The nonreceptor protein tyrosine kinase (PTK) PYK2 has been implicated in cell signaling pathways involved in left ventricular hypertrophy (LVH) and heart failure (HF), but its exact role has not been elucidated. In this study, replication-defective adenoviruses (Adv) encoding green fluorescent protein (GFP)-tagged, wildtype (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 blotting and real-time RT-PCR) to 54±4% of Adv-GFP infected cells 48h after Adv infection. Adv encoding kinase-dead (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 p38^MAPK phosphorylation 4- to 8-fold as compared to Adv-GFP. Activation of both "stress-activated" protein kinase (SAPK) cascades appeared necessary to reduce SERCA2 mRNA levels. Adv-mediated overexpression of caMKK6 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 3.8kb SERCA2 promoter-luciferase construct. Thus, a PYK2-dependent signaling cascade may have a role in abnormal cardiac Ca²⁺ handling in LVH and HF via downregulation of SERCA2 gene transcription.