Cardiomyocyte apoptosis triggered by RAFTK/pyk2 via Src kinase is antagonized by paxillin.
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 adenoviral-mediated 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 p38beta or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wild-type 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.
The Children's Hospital Research Foundation, Division of Molecular Cardiovascular Biology, Cincinnati, OH 45229, USA.
Jaime
J
Melendez
Christopher
C
Turner
Hava
H
Avraham
Susan F
SF
Steinberg
Erik
E
Schaefer
Mark A
MA
Sussman
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D001665:Binding Sites; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000494:pharmacology*; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D054638:Dual Specificity Phosphatase 1; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2; D005799:Genes, Dominant; D017874:Immediate-Early Proteins / Q000378:metabolism; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology*; D051419:Paxillin; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D016596:Vinculin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; C408142:Pag1 protein, rat; D051419:Paxillin; D010750:Phosphoproteins; C496063:Pxn protein, rat; D016596:Vinculin; D014443:Tyrosine; D004247:DNA; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat; D019061:src-Family Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine