Prohibitins and the Cytoplasmic Domain of CD86 Cooperate to Mediate CD86 Signaling in B Lymphocytes

摘要

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that promote NF-κB activation to increase Oct-2 and mature IgG1 mRNA and protein expression, as well as the rate of IgG1 transcription, without affecting class switch recombination. One of the most proximal signaling intermediates identified is phospholipase Cγ2 (PLCγ2), a protein reported to bind tyrosine residues, which are absent in the cytoplasmic domain of CD86. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins, prohibitin-1 (Phb1) and prohibitin-2 (Phb2), bind to CD86. The basal expression of Phb1/2 and association with CD86 was low in resting B cells, while the level of expression and association increased primarily after priming with CD40. The CD86-induced increase in Oct-2 and IgG1 was less when either Phb1/2 expression was reduced by shRNA or the cytoplasmic domain of CD86 was truncated or mutated at serine/threonine PKC-phosphorylation sites, which did not affect Phb1/2 binding to CD86. Using this approach, we also show that Phb1/2 and the CD86 cytoplasmic domain are required for the CD86-induced phosphorylation of IκBα, which we previously reported leads to NF-κB p50/p65 activation; whereas, only Phb1/2 was required for the CD86-induced phosphorylation of PLCγ2 and PKCα/βII, which we have previously reported leads to NF-κB (p65) phosphorylation and subsequent nuclear translocation. Together, these findings suggest that Phb1/2 and the CD86 cytoplasmic domain cooperate to mediate CD86 signaling in a B cell through differential phosphorylation of distal signaling intermediates required to increase IgG1.