FIG. 2.

PMA-induced degradation of Bcl10 depends on an intact PKC signaling pathway and is mediated by the CARD. (A) PMA but not LPS induces Bcl10 degradation in 70Z/3 cells. Bcl10 and p65 protein amounts were analyzed in extracts from LPS- or PMA-stimulated 70Z/3 cells by Western blotting. (B) PKC signaling is required for Bcl10 degradation. 70Z/3 cells as well as clones of 1.3E2 cells expressing IKKγ or PKCθ, -δ, or -βII alone or in combination, as indicated (panels 1 to 6), were treated with PMA for 10, 30, or 180 min, and NF-κB binding activity as well as Bcl10 and p65 protein amounts were determined. The migration of a nonspecific band in the EMSA is indicated by a star. (C) Schematic representation of Bcl10 and Bcl10 mutants. The amino-terminal CARD including an essential leucine at amino acid position 41 is depicted. (D) The CARD of Bcl10 mediates degradation. Stable pools of Jurkat cells transfected with pEF4 expression vectors containing wild-type XBcl10, XBcl10 L41Q, and XBcl10 1-116 were stimulated with PMA. XBcl10 amounts were analyzed by Western blotting, and the migration of the proteins is indicated by an arrow. As a control for equal loading and stimulation, p65 and IκBα levels were determined in parallel. Nonspecific bands are marked ns.