PYK2 regulates SERCA2 gene expression in neonatal rat ventricular myocytes

Abstract

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 Y(402)F phosphorylation-deficient mutants of PYK2 also significantly reduced SERCA2 mRNA (WT>KD>Y(402)F). 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 p38(MAPK) 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 p38(MAPK) 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 Ca(2+) handling in left ventricular hypertrophy and heart failure via downregulation of SERCA2 gene transcription.

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

“…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).…”

“…CRNK also did not prevent the increase in protein/DNA ratio elicited by treatment with phorbol myristate acetate (PMA; 200nM, 48h). However, CRNK overexpression increased SERCA2 mRNA levels, in keeping with PYK2's role in regulating SERCA2 gene transcription in NRVM ( Figure 4B) (5). Furthermore, CRNK significantly reduced ANF mRNA levels, suggesting a role for PYK2 in mediating the PKC and/or Ca 2+ dependence of ANF gene transcription in NRVM (33,34).…”

“…In contrast to FAK, PYK2 appears to direct downstream signals to either pro-or anti-apoptotic signaling cascades depending on which molecules bind to the phosphorylated kinase. For instance, Melendez et al (3) previously demonstrated that overexpression of wildtype PYK2 promoted cardiomyocyte apoptosis, perhaps via the preferential activation of JNKs (3,5,6,32) and direct activation of the mitochondrial apoptotic machinery (35). PYK2-dependent apoptosis could be suppressed by prior overexpression of paxillin, which binds to the C-terminal domain of PYK2 (14,36) and prevented JNK activation (3).…”

“…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, ANF, αMHC, and βMHC mRNAs were then analyzed by real-time RT-PCR, as previously described (5,28). The mixture consisted of 1µL of sample cDNA, 21µL DEPC water, 25µL Platinum Quantitative PCR SuperMix-UDG, and 3µL of a primer/dual-labeled probe combination specific for each gene of interest (Table 1).…”

“…The Ca 2+ -dependent, nonreceptor protein tyrosine kinase PYK2 has been previously implicated in cardiomyocyte cell signaling pathways leading to LVH and HF (2)(3)(4)(5)(6). PYK2 is a member of the focal adhesion kinase (FAK) family of nonreceptor PTKs.…”