Differential translocation or phosphorylation of alpha B crystallin cannot be detected in ischemically preconditioned rabbit cardiomyocytes.
Alpha B Crystallin (alpha BC) is a putative effector protein of ischemic preconditioning (IPC), that is phosphorylated on Ser 45 by ERK1/2 and Ser 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of alpha BC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Ser 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of alpha BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of alpha BC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia, D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased alpha BC phosphorylation. Neither PD98059 nor IPC altered alpha BC phosphorylation during prolonged ischemia. It is concluded that alpha BC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either alpha BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of alpha BC quantitated in this study are not protective end effectors of classical IPC.
Armstrong
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SC
Veterans Affairs Medical Center and Department of Pathology, East Tennessee State University, 37614, USA.
Shivell
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CL
Ganote
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CE
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D019157:Ischemic Preconditioning, Myocardial; D007525:Isoelectric Focusing; D007908:Lens, Crystalline / Q000737:chemistry; D008387:Marine Toxins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation; D020033:Protein Isoforms; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases
D003459:Crystallins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D008387:Marine Toxins; D010080:Oxazoles; D020033:Protein Isoforms; D011725:Pyridines; C059041:calyculin A; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one