Abstract
The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.
Highlights
The transcription factor Bcl11b plays essential roles during T-cell development
Bcl11b Is Sumoylated in Mouse Thymocytes in a Stimulationdependent Manner—Our previous studies implicated Bcl11b in the MAPK pathways [24]
We sought to determine whether stimulation of this pathway in primary thymocytes with P/A resulted in post-translational modification of Bcl11b
Summary
The transcription factor Bcl11b plays essential roles during T-cell development. Results: Bcl11b activity in thymocytes is regulated by MAPK-mediated phosphorylation and subsequent sumoylation and ubiquitination. Data presented suggest that the MAP kinase pathways dynamically regulate the PTM status of Bcl11b; treatment with a phorbol ester and a calcium ionophore resulted in the sequential and rapid phosphorylation, desumoylation, dephosphorylation, resumoylation, ubiquitination, and degradation of Bcl11b. Importantly, these PTMs modified the transcriptional regulatory activity of Bcl11b on a natural transcriptional target, the mouse Id2 promoter. This study provides a better understanding of the mechanistic basis for and the molecular consequences of cell signaling-dependent modification of Bcl11b by PTMs
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