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Am J Physiol Heart Circ Physiol 285: H1684-H1696, 2003. First published June 26, 2003; doi:10.1152/ajpheart.00016.2003
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Activation of focal adhesion kinase by protein kinase C{epsilon} in neonatal rat ventricular myocytes

Maria C. Heidkamp, Allison L. Bayer, Brian T. Scully, Diane M. Eble, and Allen M. Samarel

The Cardiovascular Institute, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153

Submitted 7 January 2003 ; accepted in final form 19 June 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2–30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 µmol/l; 1 h pretreatment). Adv-caPKC{epsilon}, but not Adv-caPKC{delta}, activated FAK compared with a control Adv encoding {beta}-galactosidase. Conversely, Adv-dnPKC{epsilon} inhibited ET-induced FAK activation. Y-27632 (10 µmol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKC{epsilon}-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 µmol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKC{epsilon}-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 µmol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKC{epsilon}, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.

heart; hypertrophy; signal transduction; actin; ERK; prolinerich tyrosine kinase 2

FOCAL ADHESIONS link the extracellular matrix to the actin cytoskeleton through integrins within the sarcolemma. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that is an important constituent of the focal adhesion complex. FAK undergoes activation and tyrosine autophosphorylation at a specific residue (Y397) after integrin engagement and stimulation by growth factors in neonatal rat ventricular myocyte (NRVM) cultures (13, 30, 41). In addition to performing a structural role in cell adhesion, this complex provides a site for bidirectional signal transduction through the activation of local signaling molecules during cardiomyocyte hypertrophy (12, 14, 20, 23, 30). We (13) have previously shown that FAK is necessary for sarcomeric assembly during endothelin (ET)-induced NRVM hypertrophy. In addition, we demonstrated that disruption of focal adhesions by adenoviral (Adv)-mediated overexpression of the COOH-terminal region of FAK (FRNK), inhibited FAK-dependent signaling and induced adhesion-dependent apoptosis or anoikis (16).

We and others (10, 11, 13, 24, 43) have shown that adhesion- and growth factor-dependent FAK signaling may require upstream activation of one or more isoenzymes of protein kinase C (PKC). However, cardiomyocytes express several PKC isoenzymes, two of which, PKC{epsilon} and PKC{delta}, are activated after ET stimulation (7, 33). Therefore, we have utilized replication-defective Adv that encodes mutant forms of PKC{epsilon} and PKC{delta} to examine which isoenzymes are involved in FAK activation and autophosphorylation at Y397 (Y397pFAK). In addition, we have used pharmacological inhibitors to further interrogate the mechanism(s) by which ET and PKCs activate FAK. Data are presented to indicate that ET stimulates a complex signaling pathway, which ultimately produces actin filament assembly that is critical for FAK activation and sarcomeric assembly during cardiomyocyte hypertrophy.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents. PC-1 tissue culture medium was purchased from BioWhittaker (Walkersville, MD). Medium 199 (M199), Ca2+-free and Mg2+-free Hanks' balanced salt solution, acid-soluble calfskin collagen, and antibiotic/antimycotic solutions were obtained from Sigma (St. Louis, MO). DMEM was obtained from GIBCO-BRL (Grand Island, NY), type II collagenase from Worthington (Lakewood, NJ), and penicillinstreptomycin from Fisher Scientific/MediaTech (Itasca, IL). The pharmacological inhibitors Y-27632, ML-7, PP2, PP3, and cytochalasin D were obtained from Calbiochem (La Jolla, CA). Anti-FAK rabbit pAb, phospho-specific cofilin pAb, and anti-cofilin pAb were obtained from Upstate (Lake Placid, NY). Phosphospecific Y397, Y576, Y577, Y861, and Y925 pFAK rabbit pAbs were purchased from Biosource (Camarillo, CA). Anti-PKC{epsilon} mouse mAb was purchased from Transduction Laboratories (Lexington, KY). Phospho-specific ERK pAb was from Promega (Madison, WI) and anti-ERK1/2 rabbit pAb was obtained from Santa Cruz Biochemical (Santa Cruz, CA). Goat anti-rabbit and goat anti-mouse secondary Abs were obtained from Molecular Probes (Eugene, OR). All other reagents were of the highest grade commercially available and were obtained from Sigma and Baxter S/P (McGaw Park, IL).

Cell culture. Animals used in these experiments were handled in accordance with the "Guiding Principles in the Care and Use of Animals," approved by the Council of the American Physiological Society. Ventricular myocytes were isolated from the hearts of 2-day-old Sprague-Dawley rats by collagenase digestion, as previously described (37). Myocytes were preplated for 1 h in serum-free PC-1 medium to reduce nonmyocyte contamination. The nonadherent NRVM were then plated at a density of 1,600 cells/mm2 onto collagen-coated chamber slides or 35-mm-thick dishes and left undisturbed in a 5% CO2 incubator for 14–18 h. Unattached cells were removed by aspiration and washed twice in Hanks' balanced salt solution, and the attached cells were maintained in a solution of DMEM/M199 (4:1) containing antibiotic/antimycotic solution. Cardiomyocytes were infected (60 min, 25°C with gentle agitation) with replication-defective Adv diluted in DMEM/M199. The medium was then replaced with virus-free DMEM/M199, and the cells were cultured for an additional 8–48 h.

Adv constructs. Replication-defective Adv encoding constitutively active (ca) rat PKC{epsilon} (Adv-caPKC{epsilon}) and caPKC{delta} were constructed as previously described (17, 40). Dominant negative (dn) PKC{epsilon} Adv was kindly provided by Dr. Peipei Ping of the University of Louisville Medical School (Louisville, KY) and constructed as previously described (31). Proline-rich tyrosine kinase 2 (PYK2) cDNA was kindly provided by Dr. Tom Parsons of the University of Virginia, and a replication-defective Adv encoding the green fluorescent protein (GFP)-tagged, wild-type (wt) kinase was constructed using the Adeno-X Expression System from BD Biosciences Clontech (Palo Alto, CA) (16). To control for Adv infection, replication-defective Adv encoding cytoplasmic (cyto) (13) or nuclear-encoded (ne) (17) {beta}-galactosidase ({beta}gal) or GFP (16) were used. The multiplicity of viral infection (MOI) was determined by viral dilution assay in human embryonic kidney-293 cells grown in 96-well clusters.

Immunolocalization. NRVM grown on chamber slides were fixed and permeabilized (16). Myocytes were stained with a polyclonal antibody specific for FAK and a monoclonal antibody that recognizes PKC{epsilon}. Appropriate FITC- or rhodamine-conjugated secondary antibodies were used to visualize the proteins of interest. Fluorescent-labeled cells were then viewed with the use of a laser scanning confocal microscope (model LSM 510, Zeiss).

Western blot analysis. NRVM were washed once in ice-cold PBS and homogenized in lysis buffer containing 1% Triton X-100 and 0.1% SDS (38). Equal amounts of extracted cellular proteins were separated on 7.5% SDS-polyacrylamide gels with 4% stacking gels. Proteins were transferred to polyvinylidene difluoride membranes with the use of the recommended transfer buffer. Western blots were probed with antibodies specific for FAK, the phosphorylated forms of FAK at Y397, Y576, Y577, Y861, and Y925, ERK1/2, cofilin, or the phosphorylated forms of ERK1/2 or cofilin. Primary antibody binding was detected with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody and visualized by enhanced chemiluminescence (Amersham; Arlington Heights, IL). Band intensity was quantified with the use of laser densitometry.

Data analysis. Results were expressed as means ± SE. Normality was assessed with the use of the Kolmogorov-Smirnov test, and homogeneity of variance was assessed with the use of Levene's test. Data were compared by one-way blocked ANOVA, followed by the Student-Newman-Keuls test. Differences among means were considered significant at P < 0.05. Data were analyzed with the use of SigmaStat statistical software (version 1.0; Jandel Scientific; San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
ET increases Y397pFAK through mechanism involving PKCs. Initial experiments were performed to examine the effect of ET on Y397pFAK. As seen in Fig. 1A, FAK was highly phosphorylated at Y397 under basal conditions. ET stimulation (10 nmol/l, 2–10 min) resulted in a further twofold increase in the level Y397pFAK, which peaked at 10 min and remained elevated for up to 30 min. The results of four experiments are summarized in Fig. 1B.



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  		Fig. 1. Endothelin (ET)-induced focal adhesion kinase (FAK) autophosphorylation at Y397 (Y397pFAK) involves protein kinase C (PKC). Neonatal rat ventricular myocytes (NRVM) were maintained in untreated control medium (UT) and treated with ET for 0–30 min (10 nmol/l) (A) or treated with ET (10 nmol/l, 10 min) (C) after being pretreated with the nonselective PKC inhibitor chelerythrine (Chel, 5 µmol/l, 1 h). Western blots (WB; 50 µg of extracted protein) were probed with an antibody specific for the Y397pFAK autophosphorylation site, an antibody that recognizes both the phosphorylated and unphosphorylated forms of FAK, an antibody that recognizes the phosphorylated forms of ERK1/2 (pERK), or an antibody that recognizes both the phosphorylated and unphosphorylated forms of ERK1/2. The numbers at the right of each blot indicate the position of molecular weight markers. B, D, and E: quantitative analysis of 4–5 Western blot experiments. Y397pFAK/FAK and pERK levels are shown normalized to those observed in UT controls. P < 0.05 vs. *UT, {dagger}ET treated, or #Chel treated, respectively.

 

To investigate the PKC dependence of basal and ET-induced Y397pFAK, NRVM were pretreated with the nonselective PKC inhibitor chelerythrine (5 µmol/l, 1 h) and then stimulated with ET (10 nmol/l, 10 min). As seen in Fig. 1C, chelerythrine markedly reduced both basal and ET-stimulated Y397pFAK. ET-induced ERK activation was also partially dependent on PKCs, consistent with a role for PKC{epsilon} in ERK activation (17, 40), although basal levels of ERK2 phosphorylation were modestly elevated after chelerythrine treatment. The results of 5–8 experiments summarizing Y397pFAK and ERK phosphorylation are shown in Fig. 1, D and E, respectively.

Overexpression of caPKC{epsilon}, but not caPKC{delta}, is sufficient to activate FAK. Previous studies have shown that ET stimulation of NRVM induces the membrane translocation of the novel PKC isoenzymes PKC{epsilon} and PKC{delta}, but not the calcium-dependent PKC isoenzyme PKC{alpha} (7, 33). Therefore, we generated replication-defective Adv encoding caPKC{epsilon} and caPKC{delta} and examined their effects on Y397pFAK at multiple time points after Adv infection. Adv-ne{beta}gal was used to control for nonspecific effects of Adv infection. As shown in Fig. 2A, caPKC{epsilon} overexpression increased Y397pFAK as early as 8 h postinfection, which continued to increase over the 48-h time period examined. In addition, total FAK levels (phosphorylated and unphosphorylated) were also significantly increased at each time point. Data summarizing the results of 7–15 experiments are depicted in Fig. 2, C and D.



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  		Fig. 2. Overexpression of constitutively active (ca)PKC{epsilon}, but not caPKC{delta}, is sufficient to activate FAK. NRVM were either maintained in control medium (uninfected; Iu) or infected with an adenovirus expressing nuclear encoded (Adv-ne{beta}gal), Adv-caPKC{epsilon} (A), Adv-caPKC{delta} (B), or Adv-ne{beta}gal alone (E) [25 multiplicity of viral infection (MOI), 8–48 h]. Western blots (50 µg of extracted protein) were probed with either an antibody specific for the Y397pFAK site or an antibody that recognizes both the phosphorylated and unphosphorylated forms of FAK. The position of molecular weight markers is indicated to the right of each blot. C and D: quantitative analysis of either 7–15 (overexpression of caPKC{epsilon} on Y397pFAK and total FAK), 5–6 (overexpression of caPKC{delta} on Y397pFAK), or 4–5 (overexpression of caPKC{delta} on total FAK) Western blot experiments. The levels of Y397pFAK and FAK are shown, normalized to those observed after Adv-ne{beta}gal infection at each time point. Data are means ± SE. *P < 0.05 vs. time-matched control.

 

In marked contrast to the effects of Adv-caPKC{epsilon}, overexpression of caPKC{delta} resulted in a time-dependent decrease in Y397pFAK and total FAK (Fig. 2B). Figure 2C summarizes the results of 5–6 experiments demonstrating that Y397pFAK was decreased to 0.8 ± 0.1- and 0.6 ± 0.1-fold of the time-matched control Adv at 24 and 48 h, respectively. Figure 2D summarizes the results of 4–5 experiments and shows that total FAK levels were decreased by 48 h to 0.6 ± 0.1-fold of time-matched control Adv.

To determine the effect of Adv infection alone on Y397pFAK and total FAK levels over time, multiple experiments were performed comparing uninfected NRVM with NRVM infected with Adv-ne{beta}gal for 8, 24, or 48 h (all cells were harvested at the same time and day). Figure 2E depicts a representative Western blot demonstrating that infection with Adv-ne{beta}gal did not substantially alter either Y397pFAK or total FAK levels over the time period examined compared with uninfected NRVM.

Overexpression of dnPKC{epsilon} reduces basal and ET-induced Y397pFAK. With the use of a replication-defective Adv encoding dnPKC{epsilon}, we next addressed whether PKC{epsilon} was necessary for FAK activation in response to ET stimulation. Adv-cyto{beta}gal was used to control for nonspecific effects of Adv infection. As shown in Fig. 3A, Adv-dnPKC{epsilon} decreased both basal and ET-induced Y397pFAK. The results of four experiments are quantitatively assessed in Fig. 3B and demonstrate that dnPKC{epsilon} overexpression reduced basal levels of Y397pFAK to 0.4 ± 0.2-fold of Adv-cyto{beta}gal-infected cells. ET-induced FAK activation was also significantly decreased from 1.4 ± 0.1-fold in Adv-cyto{beta}gal-infected NRVM to 0.5 ± 0.1-fold after dnPKC{epsilon} overexpression. Total FAK levels also decreased somewhat after dnPKC{epsilon} overexpression; however, this difference did not reach statistical significance (Fig. 3C).



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  		Fig. 3. Overexpression of dominant-negative (dn)PKC{epsilon} reduces basal and ET-induced Y397pFAK. A: NRVM were infected with either Adv-cyto{beta}gal or Adv-dnPKC{epsilon} (750 MOI, 72 h) and then treated with ET (10 nmol/l, 10 min). Western blots (50 µg of extracted protein) were probed with either an antibody specific for the Y397pFAK site or an antibody that recognizes both the phosphorylated and unphosphorylated forms of FAK. The position of molecular weight markers is indicated to the right of the blot. B and C: quantitative analysis of four Western blot experiments. The levels of Y397pFAK and FAK are shown normalized to those observed after Adv-cyto{beta}gal infection. Data are means ± SE. *P < 0.05 vs. Adv-cyto{beta}gal.

 

PKC{epsilon} and FAK colocalize to focal adhesions. Because PKC{epsilon} was both necessary and sufficient to activate FAK, we next examined whether the two kinases were localized to the same region of the cardiomyocyte. We previously showed that FAK and the focal adhesion protein paxillin were colocalized to the cell-substratum interface in control NRVM (16). As shown in Fig. 4A, double-label confocal microscopy revealed that PKC{epsilon} and FAK also colocalized to basilar regions of control cells. Although PKC{epsilon} was present in the same focal adhesion sites as FAK, additional PKC{epsilon} staining was detected in the perinuclear region as well as within cell-cell junctions. PKC{epsilon} and FAK colocalization was also apparent in cells infected with Adv-ne{beta}gal (Fig. 4B). Markedly increased levels of immunoreactive PKC{epsilon} were detected in NRVM infected with Adv-caPKC{epsilon} (Fig. 4C). caPKC{epsilon} overexpression resulted in the formation of filapodia-like projections in many of the cells, consistent with observations from our earlier studies (17, 40). caPKC{epsilon} overexpression also increased FAK staining, thus confirming the results of the Western blot experiments depicted in Fig. 2. Intense regions of FAK and PKC{epsilon} staining were noted in a banded pattern, consistent with the appearance of costameres along the lengths of the elongated cell projections. These projections terminated in FAK- (Fig. 4C) and paxillin-positive (40) focal adhesions. Thus PKC{epsilon} and FAK colocalize to focal adhesions and costameres in control and caPKC{epsilon}-overexpressing NRVM.



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  		Fig. 4. PKC{epsilon} and FAK colocalize to focal adhesions. NRVMs were maintained in control medium (A), infected with Adv-ne{beta}gal (25 MOI, 48 h) (B), or infected with Adv-caPKC{epsilon} (25 MOI, 48 h) (C). Cells were fixed and double labeled with an anti-PKC{epsilon} mAb and an anti-FAK pAb. Rhodamine-conjugated goat-anti-mouse IgG (red; PKC{epsilon}) and FITC-conjugated goat-anti-rabbit IgG (green; FAK) were used for visualization by laser confocal microscopy (1 µm optical sections obtained at the cell-substratum interface). All images were taken with identical laser and microscope settings. Areas of colocalization appear as yellow.

 

Inhibition of Rho kinase abrogates ET- and PKC{epsilon}-induced activation of Y397pFAK and cofilin. Rho belongs to a family of small GTPases that regulate actin stress fiber formation, focal adhesion assembly, and cellular contraction in response to cell adhesion and growth factors in multiple cell types (35, 36). A downstream target of GTP-bound Rho is p160ROK{beta} [Rho-associated coiled-coil-containing protein kinase (ROCK)]. ROCK, in turn, indirectly phosphorylates the actin-binding protein cofilin via activation of LIM kinase, directly phosphorylates and inactivates myosin light chain (MLC) phosphatase, and directly phosphorylates MLC2 leading to nonsarcomeric myosin-dependent cell contraction (1, 21). ET has been shown to promote significant activation of RhoA in neonatal myocytes (8). We therefore investigated ROCK as a potential intermediary in ET- and/or PKC{epsilon}-induced activation of Y397pFAK and cofilin. NRVM were pretreated with the selective ROCK inhibitor Y-27632 (10 µmol/l, 1 h) and then treated with ET (10 nmol/l, 10 m) or infected with Adv-ne{beta}gal or Adv-caPKC{epsilon} in the presence of Y-27632. Figure 5A demonstrates that both basal and ET-induced Y397pFAK were markedly reduced with Y-27632 treatment. In addition, there was a three-fold increase in ET-induced phosphorylation of cofilin. Interestingly, inhibition of ROCK completely abrogated both basal and ET-induced cofilin phosphorylation. The results of 4–8 experiments summarizing Y397pFAK and cofilin phosphorylation are shown in Fig. 5, B and C, respectively. Figure 5E demonstrates that Y-27632 treatment also reduced Y397pFAK and cofilin phosphorylation in NRVMs overexpressing ne{beta}gal and caPKC{epsilon}. In addition, overexpression of caPKC{epsilon}, like ET treatment, increased cofilin phosphorylation threefold. Figure 5, F–H, represents 3–4 experiments summarizing cofilin phosphorylation, Y397pFAK, and FAK, respectively.



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  		Fig. 5. Inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) reduces ET- and PKC{epsilon}-induced Y397pFAK and cofilin phosphorylation. NRVM were maintained in control medium and treated with ET (10 nmol/l, 10 min) (A) after pretreatment with the ROCK inhibitor Y-27632 (10 µmol/l, 1 h) or infected with Adv-ne{beta}gal or caPKC{epsilon} (25 MOI, 48 h) in the presence of Y-27632 (10 µmol/l, 48 h) (E). Western blots (50 µg of extracted protein) were probed with the following antibodies: phosphorylated coflilin (pCofilin), cofilin (both phosphorylated and unphosphorylated forms), Y397pFAK site-specific FAK (both the phosphorylated and unphosphorylated forms), phosphorylated ERK1/2 (pERK1/2), and an antibody that recognizes both the phosphorylated and unphoshorylated forms of ERK1/2. The numbers at the right of each blot indicate the position of molecular weight markers. B–D and F–H: quantitative analysis of 3–4 Western blot experiments. Y397pFAK/FAK, pERK, and pCofilin/Cofilin or Y397pFAK, FAK, and pCofilin/Cofilin levels are shown normalized to those observed without Y-27632, ET, or caPKC{epsilon} treatment. Data are means ± SE. P < 0.05 vs. *untreated, {dagger}ET or caPKC{epsilon} treated, or #Y-27632 treated, respectively.

 

We have previously shown that ET-induced ERK activation is independent of Y397pFAK in NRVM overexpressing FRNK, the autonomously expressed, COOH-terminal domain of FAK (16). Therefore, we examined ERK activation as a measure of normal cardiomyocyte responsiveness after Y-27632 treatment. As seen in Fig. 5A, ET-induced ERK activation remained unaffected by ROCK inhibition, although basal ERK activation was diminished. The results of four experiments summarizing ERK phosphorylation is shown in Fig. 5D. Similarly, in NRVM overexpressing ne{beta}gal and caPKC{epsilon}, inhibition of ROCK did not substantially alter ERK phosphorylation.

To examine the role of myosin phosphorylation in FAK activation, NRVM were pretreated with an inhibitor of MLC kinase, ML-7 (3 µmol/l, 1 h), in the presence or absence of ET (10 nmol/l, 10 min). We found that ML-7 treatment had no effect on either basal or ET-induced Y397pFAK, or ERK phosphorylation. In addition, disruption of myosin/actin cross-bridge formation with BDM (7.5 mmol/l, 1 h) also did not affect FAK phosphorylation. Furthermore, neither inhibition of calcium transients with nifedipine (10 µmol/l, 1 h) nor chelation of intracellular calcium with BAPTA-AM (50 mmol/l, 0.5-h pretreatment, 0.5-h washout) altered the phosphorylation of FAK (data not shown).

Depolymerizing actin filaments reduces ET-induced phosphorylation of cofilin and Y397pFAK. Because FAK activation was coincident with the phosphorylation of cofilin, and cofilin phosphorylation regulates F-actin assembly (27), we induced actin filament disassembly with cytochalasin D (1 µmol/l, 1 h) and examined its effects on both ET-induced phosphorylation of cofilin and Y397pFAK. As shown in Fig. 6, NRVM pretreated with cytochalasin D exhibited a marked reduction in both basal and ET-induced cofilin and FAK phosphorylation, with no significant change in ERK phosphorylation. In addition, the NRVMs were infected with Adv-caPKC{epsilon} in the presence of cytochalasin D (1 µmol/l, 1 h), and the effects on Y397pFAK and pERK were examined. As shown in Fig. 7, cytochalasin D reduced Y397pFAK in ne{beta}gal- and caPKC{epsilon}-overexpressing cells, without altering ERK phosphorylation.



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  		Fig. 6. Depolymerizing actin filaments reduces basal and ET-induced pCofilin and Y397pFAK. NRVM were maintained in control medium and treated with ET (10 nmol/l, 10 min) after pretreatment with the actin depolymerizing agent cytochalasin D (CytoD; 1 µmol/l, 1 h). Western blots (50 µg of extracted protein) were probed with the following antibodies: pCofilin and cofilin (both phosphorylated and unphosphorylated forms), Y397pFAK site-specific FAK (both the phosphorylated and unphosphorylated forms), pERK1/2, and an antibody that recognizes both the phosphorylated and unphosphorylated forms of ERK1/2. Western blots (50 µg of extracted protein) were probed with either an antibody specific for the Y397pFAK site, an antibody that recognizes both the phosphorylated and unphosphorylated forms of FAK, or an antibody that recognizes the phosphorylated forms of ERK1/2. The numbers at the right of each blot indicate the position of molecular weight markers.

 


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  		Fig. 7. Depolymerizing actin filaments reduces basal and PKC{epsilon}-induced Y397pFAK. A: NRVM were infected with Adv-ne{beta}gal or caPKC{epsilon} (25 MOI, 8 h) and then treated with CytoD (1 µmol/l, 1 h). Western blots (50 µg of extracted protein) were probed with an antibody specific for the Y397pFAK autophosphorylation site, an antibody that recognizes both the phosphorylated and unphosphorylated forms of FAK, an antibody that recognizes the phosphorylated forms of ERK1/2, or an antibody that recognizes both the phosphorylated and unphosphorylated forms of ERK1/2. The numbers at the right of each blot indicate the position of molecular weight markers. B and C: quantitative analysis of four Western blot experiments. Y397pFAK and pERK levels are shown, normalized to those observed without CytoD or caPKC{epsilon} treatment. Data are means ± SE. P < 0.05 vs. *Adv-ne{beta}gal alone and {dagger}caPKC{epsilon} alone, respectively.

 

Overexpression of PYK2 does not alter Y397pFAK. Ping and co-workers (32) have demonstrated that PKC{epsilon} forms a signaling module with multiple structural and signaling proteins in cardiomyocytes. One signaling molecule that physically associates with PKC{epsilon} is PYK2, a protein closely related to FAK (32). We have shown that PYK2 is expressed in both neonatal and adult rat ventricular myocytes and is phosphorylated in response to ET (3). We have also demonstrated that inhibition of Y397pFAK by GFP-FRNK overexpression abrogates ET-induced phosphorylation of PYK2 at Y402, its putative autoactivation site (16). Therefore, to address whether the activation of PYK2 affects FAK activation, we infected NRVM with Adv-GFP or Adv-GFP-wtPYK2 (1 MOI, 8–48 h) and examined their effects on FAK levels and Y397pFAK. By 8 h after Adv-GFP-wtPYK2 infection, an abundant amount of the exogenous protein was detected, which continued to increase over the 48-h time period examined. However, overexpression of GFP-wtPYK2 did not alter either Y397pFAK or total FAK levels over the time period examined (data not shown).

Inhibition of Src does not alter Y397pFAK, but decreases Y576pFAK, Y577pFAK, Y861pFAK, and Y925pFAK. PKC{epsilon} also forms a signaling module with Src and other Src-family nonreceptor protein tyrosine kinases in cardiomyocytes (32), and PKC{epsilon} phosphorylates and activates Src (42). In other cell types, FAK activation and autophosphorylation at Y397 provides a docking site for Src, which, in turn, phosphorylates FAK at other sites. We therefore examined whether ET-induced FAK phosphorylation at Y397 and other sites (Y576, Y577, Y861, and Y925) were dependent on the upstream activation of Src. As seen in Fig. 8A, NRVM stimulated with ET exhibited increased levels of Y397pFAK, Y576pFAK, Y577pFAK, Y861pFAK, and Y925pFAK. Treatment with the highly specific, Src-family kinase inhibitor PP2 (50 µmol/l, 1 h) decreased both basal and ET-induced phosphorylation at all sites except for Y397 compared with untreated or PP3-treated NRVM. Figure 8, B–G, summarizes the results of 5–7 experiments. Similar results were obtained in cells that overexpressed caPKC{epsilon} (data not shown).



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  		Fig. 8. Inhibition of Src does not alter Y397pFAK, but decreases Y576pFAK, Y577pFAK, Y861pFAK, and Y925pFAK. NRVM were maintained in control medium and treated with ET (10 nmol/l, 10 min) after being pretreated with the selective inhibitor of Src family protein tyrosine kinases PP2 or the negative control PP3 (50 µmol/l, 1 h) (A). Western blots (50 µg of extracted protein) were probed with either an antibody specific for the Y397pFAK site, antibodies specific for the phosphorylated forms of FAK at Y576, Y577, Y861, or Y925, or an antibody that recognizes both the phosphorylated and unphosphorylated forms of FAK. The position of molecular weight markers is indicated to the right of each blot. B–G: quantitative analysis of 6–7 Western blotting experiments. Y397pFAK, Y567pFAK, Y577pFAK, Y861pFAK, Y925pFAK, and FAK levels are shown, normalized to those observed without drug treatment. Data are means ± SE. P < 0.05 vs. *untreated, {dagger}ET treated, and #PP2 treated, respectively.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Inside-out signaling pathways initiated by Gq-coupled receptors result in FAK-dependent sarcomeric assembly (13). Agonists that activate Gq-coupled receptors increase cell spreading and formation of sarcomeres at the cell periphery, a process that requires the formation of premyofibrils (2). Premyofibrils, in turn, contain actin filaments and Z-bodies, which are precursors to Z-bands and resemble focal adhesions in protein composition (9, 34). Premyofibrils appear at the spreading edge of embryonic chick cardiomyocytes and develop into mature myofibrils when several Z-bodies fuse into a single Z-band. Interestingly, nonmuscle myosin IIB is found between Z-bodies in premyofibrils; however, only mature myofibrils contain muscle-specific myosin II. To a large extent, however, many of the signaling molecules involved in the regulation of premyofibril formation are not known. In the present study, we describe a pathway involving PKC{epsilon}, ROCK, cofilin, and actin filament assembly, which is necessary for ET-induced FAK activation and subsequent sarcomeric assembly.

Signaling through PKCs was required for Y397pFAK because the PKC inhibitor chelerythrine abrogated ET-induced FAK activation. In addition, we found that basal levels of Y397pFAK were similarly reduced by PKC inhibition, suggesting that the maintenance of signaling through focal adhesions requires PKCs. We have previously demonstrated that ET-induced FAK tyrosine phosphorylation was inhibited by chelerythrine in NRVM (13). Similarly, in other cell types, PKC inhibitors reduced FAK phosphorylation, whereas phorbol esters, which activate PKCs, increased FAK phosphorylation (10, 11, 24, 43). Despite this accumulating evidence, isoenzyme-selective PKC regulation of FAK has not yet been demonstrated in any cell type. We now show that overexpression of caPKC{epsilon} increased both Y397pFAK and total FAK, whereas overexpression of dnPKC{epsilon} reduced basal and ET-induced Y397pFAK. In contrast, overexpression of caPKC{delta} decreased both Y397pFAK and total FAK. These results are consistent with other data indicating that PKC{epsilon} and PKC{delta} have opposing effects on cardiomyocyte cell survival and injury (6, 17). Furthermore, caPKC{epsilon} overexpression elevated both Y397pFAK and total FAK levels as early as 8 h postinfection, which coincided with maximal ERK activation (17). However, ET-induced ERK activation appeared to be independent of Y397pFAK (16). Conversely, overexpression of caPKC{delta} did not decrease FAK activation until 24 h postinfection, nor did total FAK levels decrease until 48 h postinfection. We have previously shown that NRVM overexpressing caPKC{delta} undergo apoptosis within 24–48 h after Adv infection (17) as do NRVM overexpressing FRNK (16). It is interesting to speculate that the PKC{delta}-induced decrease in Y397pFAK is the initiating signal resulting in the apoptotic response. However, further experimentation would be necessary to test this hypothesis.

We demonstrated that PKC{epsilon} and FAK colocalize to regions of the cell consistent with the appearance of costameres and focal adhesions. In addition, overexpressing caPKC{epsilon} resulted in increased FAK colocalization as well as increased immunoreactive FAK. Colocalization of PKC{epsilon} and FAK has been shown in vascular smooth muscle cells after binding to fibronectin (15), and we have previously identified PKC{epsilon} within costameres of NRVMs (5). These results are also consistent with studies examining the function and physical interaction of receptor for activated C kinase (RACK1), with PKC isoenzymes (28). RACK1 was shown to directly interact with the cytoplasmic tail of {beta}-integrins in vitro (26). Furthermore, overexpression of RACK1 in NIH3T3 cells increased actin stress fiber and focal adhesion formation, and induced cell spreading. In addition, FAK and paxillin tyrosine phosphorylation increased, thereby linking activated PKCs directly to integrins (18). Although RACK1 preferentially binds Ca2+-dependent PKC isoenzymes, Pass et al. (29) have shown that transgenic mice overexpressing high levels of PKC{epsilon} in ventricular myocytes have enhanced RACK1 protein expression and increased RACK1-PKC{epsilon} interaction. Recently, Besson et al. (4) showed that PMA stimulation of human glioma cells increased focal adhesion formation and coimmunoprecipitation of PKC{epsilon}, RACK1, and {beta}1- and {beta}5-integrins. Therefore our data, which demonstrate that FAK and PKC{epsilon} are colocalized to focal adhesions and costameres even under basal conditions, provides additional evidence in support of a functional and structural link between the two signaling kinases.

Several investigators (2, 19, 22) have shown that Rho and ROCK are necessary and/or sufficient for myofibrillar reorganization and ANF expression after activation of Gq-coupled receptors in NRVM. We now show that inhibition of ROCK with Y-27632 reduced basal, and ET- and PKC{epsilon}-induced activation of Y397pFAK, demonstrating the importance of the Rho/ROCK pathway in FAK-dependent sarcomeric assembly. We then interrogated two pathways downstream of ROCK: namely, the LIM kinase/cofilin pathway, which results in F-actin assembly, and the MLC2/MLC phosphatase pathway, which results in nonsarcomeric myosin-dependent cell contraction (1, 21). We found that phophorylation of cofilin, which is an actin-depolymerizing factor that is inactivated on phosphorylation by LIM kinase (39), was increased threefold after ET treatment. ET-induced regulation of actin assembly through the phosphorylation of cofilin may occur via upstream activation of PKC{epsilon} because cofilin phosphorylation was also increased threefold after caPKC{epsilon} overexpression. We confirmed the importance of assembled actin filaments in FAK activation by depolymerizing actin filaments with cytochalasin D and demonstrated that basal, ET-, and caPKC{epsilon}-induced Y397pFAK were all reduced after this treatment. Surprisingly, we found that cytochalasin D also reduced both basal and ET-induced cofilin phosphorylation, which would indicate that one or more of components of this signaling pathway requires an intact actin cytoskeleton. This requirement may be at the level of PKC{epsilon}, rho, ROCK, or cofilin localization.

In contrast, we found that interference with ROCK-dependent myosin phosphorylation had no effect on either basal or ET-induced Y397pFAK. Similarly, neither inhibiting actin/myosin cross-bridge formation with BDM nor interference with intracellular Ca2+ concentration transients with nifedipine or BAPTA-AM reduced FAK phosphorylation. Although Izumo's group (2) has shown that MLC kinase is indeed involved in ET- and angiotensin II-mediated sarcomeric organization, our results indicate that FAK and MLC kinase reside in parallel signaling pathways. Both pathways appear necessary for sarcomeric assembly during Gq-induced cardiomyocyte hypertrophy.

It should be pointed out that in many of the experiments where inhibitors or Adv-dnPKC{epsilon} was used, ET treatment was still able to activate FAK somewhat. In most of these experiments, however, no statistical difference was found between the groups following quantitative analysis of the data (drug/Adv alone vs. drug/Adv with ET treatment). The only exception to this was the ability of ET to activate FAK slightly after inhibition of ROCK. Nevertheless, it is likely that additional pathways are involved in ET-induced FAK activation that do not require PKC{epsilon}, ROCK, or intact actin filaments.

Finally, we evaluated the potential roles of PKC{epsilon}, PYK2, and Src in ET-induced Y397pFAK. Although PKC{epsilon} was clearly an upstream regulator of FAK activation, neither PYK2 nor Src were necessary for this effect. Ping et al. (32) have previously demonstrated that PKC{epsilon} forms a signaling module with both PYK2 and Src in cardiomyocytes. However, this module does not appear to be functionally important for Y397pFAK. Nevertheless, it is conceivable that PKC{epsilon}-dependent activation of Src is required for Src binding to FAK, and phosphorylating FAK at other sites. PYK2 may also be involved in FAK phosphorylation at other sites because PYK2 phosphorylated FAK at sites other than Y397, Y576, Y577, and Y925 when coexpressed in rat liver epithelial cells (25). However, Src may be an intermediate in this process, as FRNK, a dominant negative inhibitor of FAK, prevented both FAK and PYK2 autophosphorylation in NRVM (16).

In summary, we have described a multicomponent signaling pathway that links Gq-coupled receptor activation to FAK, a critical signaling kinase involved in cell survival and sarcomeric assembly (Fig. 9). This pathway provides an important link between "inside-out" signaling via the ETA receptor and "outside-in" signaling via integrins (12, 14, 20, 23, 30) in cardiomyocytes. Both pathways converge on FAK and are critical for the hypertrophic response.



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  		Fig. 9. Hypothetical scheme for the mechanism of FAK activation by ET. Design of a potential scheme involving a multicomponent signaling pathway that links Gq-coupled receptor (Gq) activation to FAK and subsequent sarcomeric assembly. This signaling cascade that is initiated by ET stimulation involves PKC{epsilon}, ROCK, and cofilin. In addition, one or more of the components of this signaling pathway requires an intact actin cytoskeleton. This requirement may be at the level of PKC{epsilon}, Rho, ROCK, or cofilin localization. ETAR, ET type A receptor; PLC-{beta}, phospholipase C-{beta}; LIMK, LIM kinase.

 


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
These studies were supported by National Heart, Lung, and Blood Institute Grants RO1 HL-34328 and HL-63711 and a gift to the Cardiovascular Institute of Loyola University from the Ralph and Marian Falk Trust for Medical Research. M. C. Heidkamp and A. L. Bayer were recipients of National Heart, Lung, and Blood Institute Research Service Awards HL-68476 and HL-10313, respectively, and D. M. Eble received a James Beck/Patrick Scanlon, M.D., Scientist Development Award at the time the study was performed.


   ACKNOWLEDGMENTS
 
The authors thank Erika Szotek for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Heidkamp, Loyola Univ. Medical Center, 2160 S. First Ave., Maywood, IL 60153 (E-mail: mheidka{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
 DISCLOSURES
 REFERENCES
 

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