PYK2 regulates SERCA2 gene expression in neonatal rat ventricular myocytes.
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.
Heidkamp
Maria C
MC
The Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.
Scully
Brian T
BT
Vijayan
Kalpana
K
Engman
Steven J
SJ
Szotek
Erika L
EL
Samarel
Allen M
AM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D015152:Blotting, Northern; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D051418:Focal Adhesion Kinase 2; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006333:Heart Failure / Q000235:genetics; D006352:Heart Ventricles / Q000378:metabolism; D017379:Hypertrophy, Left Ventricular / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C506948:Atp2a2 protein, rat; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases