Bim is a proapoptotic member of the Bcl-2 family that shares only the BH3 domain with this family. Three Bim proteins Bim-EL, Bim-L and Bim-S are synthesized from the same transcript. We report here that Bim-EL when phosphorylated by Erk1/2 is rapidly degraded via the proteasome pathway. Using different cellular models we evidence that serine 69 is both necessary and sufficient for Erk1/2-mediated phosphorylation and degradation of Bim-EL. In K562 cells, Phorbol 12-myristate 13-acetate activates Erk1/2 and consequently increases Bim-EL phosphorylation and degradation by the proteasome, resulting in cell survival, while the Bcr-Abl inhibitor imatinib abrogates Bim-EL phosphorylation and degradation and induces caspase activation and apoptosis. We also show that Bim-EL(S69G) promotes apoptosis more efficiently than Bim-EL-WT in K562 cells. Altogether, our findings demonstrate that phosphorylation of Bim-EL by Erk1/2 on serine 69 selectively leads to its proteasomal degradation and therefore represents a new and important mechanism of Bim regulation.

Mcl-1 is an antiapoptotic member of the Bcl-2 family that can promote cell viability. We report here that Mcl-1 is a new substrate for caspases during induction of apoptosis.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA þ SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMAinduced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

Imatinib has emerged as the lead compound for clinical development against chronic myeloid leukemia. Imatinib inhibits the kinase activity of Bcr-Abl, which functions by enhancing the proliferation of hematopoietic precursors and protecting them against apoptosis. Imatinib induces apoptosis of Bcr-Abl positive cells, but how the drug effectively kills these cells remains partially understood. We show here that in K562 cells imatinib i) abolished Bcr-Abl phosphorylation and activity and as a consequence Erk1/2, JNK, and AKT activation; ii) induced mitochondrial transmembrane permeability dissipation; iii) activated caspases 3, 9, and 8, demonstrating that the effect of imatinib is integrated at the mitochondrial level; and iv) triggered caspase-dependent cleavage of Bcr-Abl. Interestingly, imatinib-mediated apoptosis was accompanied by erythroid differentiation of K562 cells. Moreover, phorbol esters inhibited imatinib-induced cell death and promoted differentiation toward the megakaryocytic lineage. Finally, we determined by c-DNA array analysis that more than 20 genes were modulated by imatinib. These genes are involved in both cell death and differentiation programs, and some of them have never been reported before to be expressed or involved in erythroid differentiation. Our results demonstrate that imatinib is responsible for a major modification of the genetic program resulting in death and/or differentiation of K562 cells.