PYK2 Expression and Phosphorylation in Neonatal and Adult Cardiomyocytes

Bayer, Allison L.; Ferguson, Alan; Lucchesi, Pamela A.; Samarel, Allen M.

doi:10.1006/jmcc.2001.1369

Cited by 40 publications

(41 citation statements)

References 42 publications

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“…In contrast, CRNK was predominantly localized to rib-like structures adjacent to the sarcoplasmic membrane of ARVM, consistent with the appearance of costameres ( Figure 1B). The difference in CRNK localization between NRVM and ARVM was reminiscent of the difference in PYK2 localization between neonatal and adult cardiomyocytes observed in our previous studies (5,9).…”

Section: Crnk Overexpression and Localization In Nrvm And Arvmsupporting

confidence: 68%

“…Adult rat ventricular myocytes (ARVM) were isolated according to Westfall et al (22). with modification as described previously (9). Myocytes were then either fixed and permeabilized, or plated in PC-1 medium onto laminincoated (15 µg/ml) chamberslides for 60 min in a 5% CO 2 incubator.…”

Section: Cell Culturementioning

confidence: 99%

“…NRVM and ARVM were fixed and permeabilized as previously described (9,24). Myocytes were stained with anti-FLAG mAb followed by rhodamine-conjugated goat anti-mouse IgG, and counterstained with FITC-phalloidin.…”

Section: Immunolocalizationmentioning

confidence: 99%

“…PYK2 coordinates integrin-, Ca 2+ -, and PKC-dependent signal transduction in a number of tissues (for review, see (7)). PYK2 expression and phosphorylation are regulated by intracellular Ca 2+ and protein kinase C (PKC) in cardiomyocytes and vascular smooth muscle cells (8)(9)(10). As examined in various cell lines, PYK2 serves as an "activatable" scaffolding protein, and transduces signals from G-protein coupled receptors to the mitogen-activated protein kinases (MAPK) and the phosphoinositol-3-kinase (PI-(3)K)-PDK1-Akt signaling pathway depending upon which adaptor proteins bind to the phosphorylated enzyme (6,(11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

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CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

Hart¹,

Heidkamp²,

Iyengar³

et al. 2008

Journal of Molecular and Cellular Cardiology

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PYK2 is a Ca 2+ -dependent, nonreceptor protein tyrosine kinase that is involved in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. We and others have previously investigated PYK2's function in vitro using cultured neonatal and adult rat ventricular myocytes as model systems. However, the function of PYK2 in the in vivo adult heart remains unclear. Here we evaluate the effect of PYK2 inhibition following myocardial infarction (MI) using adenoviral (Adv) overexpression of the C-terminal domain of PYK2, known as CRNK. First we demonstrate that CRNK functions as a dominant-negative inhibitor of PYK2-dependent signaling, presumably by displacing PYK2 from focal adhesions and costameres. Then, male Sprague-Dawley rats (~300g) underwent permanent left anterior descending coronary artery ligation. One wk post-MI, either Adv-GFP (n=34) or Adv-CRNK (n=28) was administered (10 10 pfu, 0.1ml) via catheter-based, Optison®-mediated gene transfer. LV structure and function were evaluated by echocardiography 1 and 3wk after gene transfer, and LV tissue was analyzed by real-time RT-PCR and Western blotting. CRNK overexpression was readily detected by Western blotting 1wk following gene transfer. Adv-CRNK improved overall survival (P=0.03; Logrank Test) and LV fractional shortening (23±2% vs. 31±2% for Adv-GFP vs. Adv-CRNK infected animals, respectively; P<0.05). Whereas MI hearts exhibited increased β-, and decreased α-myosin heavy chain (MHC) mRNA expression characteristic of LVH, Adv-CRNK reversed the MHC isoenzyme switch (3.3±1.4 fold increase in αMHC; 0.4±0.1 fold decrease in βMHC; P<0.05 for both). In summary, CRNK gene transfer improves survival, increases LV function, and alters MHC gene expression suggesting an attenuation of LV remodeling post-MI.Proofs and Correspondences to:

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Section: Crnk Overexpression and Localization In Nrvm And Arvmsupporting

confidence: 68%

Section: Cell Culturementioning

confidence: 99%

Section: Immunolocalizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

Hart¹,

Heidkamp²,

Iyengar³

et al. 2008

Journal of Molecular and Cellular Cardiology

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“…RAFTK/pyk2 participates in attachment to extracellular matrix, cytoskeletal rearrangement, and apoptosis (16,26,27). RAFTK/pyk2 expression is developmentally regulated in the heart and abundant in vascular smooth muscle cells where activation of ERK1/2 mitogen-activated protein kinases and phosphatidylinositol 3-kinase is influenced by RAFTK/pyk2.…”

mentioning

confidence: 99%

Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin

Meléndez

Turner

Avraham

et al. 2004

Journal of Biological Chemistry

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Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr 402 Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviralmediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38␤ or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wildtype or a phosphorylation-deficient paxillin mutated at Tyr 31 and Tyr 118 . Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.Heart failure is characterized by cellular remodeling and apoptosis of cardiomyocytes (1-3). The precipitating stimulus of chronically impaired calcium handling promotes maladaptive remodeling via activation of calcium-dependent signaling cascades (4, 5) with chronic elevation of calcium influx leading to hypertrophy and heart failure in transgenic mice (6). Earlier work from our group demonstrated activation of related adhesion focal tyrosine kinase (RAFTK, also known as pyk2) signaling in cultured cardiomyocytes treated with ionomycin (7) as well as a murine model of dilated cardiomyopathy exhibiting chronic elevation of intracellular calcium levels (8, 9). Concurrent with RAFTK/pyk2 activation, heart samples from cardiomyopathic mice showed enhanced paxillin phosphorylation (7). Thus, paxillin was implicated in the RAFTK/pyk2 signaling cascade leading to heart failure as a target substrate of RAFTK/pyk2-mediated phosphorylation.RAFTK/pyk2 signaling has been extensively characterized in nonmuscle cells where postulated effects include coordinate regulation of cytoskeletal protein phosphorylation in combination...

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Arg/Abl‐binding protein, a Z‐body and Z‐band protein, binds sarcomeric, costameric, and signaling molecules

et al. 2010

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ArgBP2 (Arg/Abl-Binding Protein) is expressed at high levels in the heart and is localized in the Z-bands of mature myofibrils. ArgBP2 is a member of a small family of proteins that also includes vinexin and CAP (c-Cbl-associated protein), all characterized by having one sorbin homology (SOHO) domain and three C-terminal SH3 domains. Antibodies directed against ArgBP2 also react with the Z-bodies of myofibril precursors: premyofibrils and nascent myofibrils. Expression in cardiomyocytes of plasmids encoding Yellow Fluorescent Protein (YFP) fused to either full length ArgBP2, the SOHO, mid-ArgBP or the SH3 domains of ArgBP2 led to Z-band targeting of the fusion proteins, whereas an N-terminal fragment lacking these domains did not target to Zbands. Although ArgBP2 is not found in skeletal muscle cells, YFP-ArgBP2 did target to Zbodies and Z-bands in cultured myotubes. GST-ArgBP2-SH3 bound actin, alpha-actinin and vinculin proteins in blot overlays, cosedimentation assays, and EM negative staining techniques. Over-expression of ArgBP2 and ArgBP2-SH3 domains, but not YFP alone, led to loss of myofibrils in cardiomyocytes. Fluorescence Recovery After Photobleaching was used to measure the rapid dynamics of both the full length and some truncated versions of ArgBP2. Our results indicate that ArgBP2 may play an important role in the assembly and maintenance of myofibrils in cardiomyocytes.

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PYK2 Expression and Phosphorylation in Neonatal and Adult Cardiomyocytes

Cited by 40 publications

References 42 publications

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

Cardiomyocyte Apoptosis Triggered by RAFTK/pyk2 via Src Kinase Is Antagonized by Paxillin

Arg/Abl‐binding protein, a Z‐body and Z‐band protein, binds sarcomeric, costameric, and signaling molecules

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