Alteration of CD40 Protein Expression by Tyrosine Phosphorylation of the NF-κB p50 Subunit.

Protein phosphorylation is critical to selective gene expression; the proteins that regulate transcription are often phosphorylated at multiple sites. Serine and threonine phosphorylation in transcription factors such as NF-κB has been studied, and specific serines are involved in transcriptional activation. Tyrosine phosphorylation of NF-κB p50 subunit can also facilitate the NF-κB-mediated expression of CD40 protein. This study seeks to determine whether tyrosine phosphorylations at positions not normally phosphorylated in vivo could nonetheless affect protein expression mediated by NF-κB. The alterations studied included p50 analogs having pTyr at positions 59 and 61, which do not contain Tyr naturally, and an analog containing a metabolically stable tyrosine methylene phosphonate at position 60. Additionally, to explore the structural basis for enhanced NF-κB binding to CD40 promoter DNA by tyrosine phosphorylation, an analog of NF-κB p50 containing pTyr at both positions 60 and 82 is prepared. The results reflected changes in the ability of the modified NF-κBs containing an altered p50 subunit to bind to CD40 promoter DNA in vitro, and to direct the synthesis of CD40 in cellulo.