ABSTRACT
p66Shc, an adaptor molecule which enhances reactive oxygen species (ROS) production by mitochondria, promotes T-cell apoptosis by inducing mitochondrial dysfunction and impairing Ca(2+) homeostasis. We have addressed the potential role of Lck, a kinase which has been implicated in T-cell apoptosis induced by a number of stimuli, in the proapoptotic activity of p66Shc. Lck deficiency in Jurkat T cells overexpressing p66Shc leads to impaired apoptotic responses to supraphysiological increases in [Ca(2+)](c). This defect could be rescued by reconstitution of Lck expression, indicating that Lck is required for p66Shc-dependent apoptosis. Furthermore, p66Shc phosphorylation on serine 36 (S36), an event on which the proapoptotic function of p66Shc depends, requires Lck. p66Shc-dependent mitochondrial dysfunction, altered Ca(2+) homeostasis and S36 phosphorylation require moreover the activity of CaMKII, a Ca(2+)/calmodulin-dependent kinase known to be implicated in the proapoptotic activity of Lck in T cells. The results suggest that increases in [Ca(2+)](c) lead to CaMKII activation and subsequent Lck-dependent p66Shc phosphorylation on S36. This event causes both mitochondrial dysfunction and impaired Ca(2+) homeostasis, which synergize in promoting Jurkat T-cell apoptosis.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Shc Signaling Adaptor Proteins/genetics , Shc Signaling Adaptor Proteins/metabolism , Signal Transduction , Apoptosis/drug effects , Calcimycin/pharmacology , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Gene Expression Regulation , Humans , Jurkat Cells , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Mitochondria/genetics , Mitochondria/metabolism , Mutation , Oxidative Stress , Phosphorylation , Reactive Oxygen Species/metabolism , Serine , Src Homology 2 Domain-Containing, Transforming Protein 1ABSTRACT
Intrinsic apoptosis defects underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL). Here, we show that the Shc family adapter p66Shc uncouples the B-cell receptor (BCR) from the Erk- and Akt-dependent survival pathways, thereby enhancing B-cell apoptosis. p66Shc expression was found to be profoundly impaired in CLL B cells compared with normal peripheral B cells. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, based on the mutational status of IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes implicated in apoptosis defects of CLL showed an alteration in the balance of proapoptotic and antiapoptotic members of the Bcl-2 family in patients with CLL. Reconstitution experiments in CLL B cells, together with data obtained on B cells from p66Shc(-/-) mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family toward proapoptotic members. The data identify p66Shc as a novel regulator of B-cell apoptosis which attenuates BCR-dependent survival signals and modulates Bcl-2 family expression. They moreover provide evidence that the p66Shc expression defect in CLL B cells may be causal to the imbalance toward the antiapoptotic Bcl-2 family members in these cells.