive evidence that ₂ CaM kinase II is able to modulate the ERK signaling pathway in VSM cells.

A novel Ca²⁺-activated soluble tyrosine kinase (PYK2) has been reported to mediate the activation of MAP kinase in cells of neuronal origin.³¹ This protein undergoes rapid phosphorylation in PC12 cells treated with Ca²⁺-mobilizing agonists or ionophores as well as with KCl depolarization. However, the protein appears to be activated equally well by phorbol ester (PKC) stimulation and may indicate the regulation of this tyrosine kinase by multiple input pathways. It was reported that PYK2 was not directly activated by Ca²⁺, implying a role for an upstream protein(s) that is regulated by Ca²⁺ or Ca²⁺/calmodulin. While the present study was in progress, Eguchi et al⁵² reported that in cultured rat aortic smooth muscle cells both a calmodulin inhibitor (W-7) and a tyrosine kinase inhibitor (genistein) completely suppress MAP kinase activation by Ca²⁺ ionophore and angiotensin II. The authors proposed that a Ca²⁺/calmodulin-dependent tyrosine kinase may mediate the activation of MAP kinase in response to elevation of [Ca²⁺]_i. However, this conclusion relies on the specificity of genistein, which exerts effects in addition to tyrosine kinase inhibition,^{55 56} including inhibition of CaM kinase II activity (Reference 57⁵⁷ and authors' unpublished data, 1997). More recently, two different groups have reported a role for Ca²⁺/calmodulin-dependent protein kinases in the activation of the MAP kinase pathway. Enslen et al⁵⁸ reported that transfection of CaM kinase IV led to the activation of ERK2, JNK-1, and p38 MAP kinase in PC12 cells. These authors proposed that the CaM kinases were able to effect gene expression via Elk-1, c-Jun, and ATF2 by activation of the different MAP kinase pathways in this cell. In another report, Muthalif et al⁵⁹ reported that norepinephrine-induced MAP kinase activity in rabbit aortic VSM cells was attenuated by treatment with KN-93 or antisense oligonucleotides against -CaM kinase II. However, unlike in the present report, the specificity of KN-93 in their system was not described. In rat aortic VSM cells, it is unlikely that -CaM kinase II is associated with MAP kinase activation, since both molecular and immunological methods demonstrate that the predominant kinase isoform expressed is the ₂ variant.³²

The data in the present study provide evidence that CaM kinase II can mediate the activation of the MAP kinase cascade in VSM cells and that it may play a significant role in the induction of this pathway by Ca²⁺-mobilizing agonists (eg, angiotensin II and thrombin). The mode of action of CaM kinase II is currently unknown but may be analogous to PKC, which is thought to activate MAP kinase by interacting with raf kinase.^{25 26 27} It is unclear what functions are regulated by the Ca²⁺-dependent activation of the ERK pathway, but there appears to be a close association between the proposed functions of CaM kinase II and MAP kinase in learning/memory,¹⁵ gene expression,^{37 60 61} and cell-cycle regulation.^{62 63} Thus, in VSM cells, CaM kinase II, via ERK1/2, may serve to regulate events such as cytoskeletal organization, cell migration, proliferation, and gene expression.

	Selected Abbreviations and Acronyms

 

CaM kinase = Ca2+/calmodulin-dependent protein kinase CMV = cytomegalovirus ERK = extracellular signal–regulated kinase MAP = mitogen-activated protein MEK = ERK kinase PCR = polymerase chain reaction PDB = phorbol 12,13-dibutyrate PDGF = platelet-derived growth factor-BB PKA, PKC = protein kinase A and C PMA = phorbol 12-myristate 13-acetate PMSF = phenylmethylsulfonyl fluoride TBS = Tris-buffered saline VSM = vascular smooth muscle

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-49426 (Dr Singer) and an American Heart Association (Pennsylvania Affiliate, Inc) Postdoctoral Fellowship (Dr Abraham). We are grateful to Dr D. VanRiper for critical review of this manuscript and to D. Cooney for performing the cell-culture work.

	Footnotes

 
Presented in part in abstract form at the 9th International Conference on Second Messengers and Phosphoproteins, Nashville, Tenn, October 27–November 1, 1995 (abstract 444) and Experimental Biology 96 (FASEB J. 1996;10:A17).

Received April 11, 1997; accepted June 23, 1997.

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				F. Li and K. U. Malik Angiotensin II-induced Akt activation is mediated by metabolites of arachidonic acid generated by CaMKII-stimulated Ca2+-dependent phospholipase A2 Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2306 - H2316. [Abstract] [Full Text] [PDF]

				P. H. Ratz, K. M. Berg, N. H. Urban, and A. S. Miner Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus Am J Physiol Cell Physiol, April 1, 2005; 288(4): C769 - C783. [Abstract] [Full Text] [PDF]

				I. H Haralambieva, I. D Iankov, P. V Ivanova, V. Mitev, and I. G Mitov Chlamydophila pneumoniae induces p44/p42 mitogen-activated protein kinase activation in human fibroblasts through Toll-like receptor 4 J. Med. Microbiol., December 1, 2004; 53(12): 1187 - 1193. [Abstract] [Full Text] [PDF]

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				E. Toth-Zsamboki, C. Oury, H. Cornelissen, R. De Vos, J. Vermylen, and M. F Hoylaerts P2X1-mediated ERK2 Activation Amplifies the Collagen-induced Platelet Secretion by Enhancing Myosin Light Chain Kinase Activation J. Biol. Chem., November 21, 2003; 278(47): 46661 - 46667. [Abstract] [Full Text] [PDF]

				T. Borbiev, A. D. Verin, A. Birukova, F. Liu, M. T. Crow, and J. G. N. Garcia Role of CaM kinase II and ERK activation in thrombin-induced endothelial cell barrier dysfunction Am J Physiol Lung Cell Mol Physiol, July 1, 2003; 285(1): L43 - L54. [Abstract] [Full Text] [PDF]

				J.-C. Schneider, D. El Kebir, C. Chereau, S. Lanone, X.-L. Huang, A. S. De Buys Roessingh, J.-C. Mercier, J. Dall'Ava-Santucci, and A. T. Dinh-Xuan Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2311 - H2319. [Abstract] [Full Text] [PDF]

				K. Mishra-Gorur, H. A. Singer, and J. J. Castellot Jr Heparin Inhibits Phosphorylation and Autonomous Activity of Ca2+/Calmodulin-Dependent Protein Kinase II in Vascular Smooth Muscle Cells Am. J. Pathol., November 1, 2002; 161(5): 1893 - 1901. [Abstract] [Full Text] [PDF]

				J. M. Lorenz, M. H. Riddervold, E. A. H. Beckett, S. A. Baker, and B. A. Perrino Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues Am J Physiol Cell Physiol, November 1, 2002; 283(5): C1399 - C1413. [Abstract] [Full Text] [PDF]

				K. Mishra-Gorur, H. A. Singer, and J. J. Castellot Jr. The S18 Ribosomal Protein Is a Putative Substrate for Ca2+/Calmodulin-activated Protein Kinase II J. Biol. Chem., September 6, 2002; 277(37): 33537 - 33540. [Abstract] [Full Text] [PDF]

				Y. Funakoshi, T. Ichiki, K. Takeda, T. Tokuno, N. Iino, and A. Takeshita Critical Role of cAMP-response Element-binding Protein for Angiotensin II-induced Hypertrophy of Vascular Smooth Muscle Cells J. Biol. Chem., May 17, 2002; 277(21): 18710 - 18717. [Abstract] [Full Text] [PDF]

				H. R. Ansari, S. Husain, and A. A. Abdel-Latif Activation of p42/p44 Mitogen-Activated Protein Kinase and Contraction by Prostaglandin F2alpha , Ionomycin, and Thapsigargin in Cat Iris Sphincter Smooth Muscle: Inhibition by PD98059, KN-93, and Isoproterenol J. Pharmacol. Exp. Ther., October 1, 2001; 299(1): 178 - 186. [Abstract] [Full Text] [PDF]

				Y. Zou, A. Yao, W. Zhu, S. Kudoh, Y. Hiroi, M. Shimoyama, H. Uozumi, O. Kohmoto, T. Takahashi, F. Shibasaki, et al. Isoproterenol Activates Extracellular Signal-Regulated Protein Kinases in Cardiomyocytes Through Calcineurin Circulation, July 3, 2001; 104(1): 102 - 108. [Abstract] [Full Text] [PDF]

				M. El Mabrouk, R. M. Touyz, and E. L. Schiffrin Differential ANG II-induced growth activation pathways in mesenteric artery smooth muscle cells from SHR Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H30 - H39. [Abstract] [Full Text] [PDF]

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				T.-R. Jan and N. E. Kaminski Role of mitogen-activated protein kinases in the differential regulation of interleukin-2 by cannabinol J. Leukoc. Biol., May 1, 2001; 69(5): 841 - 849. [Abstract] [Full Text]

				T. Kato, M. Sano, S. Miyoshi, T. Sato, D. Hakuno, H. Ishida, H. Kinoshita-Nakazawa, K. Fukuda, and S. Ogawa Calmodulin Kinases II and IV and Calcineurin Are Involved in Leukemia Inhibitory Factor-Induced Cardiac Hypertrophy in Rats Circ. Res., November 10, 2000; 87(10): 937 - 945. [Abstract] [Full Text] [PDF]

				I. Kim, H.-D. Je, C. Gallant, Q. Zhan, D. V. Riper, J. A Badwey, H. A Singer, and K. G Morgan Ca2+-calmodulin-dependent protein kinase II-dependent activation of contractility in ferret aorta J. Physiol., July 15, 2000; 526(2): 367 - 374. [Abstract] [Full Text] [PDF]

				W. Zhu, Y. Zou, I. Shiojima, S. Kudoh, R. Aikawa, D. Hayashi, M. Mizukami, H. Toko, F. Shibasaki, Y. Yazaki, et al. Ca2+/Calmodulin-dependent Kinase II and Calcineurin Play Critical Roles in Endothelin-1-induced Cardiomyocyte Hypertrophy J. Biol. Chem., May 12, 2000; 275(20): 15239 - 15245. [Abstract] [Full Text] [PDF]

				I. D. Kong, S. D. Koh, O. Bayguinov, and K. M Sanders Small conductance Ca2+-activated K+ channels are regulated by Ca2+-calmodulin-dependent protein kinase II in murine colonic myocytes J. Physiol., April 15, 2000; 524(2): 331 - 337. [Abstract] [Full Text] [PDF]

				J. Egea, C. Espinet, R. M. Soler, S. Peiro, N. Rocamora, and J. X. Comella Nerve Growth Factor Activation of the Extracellular Signal-Regulated Kinase Pathway Is Modulated by Ca2+ and Calmodulin Mol. Cell. Biol., March 15, 2000; 20(6): 1931 - 1946. [Abstract] [Full Text] [PDF]

				A. Rokolya and H. A. Singer Inhibition of CaM kinase II activation and force maintenance by KN-93 in arterial smooth muscle Am J Physiol Cell Physiol, March 1, 2000; 278(3): C537 - C545. [Abstract] [Full Text] [PDF]

				P. S. Naidu, V. Velarde, C. S. Kappler, R. C. Young, R. K. Mayfield, and A. A. Jaffa Calcium-calmodulin mediates bradykinin-induced MAPK phosphorylation and c-fos induction in vascular cells Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H1061 - H1068. [Abstract] [Full Text] [PDF]

				S. D. Koh, B. A Perrino, W. J Hatton, J. L Kenyon, and K. M Sanders Novel regulation of the A-type K+ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II J. Physiol., May 15, 1999; 517(1): 75 - 84. [Abstract] [Full Text] [PDF]

				W. C. Watt and D. R. Storm Odorants Stimulate the ERK/Mitogen-activated Protein Kinase Pathway and Activate cAMP-response Element-mediated Transcription in Olfactory Sensory Neurons J. Biol. Chem., January 12, 2001; 276(3): 2047 - 2052. [Abstract] [Full Text] [PDF]

				Y. Nishimura and T. Tanaka Calcium-dependent Activation of Nuclear Factor Regulated by Interleukin 3/Adenovirus E4 Promoter-binding Protein Gene Expression by Calcineurin/Nuclear Factor of Activated T Cells and Calcium/Calmodulin-dependent Protein Kinase Signaling J. Biol. Chem., June 1, 2001; 276(23): 19921 - 19928. [Abstract] [Full Text] [PDF]

				R. Ginnan and H. A. Singer CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle Am J Physiol Cell Physiol, April 1, 2002; 282(4): C754 - C761. [Abstract] [Full Text] [PDF]

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