PYK2 regulates SERCA2 gene expression in neonatal rat ventricular myocytes

Maria C. Heidkamp, Brian T. Scully, Kalpana Vijayan, Steven J. Engman, Erika L. Szotek, and Allen M. Samarel

The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois

Submitted 18 March 2005 ; accepted in final form 5 April 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (PYK2) has been implicated in cell signaling pathways involved in left ventricular hypertrophy and heart failure, but its exact role has not been elucidated. In this study, replication-defective adenoviruses (Adv) encoding green fluorescent protein (GFP)-tagged, wild-type (WT), and mutant forms of PYK2 were used to determine whether PYK2 overexpression activates MAPKs, and downregulates SERCA2 mRNA levels in neonatal rat ventricular myocytes (NRVM). PYK2 overexpression significantly decreased SERCA2 mRNA (as determined by Northern blot analysis and real-time RT-PCR) to 54 ± 4% of Adv-GFP-infected cells 48 h after Adv infection. Adv-encoding kinase-deficient (KD) and Y402F phosphorylation-deficient mutants of PYK2 also significantly reduced SERCA2 mRNA (WT>KD>Y402F). Conversely, the PTK inhibitor PP2 (which blocks PYK2 phosphorylation by Src-family PTKs) significantly increased SERCA2 mRNA levels. PYK2 overexpression had no effect on ERK1/2, but increased JNK1/2 and p38MAPK phosphorylation from fourfold to eightfold compared with GFP overexpression. Activation of both "stress-activated" protein kinase cascades appeared necessary to reduce SERCA2 mRNA levels. Adv-mediated overexpression of constitutively active (ca)MKK6 or caMKK7, which activated only p38MAPK or JNKs, respectively, was not sufficient, whereas combined infection with both Adv reduced SERCA2 mRNA levels to 45 ± 12% of control. WTPYK2 overexpression also significantly reduced SERCA2 promoter activity, as determined by transient transfection of a 3.8-kb SERCA2 promoter-luciferase construct. Thus a PYK2-dependent signaling cascade may have a role in abnormal cardiac Ca2+ handling in left ventricular hypertrophy and heart failure via downregulation of SERCA2 gene transcription.

signal transduction; heart failure; calcium; protein kinase C


LEFT VENTRICULAR HYPERTROPHY (LVH) and its transition to heart failure (HF) are associated with numerous changes in myocardial gene expression and protein turnover. Of particular interest, many investigators have documented a significant reduction in sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2) gene expression in ventricular tissue of experimental animals and patients with LVH and HF (for review, see Ref. 22). SERCA2 downregulation clearly has functional consequences in the HF state because even small alterations in the number of sarcoplasmic reticulum (SR) Ca2+ pumps can affect SR Ca2+ load, and thereby affect, in part, the concentration of released Ca2+ available for cardiomyocyte contraction (18, 23). SERCA2 downregulation also prolongs the intracellular [Ca2+] ([Ca2+]i) transient, and thus increases the diastolic level of [Ca2+]i, which may in turn activate Ca2+-dependent signaling pathways involved in the induction of cardiomyocyte hypertrophy (32) and apoptosis (57).

Despite the potential negative impact of SERCA2 downregulation on cardiac structure and function, relatively few studies have addressed the mechanisms and signal transduction pathways responsible for these changes. Both transcriptional (1, 2, 17, 40, 48, 49, 53) and posttranscriptional (30, 31, 37, 39, 51) mechanisms may be involved, although the specific effectors responsible for SERCA2 downregulation are only now being identified. In a previous study from our laboratory (6), we showed that decreasing [Ca2+]i by blockade of voltage-gated, L-type Ca2+ channels substantially increased basal SERCA2 mRNA and protein levels in neonatal rat ventricular myocytes (NRVM), and stimulated sarcoplasmic reticulum Ca2+ uptake, resulting in a twofold increase in the rate of [Ca2+]i decline. In contrast, cyclic stretch of noncontracting NRVM substantially downregulated SERCA2 mRNA and protein levels via a mechanism independent of Ca2+ influx via voltage-gated, L-type Ca2+ channels (12). Furthermore, we and others (15, 16, 21, 36, 37) have shown that one or more isoenzymes of protein kinase C (PKC) are critical factors regulating SERCA2 gene expression in response to neurohormonal stimuli. For instance, Ho et al. (21) previously showed that phorbol myristate acetate (PMA), a potent activator of PKC-{alpha}, -{beta}, -{delta}, and -{epsilon} in cardiomyocytes, downregulated SERCA2 and prolonged the [Ca2+]i transient via activation of the Raf-MEK1-ERK1/2 pathway. Similarly, Andrews et al. (1) demonstrated that activation of the MKK6-p38MAPK signaling pathway also caused the transcriptional downregulation of SERCA2 promoter activity. However, the specific components linking Ca2+, cyclic stretch, and PKCs to the MAPK signaling pathways that regulate SERCA2 levels in cardiomyocytes remain unknown.

The Ca2+-dependent, nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor PTKs. PYK2 coordinates Ca2+-, integrin-, and PKC-dependent signaling pathways in some tissues (for review, see Ref. 3). PYK2 expression and/or phosphorylation are regulated by [Ca2+]i and PKCs in cardiomyocytes and vascular smooth muscle cells (7, 8, 41). As examined in various cell types, PYK2 acts as an important scaffolding protein, and transduces signals from G protein-coupled receptors to downstream MAPK signaling pathways depending on which signaling kinases and adapter proteins bind to the phosphorylated enzyme (10, 34). PYK2 has also been shown to link a variety of stressful stimuli, including Ca2+ overload, UV irradiation, and TNF-{alpha} treatment to MAPK activation in several cell types (50). Recently, Hirotani et al. (20) demonstrated that PYK2 is an essential signaling component in endothelin- and phenylephrine-induced NRVM hypertrophy, perhaps acting via the Ca2+- and/or PKC-dependent activation of Rac1.

We (9) previously demonstrated that PYK2 expression and phosphorylation were significantly increased in adult rat ventricular myocytes in vivo during the transition from LVH to HF coincident with SERCA2 downregulation. Similarly, Melendez et al. (29) showed that PYK2 expression and phosphorylation were increased in a mouse model of dilated cardiomyopathy, but its exact role in these conditions has not been elucidated. In this study, we utilized replication-defective adenoviruses (Adv) encoding wild-type and mutant forms of PYK2 to interrogate their effects on cell growth, MAPK activation, and SERCA2 gene expression in NRVM. These data are presented to suggest a novel, and potentially important negative feedback mechanism regulating [Ca2+]i levels in cardiomyocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. PC-1 tissue culture medium was obtained from BioWhittaker (Walkersville, MD). Dulbecco's modified Eagle’s medium (DMEM) and medium 199 were obtained from GIBCO-BRL (Grand Island, NY). PYK2 and paxillin monoclonal antibodies (mAb) were obtained from Transduction Laboratories (Lexington, KY) and Upstate Biotechnologies (Lake Placid, NY). Rhodamine-conjugated goat anti-mouse IgG was obtained from Molecular Probes (Eugene, OR). Phosphospecific PYK2-Y402 mAb was purchased from Biosource (Camarillo, CA). Phospho-specific ERK and JNK polyclonal antibodies (pAb) were from Promega (Madison, WI) and phospho-specific p38MAPK, total SAPK/JNK, and total p38MAPK pAb were from Cell Signaling (Beverly, MA). Antibodies for total ERK1/2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Real-time RT-PCR reagents were obtained from Amersham Biosciences and Invitrogen (Carlsbad, CA). cDNA primers and probes were obtained from Integrated DNA Technologies (Coralville, IA). 4-Amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) was obtained from Calbiochem (La Jolla, CA). All other reagents were of the highest grade commercially available and were obtained from Sigma (St. Louis, MO) and Baxter S/P (McGaw Park, IL).

Cell culture. The animals were handled in accordance with the American Physiological Society "Guiding Principles in the Care and Use of Animals." All procedures involving the animals were approved by the Institutional Animal Care and Use Committee of Loyola University Medical Center. NRVM were isolated as previously described (42). NRVM were plated at a density of 400, 1,000, or 1,600 cells/mm2 onto collagen-coated 35, 60, or 100 mm dishes, or one-well Permanox chamberslides, and left undisturbed in a 5% CO2 incubator for 18–24 h. Attached cells were maintained in DMEM/medium 199 (4:1) containing antibiotic/antimycotic solution. Under these serum-free culture conditions, NRVM displayed synchronous [Ca2+]i transients and beating activity (~100–150 beats/min) within 24 h after being plated.

Adenoviral constructs. Replication-deficient Adv encoding WT and mutant forms of PYK2 were generated as previously described (27). Briefly, PYK2 cDNAs (kindly provided by J. T. Parsons, University of Virginia) were subcloned in frame into pEGFP-C1 vector (BD Biosciences-Clontech, Palo Alto, CA). The WTPYK2 contained the entire 1,009 amino acid coding sequence of rat PYK2 (43). The kinase-defective (KD) mutant consisted of a single amino acid substitution (K457A) within the central catalytic domain. The autophosphorylation site mutant consisted of a single amino acid substitution (Y402F), which is required for Src-family kinase binding. The green fluorescent protein (GFP)-tagged PYK2 inserts were then sequentially subcloned into pShuttle-CMV plasmid, and then pAdeno-X viral DNA (BD Biosciences-Clontech). Linearized pAdeno-X+GFP-PYK2 sequences were transfected into HEK-293 cells, and the resulting WT and mutant PYK2 Adv were amplified and purified by double CsCl gradient centrifugation. Replication-defective Adv encoding constitutively active (ca)MKK6 and caMKK7 were kindly provided by Dr. Yibin Wang (University of California at Los Angeles) (54, 55), whereas a replication-defective Adv encoding caMEK1 was kindly provided by Dr. Jeffrey Molkentin, University of Cincinnati (11). An Adv-expressing GFP (Adv-GFP) and an Adv-expressing nuclear-encoded (ne) {beta}-galactosidase (Adv-ne{beta}gal) were used to control for nonspecific effects of Adv infection. The multiplicity of viral infection (MOI) was determined by dilution assay in human embryonic kidney (HEK)-293 cells grown in 96 well clusters. Cardiomyocytes were infected (60 min, 25°C with gentle agitation) with each Adv diluted in DMEM/medium 199. The medium was then replaced with virus-free DMEM/medium 199, and the cells cultured for an additional 8, 24, or 48 h.

Protein content and cell size. For the analysis of NRVM protein content, cells were quantitatively scraped from dishes in 0.2 N perchloric acid (1 ml) and collected by centrifugation (10,000 g, 10 min). The precipitate was redissolved by incubation (60°C, 20 min) in 250 µl of 0.3 N KOH. Aliquots were used for analysis of total protein by the Lowry method with crystalline human serum albumin as standard, and for DNA using 33258 Hoechst dye and salmon sperm DNA as standard (46). For determination of cell surface area, cardiomyocytes were first loaded with 2'7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM [2 µM in a modified Krebs medium composed of (in mM) 135 NaCl, 5.9 KCl, 1.5 CaCl2, 1.2 MgCl2, 11.5 glucose, and 11.6 HEPES, pH 7.3, supplemented with 0.1% BSA and 0.2% Pluronic F-127 detergent] for 1 h, followed by 1-h incubation in BCECF-free Krebs buffer. Cells were viewed with a laser scanning confocal microscope (model LSM 510, Zeiss). Optical sections through the base of the cells (~20 cells/field) were stored as digital images and analyzed with Image-1 software (Universal Imaging, West Chester, PA). A binary mask was created by setting the threshold brightness that distinguished the fluorescent cells from the black background. Cell area was determined as an exact count of the number of pixels that made up the object's binary mask multiplied by the area of a unit pixel (46).

Cell fixation and confocal microscopy. NRVM grown on chamberslides were infected with Adv-GFP or Adv-GFPWTPYK2 (10 MOI, 48 h). Cells were fixed, permeabilized, and stained with anti-paxillin mAb, as previously described (13, 14). Fluorescently labeled cells were viewed with a Zeiss LSM 510 confocal microscope.

Western blot analysis. NRVM were homogenized in lysis buffer (44). Equal amounts of extracted proteins (50 µg) were separated by SDS-PAGE and Western blot analysis on 7.5% or 10% polyacrylamide gels. Primary antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody, and visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL).

mRNA analysis. Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA). RNA was quantified by absorbance at 260 nm, and its integrity was determined by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels. SERCA2 and GAPDH mRNA levels were then analyzed with Northern blots or by real-time RT-PCR, as previously described (12, 36).

SERCA2 promoter activity. NRVM plated at a density of ~1,000 cells/mm2 were transiently transfected with a SERCA2 promoter plasmid construct pGL3-b-S2-HindIII/HincII (kindly provided by Dr. Warner Simonides, VU University Medical Center, Amsterdam) (53). The plasmid contained a fragment from the rat SERCA2 promoter (-3,263/+550 bp from the transcriptional start site) linked to the firefly luciferase reporter gene. A control plasmid, containing the Rous sarcoma virus-long terminal repeat (RSV-LTR), linked to a bacterial {beta}gal reporter gene (kindly provided by Dr. Edward Kislauskis, University of Massachusetts Medical Center) was used in conjunction with the SERCA2 plasmid to control for transfection efficiency. Cells were transfected with 5 µg of the SERCA2 promoter plasmid along with 0.5 µg of control RSV-LTR {beta}gal. The plasmids were added to 4:1 medium free of both serum, as well as antibiotic/antimycotic agents, in the presence of 10 µl/ml Lipofectamine 2000 reagent (Invitrogen Life Technologies, Carlsbad, CA). Cells were treated with the transfection cocktail for 6 h, and were then maintained in serum-free, 4:1 medium containing antibiotic/antimycotic agents for up to 72 h.

SERCA2 promoter activity was assessed using the protocol described in the Luciferase Assay System (Promega, Madison, WI). Briefly, 4:1 medium were removed and 300 µl of 1 x reporter lysis buffer were added to the cells. The cells were then quick-frozen in a methanol/dry ice bath and allowed to thaw on ice. Cells were scraped into prechilled 1.5-ml Eppendorf tubes, briefly vortexed, and then centrifuged at 12,000 g for 2 min at 4°C. Supernatants were transferred into a fresh set of tubes and maintained on ice. A manual luminometer (Lumat LB 9501, Berthold Systems, Aquilippa, PA) was used to measure luciferase activity. For each sample, 100 µl of luciferase assay reagent were added to 50 µl of cell lysate, and luminescence was measured over a 30-s period. Detection of RSV-LTR {beta}gal activity in cell lysates was performed using a {beta}gal detection kit (catalog no. MO255, Marker Gene Technologies). Briefly, 20-µl aliquots of cell lysates were pipetted into individual wells in opaque 96-well plates. One hundred microliters of reaction buffer (100 mM NaPO4, 1 mM MgCl2, 10 mM {beta}-mercaptoethanol, and 0.1% Triton X-100, pH 7.0) were added to each well. The fluorogenic substrate reagent 3-carboxyumbellifery {beta}-D-galactopyranoside (50 µl, 5 mM) was added and samples were allowed to incubate (20 min) at room temperature. Stop buffer (500 mM glycine and 10 mM EDTA, pH 12.0; 100 µl) was then added and samples were allowed to incubate (10 min) at room temperature. Fluorescence values were measured on a Synergy HT Multi-Detection Plate Reader (Bio-Tek Instruments, Winooski, VT) using 390-nm excitation and 460-nm emission wavelengths. Promoter activity was reported as the ratio of luciferase luminescence values to {beta}-galactosidase fluorescence values.

Data analysis. Results were expressed as means ± SE. Normality was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was assessed using Levene's test. Data from multiple groups were compared with one-way ANOVA, or one-way ANOVA on ranks, followed by Dunnett's test, or Student-Newman-Keuls test, where appropriate. Data from two groups were compared by paired t-test, or Wilcoxon's signed-rank test where appropriate. Differences among means were considered significant at P < 0.05. Data were analyzed using SigmaStat version 1.0 (Jandel Scientific, San Rafael, CA).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
GFPWTPYK2 localization and expression in NRVM. NRVM were infected (10 MOI, 48 h) with Adv-GFPWTPYK2, and then fixed, permeabilized, counterstained with a mAb specific for paxillin, followed by rhodamine-conjugated goat anti-mouse IgG, and viewed under a laser-scanning confocal microscope. As shown in Fig. 1A, GFPWTPYK2 was predominantly distributed diffusely throughout the cytoplasm and was excluded from the cell nucleus, which was similar to the localization of endogenous PYK2 in neonatal and adult cardiomyocytes (7). However, PYK2 was also detected in some centrally arranged focal adhesions, as indicated by co-localization with the cytoskeletal adaptor protein paxillin.



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Fig. 1. Green fluorescent protein (GFP) wild-type (WT) proline-rich tyrosine kinase 2 (PYK2) localization and expression in neonatal rat ventricular myocytes (NRVM). A: NRVM were infected with adenovirus (Adv)-GFPWTPYK2 [10 multiplicity of infection (MOI), 48 h], fixed, counterstained with a mouse monoclonal antibody (mAb) specific for paxillin (1:1,000), followed by rhodamine-conjugated goat-anti mouse IgG (1:30), and viewed for rhodamine (red; left), or GFP (green; middle) fluorescence by confocal microscopy. Regions of colocalization appear yellow (right). B: NRVM were infected with either Adv-GFP or Adv-GFPWTPYK2 (8–48 h, 10 MOI; 8–48 h). Western blots (50 µg of extracted protein) were probed with an antibody that recognizes total PYK2 (phosphorylated + unphosphorylated) (top blot), or an antibody that recognizes PYK2 phosphorylated at Y402 (pPYK2-Y402; bottom blot). The position of molecular weight markers is indicated to the right of each blot.

 
Cell extracts from comparably infected NRVM were then examined for PYK2 expression and tyrosine phosphorylation at Y402. Endogenous PYK2 (106–110 kDa) was detected at relatively low levels in cells infected with Adv-GFP, whereas a prominent protein band of ~140 kDa was detected in cells as early as 8 h after infection with Adv-GFPWTPYK2 (Fig. 1B, top blot). Additional protein bands were detected at later time points after Adv infection. The same samples were then probed with an antibody specific for PYK2 phosphorylated at Y402 (Fig. 1B, bottom blot). Relatively little phosphorylated, endogenous PYK2 was detected in NRVM infected with the control Adv-GFP. However, the overexpressed, ~140 kDa protein was readily detected using the phosphospecific antibody. This polypeptide also cross-reacted with an anti-GFP antibody (data not shown). These results suggested that WTPYK2 overexpression substantially increased the amount of active PYK2 in cardiomyocytes, and thereby stimulated PYK2-dependent signaling.

GFPWTPYK2 expression alone does not cause NRVM hypertrophy. The effect of WTPYK2 overexpression on NRVM protein content and cell size were then examined. Total protein (µg/dish), DNA (µg/dish), and total protein/DNA ratio (Fig. 2A) were similar in uninfected, Adv-GFP-infected, and Adv-GFPWTPYK2-infected NRVM. Cell surface area (µm2/cell; Fig. 2B) was also unaffected. In contrast, the hypertrophic agent phorbol myristate acetate (PMA; 200 nM, 24 h) increased total protein/DNA ratio and cell size to a similar extent in each group. These data indicate that WTPYK2 overexpression alone was not sufficient to induce NRVM hypertrophy, but WTPYK2 overexpressing cells were still capable of undergoing hypertrophy in response to a hypertrophic stimulus.



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Fig. 2. GFP-WT-PYK2 expression alone does not cause NRVM hypertrophy. A: NRVM were maintained under control conditions (uninfected; UI) or infected (10 MOI) with either Adv-GFP or Adv-GFPWTPYK2. After 24 h, the dishes were then maintained in either control medium lacking phorbol myristate acetate (–PMA) or stimulated with the hypertrophic agent PMA (+PMA, 200 nM). Total protein/DNA ratio (µg/µg) was then analyzed in cell extracts 24 h later. Data are the means ± SE for triplicate determinations of total protein/DNA ratio from 6 experiments. B: similarly treated NRVM were dye loaded with BCECF-AM, and cell surface area (µm2) analyzed by confocal microscopy and image analysis. Data are means ± SE of 1,135–1,156 cells derived from 3 different cell isolations. There was no significant difference in protein/DNA ratio or surface area between the UI, GFP-treated, or WT-PYK2-treated groups. However, +PMA significantly (+P < 0.05) increased protein/DNA and surface area vs. –PMA cells in UI, GFP-, and WTPYK2-expressing cells.

 
GFPWTPYK2 overexpression downregulates SERCA2 mRNA levels. We next examined the effects of GFPWTPYK2 overexpression on SERCA2 mRNA levels in Northern blotting experiments. As seen in Fig. 3A, Adv-GFPWTPYK2 (10 MOI, 48 h) substantially reduced SERCA2 mRNA levels, compared with cells infected with Adv-GFP (10 MOI, 48 h). At this relatively low MOI, Adv infection alone had no apparent effect, as SERCA2 mRNA levels were similar in uninfected, control cells, and cells infected with Adv-GFP. The extent of SERCA2 downregulation was similar to that observed with PMA (16, 36, 38).



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Fig. 3. GFP-WT-PYK2 overexpression decreases sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2) mRNA levels. A: NRVM were maintained in control medium (UI), or medium containing PMA (48 h, 200 nM). Similar cultures were infected with Adv-GFP or Adv-GFPWTPYK2 (48 h, 10 MOI). A Northern blot (5 µg total RNA/lane) was sequentially probed with 32P-labeled cDNA probes specific for SERCA2 and GAPDH mRNAs. B: SERCA2 mRNA was quantitatively analyzed by real-time RT-PCR. Relative quantities of amplified SERCA2 cDNA from each sample were standardized to GAPDH cDNA and then normalized to levels in UI cells. Data are means ± SE of 5 experiments; *P < 0.05 vs. time-matched UI cells. C: NRVM were infected with either Adv-GFP or Adv-GFPWTPYK2. RNA was analyzed at 8, 24, and 48 h after infection. Data are means ± SE of 4 experiments; *P < 0.05 vs. time-matched control.

 
To obtain a more sensitive and quantitative assessment of these relatively modest changes in gene expression, the effects WTPYK2 overexpression on SERCA2 mRNA levels (relative to GAPDH mRNA) were analyzed by real-time RT-PCR, and the results of five experiments are depicted in Fig. 3B. Both PMA and Adv-GFPWTPYK2 significantly decreased SERCA2 mRNA levels to 42 ± 9% and 54 ± 4% of uninfected, control NRVM, respectively. In four additional experiments, we analyzed the time course of SERCA2 downregulation after Adv infection (5 MOI, 8–48 h) with either Adv-GFP or Adv-GFP-WTPYK2. As seen in Fig. 3C, SERCA2 mRNA levels progressively decreased over time coincident with the time course of GFPWTPYK2 overexpression and phosphorylation (Fig. 1B).

Inhibition of PYK2 increases basal SERCA2 mRNA levels. We (8) previously demonstrated that the highly specific Src-family PTK inhibitor PP2 reduced basal, endothelin- and caPKC-{epsilon}-induced PYK2 activation in NRVM. Therefore, we next examined whether inhibition of Src-family PTKs affected SERCA2 mRNA levels in these high-density, spontaneously contracting NRVM cultures. As seen in Fig. 4, PP2 (50 µM; 48 h) significantly increased SERCA2 mRNA levels 2.9-fold compared with untreated NRVM. However, PMA was still able to downregulate SERCA2 mRNA levels to 53 ± 11% of untreated NRVM, which is similar to the level of SERCA2 mRNA downregulation observed with WTPYK2 overexpression in the absence of PP2, or with PMA (Fig. 3).



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Fig. 4. Inhibition of PYK2 phosphorylation increases basal SERCA2 mRNA levels. NRVM were maintained in control medium, or treated with the Src-family protein kinase inhibitor PP2 (50 µM). After 24 h, dishes were then switched to control or PP2-containing medium in the absence (open bars) or presence (solid bars) of phorbol myristate acetate (PMA; 200 nM). Total RNA was then isolated 24 h later, and quantitatively analyzed for SERCA2 and GAPDH mRNA by real-time RT-PCR. Data are the means ± SE from 13 experiments. *P < 0.05 vs. unstimulated, PP2-treated NRVM; +P < 0.05 for corresponding PMA-stimulated (+PMA) vs. unstimulated (–PMA) cells.

 
Effects of mutant forms of PYK2 on SERCA2 mRNA levels. PYK2 contains a central catalytic domain, as well as an NH2 terminal autophosphorylation site (Y402) required for Src-family PTK binding. When activated in response to an increase in [Ca2+]i in PC12 cells, PYK2 undergoes a bimolecular, transautophosphorylation reaction, leading to the phosphorylation of the kinase at Y402, subsequent recruitment and activation of Src, which in turn phosphorylates the kinase domain and enhances PYK2 kinase activity (35). Therefore, we examined whether mutations within the kinase domain or the Y402 site affected SERCA2 gene expression in NRVM. As seen in Fig. 5A, overexpression of kinase-inactive, GFPKDPYK2 increased the amount of PYK2 immunoreactive protein. Interestingly, the overexpressed GFP-KDPYK2 mutant underwent efficient tyrosine phosphorylation at Y402, despite the fact that the catalytic domain was rendered inactive by a point mutation (K457A) within its active site. Adv-GFP-Y402FPYK2 also caused the overexpression of a ~140-kDa PYK2 transgene. However, the transgene was not detected with the phosphospecific PYK2-Y402 antibody, consistent with the fact that this site was indeed mutated. Nevertheless, WTPYK2 and both mutants significantly reduced SERCA2 mRNA levels (WT>KD>Y402F) (Fig. 5, B and C), indicating that neither the Y402 site, nor the kinase domain, were critical in PYK2-dependent SERCA2 downregulation.



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Fig. 5. Effects of mutant forms of PYK2 on SERCA2 mRNA levels. A: NRVM were infected with Adv-GFP, Adv-GFP-WTPYK2, Adv-GFP kinase deficient (KD)-PYK2, or Adv-GFPY402FPYK2 (8–48 h, 10 MOI). Western blots (50 µg of extracted protein) were probed with antibodies that recognized PYK2 phosphorylated at Y402 (pPYK2-Y402; top blot) or total PYK2 (i.e., phosphorylated + unphosphorylated PYK2; bottom blot). The position of molecular weight standards is indicated to the right of each blot. B: total RNA (5 µg) from cells infected with Adv-GFP, Adv-GFP-WTPYK2, Adv-GFPKDPYK2, or Adv-GFPY402FPYK2 (10 MOI, 48 h) was analyzed by Northern blotting with probes specific for SERCA2 and GAPDH mRNAs. C: total RNA from a total of 8 similarly infected NRVM experiments was analyzed by real-time RT-PCR. Data are means ± SE; *P < 0.05 vs. GFP-infected NRVM.

 
PYK2 overexpression activates the JNK1/2 and p38MAPK cascades. PYK2 has been shown to link G protein-coupled receptors to activation of the ERK cascade in multiple cell types (3). Melendez et al. (28) recently demonstrated that PYK2 overexpression activates the stress-activated protein kinases (SAPKs) JNK1/2 and p38MAPK in NRVM, and both ERKs and p38MAPK activation have been implicated in regulating SERCA2 gene expression in cardiomyocytes (1, 21). Therefore, we next examined the effects of PYK2 overexpression on ERKs, JNKs, and p38MAPK in NRVM. Adv-GFPWTPYK2 had no significant effect on ERK1/2 phosphorylation (Fig. 6A), but increased the activation of JNK1/2 (Fig. 6B) and p38MAPK (Fig. 6C), as detected by Western blot analysis with their corresponding phosphospecific antibodies. Steady-state protein levels of all three MAPKs were unaffected compared with NRVM infected with Adv-GFP. The quantitative analysis of 10 Western blotting experiments is also depicted. As is evident from the figure, both SAPKs were substantially activated (4 to 8 fold) in response to WTPYK2 overexpression.



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Fig. 6. Overexpression of GFP-WTPYK2 activates JNKs and p38MAPK, but not ERKs. NRVM were infected with either Adv-GFP or Adv-GFPWTPYK2 (8, 24, or 48 h; 10 MOI). Western blots (WB; 50 µg of extracted protein) were probed with antibodies that recognize either the phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, or total (i.e., the phosphorylated + unphosphorylated) ERK1/2, JNK1/2, and 38PMAPK (A, B, and C, respectively). The position of molecular weight markers is indicated to the right of each blot. The quantitative analysis of 10 Western blotting experiments is summarized to the right of each set of Western blots. Levels of pERK2, pJNK1, and pp38MAPK after Adv-GFP-WTPYK2 infection were normalized to the respective levels after Adv-GFP infection at each time point. Data are means ± SE; *P < 0.05 vs. time-matched control.

 
Because PYK2 overexpression activated JNKs and p38MAPK, we examined whether activation of either or both SAPKs was sufficient to downregulate SERCA2 mRNA levels. NRVM were infected with a control Adv (Adv-ne{beta}gal; 10 MOI, 48 h), and either Adv-caMKK6 or Adv-caMKK7 (10 MOI, 48 h), which are upstream regulators of p38MAPK and JNK1/2, respectively. An Adv-expressing caMEK1, an upstream activator of ERK1/2, was used as a positive control for MAPK-dependent SERCA2 downregulation in NRVM (21). Cells were then examined for MAPK activation with Western blot analysis (Fig. 7A) and for SERCA2 mRNA levels by real-time RT-PCR (Fig. 7B). As shown in the figure, caMEK1 activated ERKs (Fig. 7A) and was sufficient to downregulate SERCA2 mRNA levels to 61 ± 10% of Adv-ne{beta}gal infected NRVM (Fig. 7B). Neither caMKK6 nor caMKK7 activated ERKs in NRVM, but each caMKK activated its appropriate SAPK (Fig. 7A). However, neither SAPK alone was sufficient to downregulate SERCA2 mRNA levels (Fig. 7B). In contrast, infection with both Adv-caMKK6 and Adv-caMKK7 (10 MOI for each Adv, 48 h) concomitantly activated both p38MAPK and JNK1/2 (Fig. 7A), and also significantly downregulated SERCA2 mRNA levels to 45 ± 12% of Adv-ne{beta}gal-infected, control cells (20 MOI, 48 h; Fig. 7C).



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Fig. 7. PYK2-dependent activation of both JNKs and p38MAPK is sufficient to downregulate SERCA2 mRNA levels. A: NRVM were infected with Adv-constitutively active (ca)MEK1, Adv-caMKK6, Adv-caMKK7, or Adv-nuclear encoded (ne){beta}-galactosidase ({beta}gal) (48 h, 10 MOI). NRVM were also infected with the combination of Adv-caMKK6+Adv-caMKK7 (48 h, 10 MOI for each virus) or Adv-ne{beta}gal (48 h, 20 MOI). Western blots (50 µg of extracted protein) were probed with antibodies that recognize either the phosphorylated forms of ERK1/2, JNK, and p38MAPK or the phosphorylated+unphosphorylated forms of ERK1/2, JNK, and p38MAPK. The position of molecular weight markers is indicated to the right of each blot. B and C: SERCA2/GAPDH mRNA ratios were analyzed by real-time RT-PCR. Data are means ± SE of 8–12 experiments; *P < 0.05 vs. ne{beta}gal-expressing control cells.

 
Effects of PYK2 and PMA on SERCA2 promoter activity. Previous studies (1, 21) have indicated that MAPKs downregulate SERCA2 gene expression at the level of gene transcription. To determine whether PYK2 overexpression mediates its effect on SERCA2 mRNA levels by decreasing SERCA2 promoter activity, NRVM were transfected with a 3.8-kb SERCA2 promoter-luciferase reporter plasmid (along with an RSV-LTR {beta}gal reporter construct to control for any differences in transfection efficiency), and were then infected with either Adv-GFP or Adv-GFPWTPYK2 (10 MOI, 24 h). Paired plates were then maintained in control medium, or medium containing PMA (200 nM) for an additional 24 h before analysis of cellular extracts for luciferase and {beta}gal activities. As shown in Fig. 8, NRVM overexpressing WTPYK2 had significantly decreased SERCA2 promoter activity, compared with cells infected with Adv-GFP. PMA reduced SERCA2 promoter activity in Adv-GFP infected cells, but did not further reduce SERCA2 promoter activity in cells overexpressing WTPYK2.



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Fig. 8. Effects of PYK2 and PMA on SERCA2 promoter activity. NRVM were transfected (6 h) with a 3.8-kb SERCA2 promoter-luciferase construct (5 µg) along with a Rous sarcoma virus (RSV)-long terminal repeat (LTR) {beta}gal plasmid (0.5 µg) to control for differences in transfection efficiency. Thereafter, NRVM were infected (10 moi) with either Adv-GFP or Adv-GFPWTPYK2. After 18 h, cells were maintained in control medium (–PMA), or medium supplemented with PMA (+PMA; 200 nM). Cells were harvested 24 h later for analysis of luciferase and {beta}gal activities. Data are means ± SE of normalized SERCA2 promoter activity derived from 6–11 transfection experiments. *P < 0.05 vs. GFP-infected cells maintained in the absence of PMA.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In this report, we have identified a novel, and potentially important signaling pathway that regulates SERCA2 gene expression and [Ca2+]i handling in cardiomyocytes. When overexpressed, WTPYK2, a Ca2+-dependent, nonreceptor PTK, was sufficient to downregulate SERCA2 gene expression in NRVM. Conversely, the PTK inhibitor PP2 was sufficient to significantly increase basal SERCA2 mRNA levels in spontaneously contracting NRVM. PYK2 is known to integrate signals arising from a variety of different stimuli in cardiomyocytes via alterations in [Ca2+]i and/or activation of PKCs (7, 8, 25, 29). Here we demonstrate that PYK2 may also regulate the expression of a critical, membrane Ca2+ pump, thereby affecting [Ca2+]i regulation and adding to the complexity of this signaling web.

In previous studies (6, 12, 36, 37), we demonstrated that mechanical loading, [Ca2+]i, and PKCs are all important regulators of SERCA2 gene expression. Ca2+ influx via L-type Ca2+ channels substantially reduced SERCA2 mRNA and protein levels, and prolonged the time constant for relaxation of the [Ca2+]i transient compared with verapamil-treated NRVM (6). Our present results now provide a potential explanation for these findings, in that Ca2+ channel blockade markedly decreased PYK2 phosphorylation (8) and expression (7), thus eliminating a key component of a signaling pathway that may be important in regulating SERCA2 levels. Similarly, SERCA2 mRNA levels were increased by treating spontaneously contracting NRVM with the PKC inhibitors staurosporine and chelerythrine (37), and by overexpressing dominant-negative inhibitors of individual PKC isoenzymes (36), thereby inhibiting potential activators of a PYK2-dependent signal transduction pathway (8). It is also interesting to note that increasing Ca2+ influx/release activates PYK2 in many cell types, including NRVM (8, 29). Electrical stimulation of contraction also caused increased [Ca2+]i, and caused the membrane translocation of PKC-{epsilon} and PKC-{delta} (47), thus providing additional links to the SERCA2 regulatory pathway.

PYK2 overexpression appeared to mediate SERCA2 downregulation via activation of the JNK and p38MAPK cascades. PYK2 has been identified as a potential upstream regulator of both SAPKs in NRVM (28), as well as in other cell types. For example, Tokiwa et al. (50) showed that WTPYK2 overexpression activated JNKs in PC12 cells, whereas a dominant-negative mutant of PYK2 interfered with UV light- or osmotic shock-induced activation of JNKs. Furthermore, Kodama et al. (24) demonstrated that ET-induced JNK activation in NRVM was mediated primarily via the PKC- and Ca2+-dependent activation of PYK2. PYK2 was found to associate with active Src, and the adapter proteins p130Cas and Crk, which are involved in SAPK activation in a variety of cells. In cardiac fibroblasts, angiotensin II-induced JNK activation was also dependent upon PYK2 via GTP-loading of Rac1 (33). A similar PYK2-dependent pathway was necessary for endothelin-induced activation of p38MAPK in human and rat mesangial cells (45). Finally, we (27) recently demonstrated in cultured chondrocytes that soluble fibronectin fragments activated a {alpha}5{beta}1-integrin-dependent signaling pathway involving PKC-{delta}, PYK2, JNKs, and p38MAPK, leading to stimulation of MMP-13 promoter activity and expression. Thus it seems likely that PYK2 activates the JNK and p38MAPK cascades via similar mechanisms in NRVM.

We found that specific functional domains within PYK2 were important in downregulating SERCA2 mRNA levels. We found that mutations at the putative autophosphorylation site and within the kinase domain decreased (but did not eliminate) the ability of PYK2 to elicit these effects. In fact, the kinase activity of the overexpressed transgene appeared to contribute relatively little to SERCA2 downregulation, as the GFPKDPYK2 mutant underwent efficient phosphorylation at Y402 despite a mutation that rendered the kinase catalytically inactive. Similar results were obtained by Lakkakorpi et al. (26), who found that in osteoclasts, PYK2 kinase activity was dispensable for efficient tyrosine phosphorylation at Y402 as well as at other sites. These data suggest that in some cell types, PYK2 can be phosphorylated at Y402 by another kinase, perhaps a member of the Src-family of PTKs. Of note, we recently demonstrated that basal and endothelin-induced phosphorylation of PYK2 at Y402 was inhibited by PP2, a relatively specific Src-family PTK inhibitor (8). It is also possible that the catalytically inactive, GFPKDPYK2 transgene underwent Y402 phosphorylation via bimolecular autophosphorylation involving the endogenous enzyme, as was recently demonstrated to occur in PC12 cells and Src/Yes/Fyn-deficient fibroblasts (35). In contrast to the kinase-deficient mutant, SERCA2 downregulation were nearly prevented by overexpression of PYK2 bearing the Y402F mutation. Phosphorylation of Y402 provides a docking site for Src-family PTKs to bind to and phosphorylate PYK2 at other sites, including Y579, Y580, and Y881. Lakkakorpi et al. (26) also showed that overexpressed Y402FPYK2 was only weakly phosphorylated at these additional sites, and therefore functioned as a dominant-negative inhibitor of PYK2-dependent cell spreading and bone resorption in osteoclasts. Furthermore, PP2 (which blocks PYK2 phosphorylation at Y402) substantially increased basal SERCA2 mRNA levels in NRVM (Fig. 4). Taken together, these results suggest that the PYK2/Src complex is both necessary and sufficient to generate downstream signals that are important in regulating SERCA2 gene expression in cardiomyocytes.

Our results differ somewhat from a recent study by Andrews et al. (1), which demonstrated that activation of the MKK6-p38MAPK pathway regulated cardiomyocyte [Ca2+]i via downregulation of SERCA2 gene transcription. They showed that overexpression of caMKK6 reduced SERCA2 promoter activity, and lowered steady-state levels of SERCA2 mRNA and protein resulting in prolongation of the [Ca2+]i transient. Although we found that p38MAPK activation alone was not sufficient to downregulate SERCA2 mRNA (Fig. 7), basal levels of JNK1/2 activation in their cells may have been relatively higher than in our experiments, therefore accounting for this difference. Nevertheless, our results indicating that PYK2 can mediate activation of the JNK and p38MAPK cascades support other studies, and confirm recently published work by Melendez et al. (28) indicating that PYK2 activates JNKs and p38MAPK in cardiomyocytes. Taken together, these studies provide a potential upstream link between integrin-, Ca2+-, and PKC-dependent pathways leading to SAPK activation and SERCA2 downregulation in cardiomyocytes.

PYK2 overexpression substantially reduced SERCA2 promoter activity, suggesting that PYK2-dependent signaling pathways reduced SERCA2 gene expression by decreasing the rate of SERCA2 transcription. PMA, a potent activator of both Ca2+-dependent (i.e., PKC-{alpha} and PKC-{beta}) and Ca2+-independent (i.e., PKC-{delta} and PKC-{epsilon}) PKC isoenzymes, also reduced SERCA2 promoter activity. Of note, PMA substantially activates all three MAPK cascades in NRVM, although their degree of activation by physiological stimuli is in part dependent on which PKC isoenzymes are differentially activated (19, 47, 52). However, PP2 did not prevent PMA-induced SERCA2 downregulation, suggesting that there is a PKC-dependent, but PYK2-independent pathway that also regulates SERCA2 promoter activity in NRVM. The most likely explanation for these data is that PMA activates a PKC-{epsilon}/MEK1/ERK signaling cascade independently of PYK2 (19, 36, 46). However, additional studies using physiological agonists and highly specific inhibitors of the individual PKCs and MAPKs will be necessary to fully evaluate these complex signaling pathways.

A potential concern of our studies is that PYK2- and SAPK-mediated SERCA2 downregulation may be part of a more generalized change in mRNA expression in a compromised cardiomyocyte preparation. Indeed, prolonged PYK2 overexpression can trigger apoptosis in some cell culture systems (56), including NRVM (28), and both p38MAPK and JNKs have been implicated in the induction of cardiomyocyte apoptosis (5). We found that WTPYK2 overexpression had no significant effect on cardiomyocyte attachment, protein and DNA content, or the ability of the cells to respond to a hypertrophic stimulus, at least over the time course of these relatively brief cell culture experiments. Quantitative analysis of SERCA2 mRNA was consistently compared with that of another mRNA (GAPDH mRNA), and GAPDH mRNA levels did not systematically change relative to total RNA. Nevertheless, much of the data presented above provide only circumstantial evidence that PYK2 and SAPKs are important regulators of SERCA2 gene expression in NRVM. Although experiments utilizing PP2 suggest that PYK2 and Src-family PTKs are indeed involved, these inhibitors may be nonspecific. Furthermore, the majority of our studies utilize a "gain-of-function" approach to analyze potential, interacting signaling pathways. It is conceivable that overexpression of wild-type or constitutively active signaling kinases induces promiscuous, nonphysiological responses by causing phosphorylation reactions and complexation of signaling proteins that may not normally occur in response to the intricate, redundant signaling pathways operative in the intact heart. Overexpression of kinase-inactive, and phosphorylation-site mutant forms of PYK2, which function as dominant-negative inhibitors of PYK2 signaling in other cell types (4, 27, 50), is also problematic in NRVM because these molecules appear to still generate specific (or nonspecific) signals via their scaffolding function in the cell. Additional studies using highly specific, pharmacological inhibitors of PYK2 autophosphorylation, or "knockdown/knockout" strategies will be needed to fully define the role of this signaling kinase in the altered gene expression that accompanies LVH and HF.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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These studies were supported by National Heart, Lung, and Blood Institute (NHLBI) Grants RO1-HL-34328 and RO1-HL-63711, and a grant to The Cardiovascular Institute from the Ralph and Marian Falk Trust for Medical Research. M. C. Heidkamp was a recipient of NHLBI National Research Service Award HL-68476, and S. J. Engman was a recipient of an American Heart Association Predoctoral Fellowship during the time these studies were performed.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Samarel, Loyola Univ. Medical Center, Bldg. 110, Rm. 5222, 2160 S. First Ave., Maywood, IL 60153 (e-mail: asamare{at}lumc.edu)

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. Andrews C, Ho PD, Dillmann WH, Glembotski CC, and McDonough PM. The MKK6-p38 MAPK pathway prolongs the cardiac contractile calcium transient, downregulates SERCA2, and activates NF-AT. Cardiovasc Res 59: 46–56, 2003.[CrossRef][ISI][Medline]

2. Aoyagi T, Yonekura K, Eto Y, Matsumoto A, Yokoyama I, Sugiura S, Momomura S, Hirata Y, Baker DL, and Periasamy M. The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene promoter activity is decreased in response to severe left ventricular pressure-overload hypertrophy in rat hearts. J Mol Cell Cardiol 31: 919–926, 1999.[CrossRef][ISI][Medline]

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

4. Avraham HK, Lee TH, Koh Y, Kim TA, Jiang S, Sussman M, Samarel AM, and Avraham S. Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase. J Biol Chem 278: 36661–36668, 2003.[Abstract/Free Full Text]

5. Baines CP and Molkentin JD. Stress signaling pathways that modulate cardiac myocyte apoptosis. J Mol Cell Cardiol 38: 47–62, 2005.[CrossRef][ISI][Medline]

6. Bassani JW, Qi M, Samarel AM, and Bers DM. Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells. Circ Res 74: 991–997, 1994.[Abstract]

7. Bayer AL, Ferguson AG, Lucchesi PA, and Samarel AM. Pyk2 expression and phosphorylation in neonatal and adult cardiomyocytes. J Mol Cell Cardiol 33: 1017–1030, 2001.[CrossRef][ISI][Medline]

8. Bayer AL, Heidkamp MC, Howes AL, Heller Brown J, Byron KL, and Samarel AM. Protein kinase C{epsilon}-dependent activation of proline-rich tyrosine kinase 2 in neonatal rat ventricular myocytes. J Mol Cell Cardiol 35: 1121–1133, 2003.[CrossRef][ISI][Medline]

9. Bayer AL, Heidkamp MC, Patel N, Porter MJ, Engman SJ, and Samarel AM. PYK2 expression and phosphorylation increase in pressure overload-induced left ventricular hypertrophy. Am J Physiol Heart Circ Physiol 283: H695–H706, 2002.[Abstract/Free Full Text]

10. Blaukat A, Ivankovic-Dikic I, Gronroos E, Dolfi F, Tokiwa G, Vuori K, and Dikic I. Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades. J Biol Chem 274: 14893–14901, 1999.[Abstract/Free Full Text]

11. Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R, Hewett TE, Jones SP, Lefer DJ, Peng CF, Kitsis RN, and Molkentin JD. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 19: 6341–6350, 2000.[Abstract/Free Full Text]

12. Cadre BM, Qi M, Eble DM, Shannon TR, Bers DM, and Samarel AM. Cyclic stretch down-regulates calcium transporter gene expression in neonatal rat ventricular myocytes. J Mol Cell Cardiol 30: 2247–2259, 1998.[CrossRef][ISI][Medline]

13. Eble DM, Spragia ML, Ferguson AG, and Samarel AM. Sarcomeric myosin heavy chain is degraded by the proteasome. Cell Tissue Res 296: 541–548, 1999.[CrossRef][ISI][Medline]

14. Eble DM, Strait JB, Govindarajan G, Lou J, Byron KL, and Samarel AM. Endothelin-induced cardiac myocyte hypertrophy: role for focal adhesion kinase. Am J Physiol Heart Circ Physiol 278: H1695–H1707, 2000.[Abstract/Free Full Text]

15. Giordano FJ, He H, McDonough P, Meyer M, Sayen MR, and Dillmann WH. Adenovirus-mediated gene transfer reconstitutes depressed sarcoplasmic reticulum Ca2+-ATPase levels and shortens prolonged cardiac myocyte Ca2+ transients. Circulation 96: 400–403, 1997.[Abstract/Free Full Text]

16. Hartong R, Villarreal FJ, Giordano F, Hilal-Dandan R, McDonough PM, and Dillmann WH. Phorbol myristate acetate-induced hypertrophy of neonatal rat cardiac myocytes is associated with decreased sarcoplasmic reticulum Ca2+ ATPase (SERCA2) gene expression and calcium reuptake. J Mol Cell Cardiol 28: 2467–2477, 1996.[CrossRef][ISI][Medline]

17. Hartong R, Wang N, Kurokawa R, Lazar MA, Glass CK, Apriletti JW, and Dillmann WH. Delineation of three different thyroid hormone-response elements in promoter of rat sarcoplasmic reticulum Ca2+ATPase gene. Demonstration that retinoid X receptor binds 5' to thyroid hormone receptor in response element 1. J Biol Chem 269: 13021–13029, 1994.[Abstract/Free Full Text]

18. He H, Giordano FJ, Hilal-Dandan R, Choi DJ, Rockman HA, McDonough PM, Bluhm WF, Meyer M, Sayen MR, Swanson E, and Dillmann WH. Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation. J Clin Invest 100: 380–389, 1997.[Abstract/Free Full Text]

19. Heidkamp MC, Bayer AL, Martin JL, and Samarel AM. Differential activation of mitogen-activated protein kinase cascades and apoptosis by protein kinase C{epsilon} and {delta} in neonatal rat ventricular myocytes. Circ Res 89: 882–890, 2001.[Abstract/Free Full Text]

20. Hirotani S, Higuchi Y, Nishida K, Nakayama H, Yamaguchi O, Hikoso S, Takeda T, Kashiwasi K, Watanabe T, Asahi M, Taniike M, Tsujimoto I, Matsumura Y, Sasaki T, Hori M, and Otsu K. Ca2+-sensitive tyrosine kinase Pyk2/CAK{beta}-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy. J Mol Cell Cardiol 36: 799–807, 2004.[CrossRef][ISI][Medline]

21. Ho PD, Zechner DK, He H, Dillmann WH, Glembotski CC, and McDonough PM. The Raf-MEK-ERK cascade represents a common pathway for alteration of intracellular calcium by Ras and protein kinase C in cardiac myocytes. J Biol Chem 273: 21730–21735, 1998.[Abstract/Free Full Text]

22. Houser SR, Piacentino V 3rd, and Jutta W. Abnormalities of calcium cycling in the hypertrophied and failing heart. J Mol Cell Cardiol 32: 1595–1607, 2000.[CrossRef][ISI][Medline]

23. Ji Y, Lalli MJ, Babu GJ, Xu Y, Kirkpatrick DL, Liu LH, Chiamvimonvat N, Walsh RA, Shull GE, and Periasamy M. Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function. J Biol Chem 275: 38073–38080, 2000.[Abstract/Free Full Text]

24. Kodama H, Fukuda K, Takahashi E, Tahara S, Tomita Y, Ieda M, Kimura K, Owada KM, Vuori K, and Ogawa S. Selective involvement of p130Cas/Crk/Pyk2/c-Src in endothelin-1-induced JNK activation. Hypertension 41: 1372–1379, 2003.[Abstract/Free Full Text]

25. Kodama H, Fukuda K, Takahashi T, Sano M, Kato T, Tahara S, Hakuno D, Sato T, Manabe T, Konishi F, and Ogawa S. Role of EGF receptor and Pyk2 in endothelin-1-induced ERK activation in rat cardiomyocytes. J Mol Cell Cardiol 34: 139–150, 2002.[CrossRef][ISI][Medline]

26. Lakkakorpi PT, Bett AJ, Lipfert L, Rodan GA, and Duong Le T. PYK2 autophosphorylation, but not kinase activity, is necessary for adhesion-induced association with c-Src, osteoclast spreading, and bone resorption. J Biol Chem 278: 11502–11512, 2003.[Abstract/Free Full Text]

27. Loeser RF, Forsyth CB, Samarel AM, and Im HJ. Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway. J Biol Chem 278: 24577–24585, 2003.[Abstract/Free Full Text]

28. Melendez J, Turner C, Avraham H, Steinberg SF, Schaefer E, and Sussman MA. Cardiomyocyte apoptosis triggered by RAFTK/pyk2 via Src kinase is antagonized by paxillin. J Biol Chem 279: 53516–53523, 2004.[Abstract/Free Full Text]

29. Melendez J, Welch S, Schaefer E, Moravec CS, Avraham S, Avraham H, and Sussman MA. Activation of pyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling. J Biol Chem 277: 45203–45210, 2002.[Abstract/Free Full Text]

30. Misquitta CM, Iyer VR, Werstiuk ES, and Grover AK. The role of 3'-untranslated region (3'-UTR) mediated mRNA stability in cardiovascular pathophysiology. Mol Cell Biochem 224: 53–67, 2001.[CrossRef][ISI][Medline]

31. Misquitta CM, Mwanjewe J, Nie L, and Grover AK. Sarcoplasmic reticulum Ca2+ pump mRNA stability in cardiac and smooth muscle: role of the 3'-untranslated region. Am J Physiol Cell Physiol 283: C560–C568, 2002.[Abstract/Free Full Text]

32. Molkentin JD and Dorn IG 2nd. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu Rev Physiol 63: 391–426, 2001.[CrossRef][ISI][Medline]

33. Murasawa S, Matsubara H, Mori Y, Masaki H, Tsutsumi Y, Shibasaki Y, Kitabayashi I, Tanaka Y, Fujiyama S, Koyama Y, Fujiyama A, Iba S, and Iwasaka T. Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase. J Biol Chem 275: 26856–26863, 2000.[Abstract/Free Full Text]

34. Pandey P, Avraham S, Kumar S, Nakazawa A, Place A, Ghanem L, Rana A, Kumar V, Majumder PK, Avraham H, Davis RJ, and Kharbanda S. Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism. J Biol Chem 274: 10140–10144, 1999.[Abstract/Free Full Text]

35. Park SY, Avraham HK, and Avraham S. RAFTK/Pyk2 activation is mediated by trans-acting autophosphorylation in a Src-independent manner. J Biol Chem 279: 33315–33322, 2004.[Abstract/Free Full Text]

36. Porter MJ, Heidkamp MC, Scully BT, Patel N, Martin JL, and Samarel AM. Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes. Am J Physiol Cell Physiol 285: C39–C47, 2003.[Abstract/Free Full Text]

37. Qi M, Bassani JW, Bers DM, and Samarel AM. Phorbol 12-myristate 13-acetate alters SR Ca2+-ATPase gene expression in cultured neonatal rat heart cells. Am J Physiol Heart Circ Physiol 271: H1031–H1039, 1996.[Abstract/Free Full Text]

38. Qi M, Shannon TR, Euler DE, Bers DM, and Samarel AM. Downregulation of sarcoplasmic reticulum Ca2+-ATPase during progression of left ventricular hypertrophy. Am J Physiol Heart Circ Physiol 272: H2416–H2424, 1997.[Abstract/Free Full Text]

39. Ribadeau-Dumas A, Brady M, Boateng SY, Schwartz K, and Boheler KR. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene products are regulated post-transcriptionally during rat cardiac development. Cardiovasc Res 43: 426–436, 1999.[CrossRef][ISI][Medline]

40. Ribadeau Dumas A, Wisnewsky C, Boheler KR, Ter Keurs H, Fiszman MY, and Schwartz K. The sarco(endo)plasmic reticulum Ca2+-ATPase gene is regulated at the transcriptional level during compensated left ventricular hypertrophy in the rat. CR Acad Sci III 320: 963–969, 1997.

41. Sabri A, Govindarajan G, Griffin TM, Byron KL, Samarel AM, and Lucchesi PA. Calcium- and protein kinase C-dependent activation of the tyrosine kinase PYK2 by angiotensin II in vascular smooth muscle. Circ Res 83: 841–851, 1998.[Abstract/Free Full Text]

42. Samarel AM and Engelmann GL. Contractile activity modulates myosin heavy chain-{beta} expression in neonatal rat heart cells. Am J Physiol Heart Circ Physiol 261: H1067–H1077, 1991.[Abstract/Free Full Text]

43. Sasaki H, Nagura K, Ishino M, Tobioka H, Kotani K, and Sasaki T. Cloning and characterization of cell adhesion kinase {beta}, a novel protein-tyrosine kinase of the focal adhesion kinase subfamily. J Biol Chem 270: 21206–21219, 1995.[Abstract/Free Full Text]

44. Schlaepfer DD and Hunter T. Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases. Mol Cell Biol 16: 5623–5633, 1996.[Abstract]

45. Sorokin A, Kozlowski P, Graves L, and Philip A. Protein-tyrosine kinase Pyk2 mediates endothelin-induced p38MAPK activation in glomerular mesangial cells. J Biol Chem 276: 21521–21528, 2001.[Abstract/Free Full Text]

46. Strait JB, 3rd Martin JL, Bayer A, Mestril R, Eble DM, and Samarel AM. Role of protein kinase C-{epsilon} in hypertrophy of cultured neonatal rat ventricular myocytes. Am J Physiol Heart Circ Physiol 280: H756–H766, 2001.[Abstract/Free Full Text]

47. Strait JB and Samarel AM. Isoenzyme-specific protein kinase C and c-Jun N-terminal kinase activation by electrically stimulated contraction of neonatal rat ventricular myocytes. J Mol Cell Cardiol 32: 1553–1566, 2000.[CrossRef][ISI][Medline]

48. Takizawa T, Arai M, Yoguchi A, Tomaru K, Kurabayashi M, and Nagai R. Transcription of the SERCA2 gene is decreased in pressure-overloaded hearts: a study using in vivo direct gene transfer into living myocardium. J Mol Cell Cardiol 31: 2167–2174, 1999.[CrossRef][ISI][Medline]

49. Thuerauf DJ, Hoover H, Meller J, Hernandez J, Su L, Andrews C, Dillmann WH, McDonough PM, and Glembotski CC. Sarco/endoplasmic reticulum calcium ATPase-2 expression is regulated by ATF6 during the endoplasmic reticulum stress response: intracellular signaling of calcium stress in a cardiac myocyte model system. J Biol Chem 276: 48309–48317, 2001.[Abstract/Free Full Text]

50. Tokiwa G, Dikic I, Lev S, and Schlessinger J. Activation of Pyk2 by stress signals and coupling with JNK signaling pathway. Science 273: 792–794, 1996.[Abstract]

51. Van Heugten HA, van Setten MC, Eizema K, Verdouw PD, and Lamers JM. Sarcoplasmic reticulum Ca2+ ATPase promoter activity during endothelin-1 induced hypertrophy of cultured rat cardiomyocytes. Cardiovasc Res 37: 503–514, 1998.[CrossRef][ISI][Medline]

52. Vijayan K, Szotek EL, Martin JL, and Samarel AM. Protein kinase C{alpha}-induced hypertrophy of neonatal rat ventricular myocytes. Am J Physiol Heart Circ Physiol 287: H2777–H2789, 2004.[Abstract/Free Full Text]

53. Vlasblom R, Muller A, Musters RJ, Zuidwijk MJ, Van Hardeveld C, Paulus WJ, and Simonides WS. Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes. Cardiovasc Res 63: 537–544, 2004.[CrossRef][ISI][Medline]

54. Wang Y, Huang S, Sah VP, Ross J Jr, Brown JH, Han J, and Chien KR. Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem 273: 2161–2168, 1998.[Abstract/Free Full Text]

55. Wang Y, Su B, Sah VP, Brown JH, Han J, and Chien KR. Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a specific activator for c-Jun NH2-terminal kinase in ventricular muscle cells. J Biol Chem 273: 5423–5426, 1998.[Abstract/Free Full Text]

56. Xiong W and Parsons JT. Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase. J Cell Biol 139: 529–539, 1997.[Abstract/Free Full Text]

57. Zhu WZ, Wang SQ, Chakir K, Yang D, Zhang T, Brown JH, Devic E, Kobilka BK, Cheng H, and Xiao RP. Linkage of {beta}1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II. J Clin Invest 111: 617–625, 2003.[Abstract/Free Full Text]