The αNAC Transcriptional Coregulator is a Substrate of p38α MAP Kinase
Dept. of Human Genetics, McGill University and Shriners Hospitals for Children – Canada, Montreal (Quebec) Canada H3G 1A6
The alpha chain of the nascent polypeptide associated complex, abridged to αNAC, plays an important role as a transcriptional coactivator in bones. Its activity is controlled by post-translational modifications that lead to various specific outcomes. Post-translational modifications of αNAC have been shown to be important for proper bone development. We have looked for additional kinases that could phosphorylate αNAC to regulate its subcellular localization and activity.
In silico analysis predicted that P38α could phosphorylate αNAC. The analysis identified the most probable phosphoacceptor sites on αNAC as serine 114 (S114) and threonine 159 (T159). To validate this analysis, a radioactive in vitro kinase assay was performed using purified P38α and recombinant αNAC protein. This confirmed phosphorylation of αNAC by P38α. The next step was to determine the phosphoacceptor site(s). Using site-specific or truncation mutants of the αNAC protein, we narrowed down the phosphorylation region between residues 88 and 129. There are eight probable sites in this region. We generated site-specific mutants for all eight probable sites and tested them in the in vitro kinase assay. The level of phosphorylation of the Threonine 89 (T89) and Serine 114 (S114) was decreased when compared to wild-type αNAC. We thus generated the double mutant T89A/S114A which was not phosphorylated by the kinase. These data show that the phosphoacceptor sites for the P38α activity on the αNAC protein are T89 and S114.
In order to assess the impact of the P38α-induced modification on the subcellular localization of αNAC, MC3T3-E1 cells were transiently transfected with constitutively active MKK6, an upstream activator of P38 kinases. In the MKK6 transfected cells, αNAC translocated to the nucleus. Interestingly, the subcellular localization of the unphosphorylatable αNAC mutant T89A/S114A was cytosolic.
These results show that αNAC is a substrate of the P38α kinase. P38α-phosphorylated αNAC translocates to the nucleus of osteoblasts. Future experiments will address the impact of P38α-dependent post-translational modification on αNAC’s activity and osteoblast differentiation and function.