Differential Translocation or Phosphorylation of Alpha B Crystallin Cannot be Detected in Ischemically Preconditioned Rabbit Cardiomyocytes

Abstract

Alpha B Crystallin (α 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 α 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 α BC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of α 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 decreasedα BC phosphorylation. Neither PD98059 nor IPC altered α BC phosphorylation during prolonged ischemia. It is concluded that α 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 α BC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of α BC quantitated in this study are not protective end effectors of classical IPC.