FIG. 6.

CYLD phosphorylation is required for signal-induced TRAF2 ubiquitination and optimal JNK activation. (A) Signal-induced TRAF2 ubiquitination requires CYLD phosphorylation. HeLa-shCYLD cells reconstituted with RNAi-resistant CYLD (WT^R) or M4 (M4^R) were transfected with HA-tagged ubiquitin. The cells were either not treated (NT) or stimulated with TNF-α. Ubiquitin-conjugated TRAF2 were isolated by IP using anti-TRAF2 followed by detection by IB using anti-HA-HRP (upper panel). The level of total ubiquitinated cellular proteins was analyzed by direct IB (lower panel). (B) Phospho-mimetic CYLD loses TRAF2-deubiquitinating activity. 293 cells were transfected with TRAF2 together with empty vector or expression vectors encoding wild-type CYLD (WT), M4, or a phospho-mimetic CYLD harboring serine/glutamic acid substitutions at the phosphorylation sites (M4 S/E). Ubiquitin-conjugated (Ub Conj) TRAF2 was isolated by IP using anti-TRAF2 and detected by IB using anti-HA-HRP (top panel). The expression of CYLD and TRAF2 proteins was monitored by IB using anti-CYLD (middle panel) and anti-TRAF2 (bottom panel). (C) coIP assays to detect the association of CYLD mutants with TRAF2. 293 cells were transfected with HA-tagged TRAF2 together with either an empty vector or expression vectors encoding HA-tagged wild-type (WT) CYLD, CYLD M4, or CYLD M4 S/E. The CYLD complexes were isolated by IP using anti-CYLD followed by detection of the associated HA-TRAF2 by IB using HRP-conjugated anti-HA (upper panel). The protein expression level was monitored by direct IB using HRP-conjugated anti-HA (lower panels). (D) Diminished activation of JNK in cells expressing the phosphorylation-defective CYLD mutant. CYLD-knockdown HeLa (HeLa-shCYLD) or Jurkat (Jurkat-shCYLD) cells were reconstituted with the RNAi-resistant form of wild-type CYLD (WT^R) or the CYLD M4 mutant (M4^R). Following TNF-α stimulation, JNK kinase activity and expression were determined by kinase assays (upper panel) and IB (lower panel), respectively.