Prohibitins and the cytoplasmic domain of CD86 are necessary to mediate signaling in B lymphocytes

CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that lead to an increase in Oct-2 and mature IgG1 mRNA and protein expression, without affecting class switch recombination. The most proximal signaling intermediates identified thus far are Lyn and phospholipase Cγ2α/β, which are usually activated after recruitment and binding to phospho-tyrosine residues on receptors, but the cytoplasmic domain of CD86 lacks tyrosines. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins, prohibitin-1 (Phb1) and Phb2, bind to CD86. The basal association is low in resting cells, but increased after priming with CD40L/IL-4. The level of total Phb1/2 expression increased primarily via a CD40-dependent mechanism. Phb1 protein expression and binding to CD86 were consistently greater than that for Phb2 in CD40L/IL-4-primed B cells. The CD86-induced increase in Oct-2 and IgG1 was reduced when either Phb1/2 expression was silenced by shRNA or when the cytoplasmic domain of CD86 was truncated or mutated at the serine/threonine protein kinase C-phosphorylation sites. Truncation of the CD86 cytoplasmic domain did not affect the binding of Phb1/2 to CD86, or the level of T cell activation and class switch recombination. These findings suggested that Phb1/2 and the CD86 cytoplasmic domain are each necessary to mediate the CD86-induced signaling in B cells.