| Summary: | The granulocyte/macrophage colony stimulating factor (GM-CSF) exerts its antiapoptotic effects on hemopoietic cells by activating multiple cellular signaling pathways including the phosphatidylinositol 3'-OH kinase (PI3K) cell survival pathway and the Ras-Raf-MEK-ERK kinase cascade. I hypothesized that cytokines activate each of these pathways to regulate components of the apoptotic machinery, including members of the Bcl-2 family. The mechanism by which cytokines such as GM-CSF regulate expression of Bcl-2 family members was examined in several models, including primary human eosinophils and basophils, and the hemopoietic cell lines TF-1 and MC/9. Protein expression of one pro-survival Bcl-2 family member, Mcl-1, was found to be dependent upon phosphatidylinositol (PI) 3-kinase. The cytokine-induced increase in Mcl-1 mRNA transcription was not dependent upon PI3K, thus dissociating the immediate-early transcription factors responsible for Mcl-1 transcription from the PI3K signaling pathway. In contrast, Mcl-1 mRNA levels were dependent upon MEK activation, suggesting a role for the Ras-MEK-ERK pathway in Mcl-1 transcription. However, activation of PI3K was shown to be necessary for GM-CSF to stimulate Mcl-1 protein translation. This was not due to any effect on prolonging the half-life of the protein. I was also interested in other pathways that may regulate PI3K and Mcl-1 to affect cell survival. The second messenger ceramide has been implicated in a host of cellular processes including growth arrest and apoptosis. The relationship between ceramide signaling and the activation of PI3K and its downstream target, protein kinase B (PKB) was examined in detail. PKB activation was observed following stimulation of cells with GM-CSF. Addition of cell-permeable analogs, C₂- or C₆-ceramide, caused a partial loss (50-60%) of PKB activation and resulted in reduced Mcl-1 translation. These reductions were not a result of a decrease in PI(3,4,5)P₃ or PI(3,4)P₂ generation by PI3K. Two residues of PKB (threonine 308 and serine 473) require phosphorylation for maximal PKB activation. Ser⁴⁷³ phosphorylation was consistently reduced by treatment with ceramide, whereas Thr³⁰⁸ phosphorylation remained unaffected. In further experiments, ceramide appeared to accelerate Ser⁴⁷³ dephosphorylation, suggesting the activation of a phosphatase. Consistent with this, the reduction in Ser⁴⁷³ phosphorylation was inhibited by the phosphatase inhibitors okadaic acid and calyculin A. Surprisingly, Thr³⁰⁸ phosphorylation was abolished in cells treated with these inhibitors, revealing a novel mechanism of regulation of Thr³⁰⁸ phosphorylation. These results demonstrate that PDK2-catalyzed phosphorylation of Ser⁴⁷³ is the principal target of a ceramide-activated phosphatase, which may regulate cell survival by reducing the level of pro-survival proteins such as Mcl-1. === Medicine, Faculty of === Medicine, Department of === Experimental Medicine, Division of === Graduate
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