Phosphorylation of αB-crystallin at serines 19, 45 and 59 plays a pivotal role in preventing MGO-induced ARPE19 apoptosis.

<p>ARPE-19 cells were transfected with constructs containing wild-type or nonphosphorylatable αB-crystallin mutants (S19A, S45A or S59A). (A) A co-immunoprecipitation assay indicating the interactions between αB-crystallin and caspase-3 or -7 as well as between PIDD and RAIDD in mutant cells. While the interactions between αB-crystallin and caspase-3 or -7 were decreased, the interaction between RAIDD and PIDD was increased in mutant-expressing cells (Flag, Flag antibody used to detect the wild type and mutant αB-crystallin). (B) The viability of mutant cells treated with 2 mM MGO for 24 h. All mutant-expressing cells showed a significant reduction in viability at 24 h post-MGO treatment. ** <i>P</i><0.01. (C) A western blot indicating the effects of mutant αB-crystallin expression in response to MGO treatment. MGO upregulated the expression of caspase-2L in mutant-expressing cells. The cleavage products of caspase-2L, -7 and -3, PARP, and PIDD-CC were also increased in the mutant cells (Flag, Flag antibody used to detect the transfected wild-type and mutant αB-crystallin). (D) RT-PCR assays showing the expression of caspase-2L in mutant-expressing cells. Caspase-2L mRNA transcripts were increased in the mutant cells treated with MGO.