Fig. 5.

The Itch HECT domain is inhibited by N-terminal sequences. (A) Self-ubiquitylation activity of different Itch fragments was tested in the presence (lanes 5–8) or absence (lanes 1–4) of E1 and E2 (Ubc7). HECT(C-A) is a catalytically inactive mutant used to demonstrate the dependence of polyubiquitylation on Itch activity. Reaction mixtures were analyzed by immunoblotting with anti-Ub (Upper) or anti-GST (Lower) antibodies. (B) The enzymatic activity of the Itch HECT domain is inhibited by the WW domain. GST-ItchHECT was incubated with increasing quantities of H₆-ItchWW (0, 0.25, 0.5, and 1 μg) together with E1 and E2. Reaction mixtures were analyzed by immunoblotting with anti-Ub (Top), anti-H₆ (Middle), or anti-GST (Bottom) antibodies. (C) Suppression of HECT activity is relieved by JNKK2-JNK1-induced phosphorylation. GST-ItchΔC2 or GST-ItchHECT were incubated in an ubiquitylation assay with (lanes 2 and 4) or without (lanes 1 and 3) JNKK2-JNK1. ItchΔC2 and HECT domain ubiquitylation was analyzed as described above.