Activation of the P38 MAPK Pathway Results in Ubiquitin-Proteasome Degradation of the PML Tumor Suppressor Protein.

The promyelocytic leukemia gene (PML) is involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL). PML is a tumor suppressor whose inactivation is causal in leukemogenesis. PML exerts its function by interacting with several tumor suppressor proteins such as p53, pRb, Smad2, Smad3 and CBP/p300. We have recently demonstrated that PML protein is frequently lost in a large portion of hematologic and solid tumors. In these tumors, PML gene sequence and RNA expression were wild type. These findings suggest that PML protein loss is mediated by a post-translational mechanism. We investigated whether PML protein loss is due to degradation via the ubiquitin/proteasome system. Several tumor cell lines have absent or reduced levels of PML protein when compared to untransformed primary cells. We found that in these cases, PML protein half-life is reduced as a result of increased protein degradation. In addition, PML ubiquitinylation is markedly increased in tumor derived cell lines. Incubation of PML negative tumor cell lines with proteasome inhibitors restored wild type PML protein levels. We also defined the minimal PML sequence that is required for ubiquitinylation. This region is in the immediate proximity to p38 mitogen-activated protein kinase consensus sites (p38 MAPK). The p38 MAPK kinase responds to cellular stress, such as osmotic shock, increased free radicals and hypoxia. Notably, p38 MAPK activation has been associated with poor prognosis in breast cancer and follicular lymphoma (Esteva et al. Cancer, 2003. Elenitoba-Johnson et al. Proc Natl Acad Sci U S A. 2003). We therefore, tested whether PML and p38 MAPK functionally interact. Activation of p38 MAPK signaling resulted in dramatic PML degradation due to increased ubiquitinylation. Furthermore, activation of P38 MAPK resulted in increased PML phosphorylation and sumoylation. Treatment with specific inhibitors of p38 MAPK or expression of a p38 dominant negative mutant also inhibited p38 induced PML degradation. In addition, we demonstrated that PML is a direct target of p38 MAPK activity. Bacterially expressed PML is a direct substrate of p38 MAPK in an in vitro kinase assay. The p38 MAPK phosphorylation sites present in the PML protein were mapped. Alanine to serine substitutions of predicted p38 MAPK sites abolished PML phosphorylation by p38 MAPK in vitro and abrogated p38 MAPK dependent PML degradation within the cell. In addition, co-immunoprecipitation experiments clearly demonstrated that PML binds to the active form of p38 MAPK. Confocal immunofluorescence analysis revealed that both activated p38 MAPK colocalize with PML in the nuclear body. These data indicate that the p38 MAPK signaling pathway results in ubiquitin/proteasome mediated PML degradation and imply that MAPK signaling pathways may contribute to oncogenesis by inducing the degradation of the PML tumor suppressor protein. Pharmacological manipulation of this pathway may prove useful for the treatment of tumors that lose PML protein.