Figure 2.

TRAF6 mediates effects of IL-1β on CRTC2 activity. (a) Diagram of CRTC2 and amino acid sequence alignment showing conserved TRAF6-binding motifs (PXEXXAr/Ac) in CRTC2 from different species. (b) Immunoblot of CRTC2 recovered from immunoprecipitates of TRAF6 prepared from wild type, TRAF6^−/− or CRTC2 ^−/− MEFs. Effect of IL-1β (10 μg l⁻¹) treatment shown. (c) Effect of IL-1β (10 μg l⁻¹) on CRTC2 target gene Nr4a2 expression in wild type and TRAF6^−/− or CRTC2^−/− MEFs co-treated with FSK (10 μm) (*P<0.05). (d) Immunoblot showing amounts of ubiquitinated CRTC2 recovered from IPs of CRTC2 prepared from wild type and TRAF6^−/− MEFs. (e) Effect of TRAF6 wild type or catalytically inactive (C70A) overexpression on CRTC2 ubiquitination in primary hepatocytes exposed to IL-1β (10 μg l⁻¹). (f) Effect of RNAi-mediated depletion of TRAF6 on CRTC2 ubiquitination in primary hepatocytes exposed to IL-1β (10 μg l⁻¹). (g) Immunoblot showing effect of TRAF6 on amounts of ubiquitinated CRTC2 in HEK293T cells co-expressing mutant ubiquitins with mutation of all lysines except K48 or K63. (h) Immunoblots showing effect of mutations in the TRAF6-binding motifs (E2A) in CRTC2 on its association with TRAF6 in HEK293T cells. (i) Immunoblots showing effect of mutations in the TRAF6-binding motifs (E2A) in CRTC2 on its ubiquitination in HEK293T cells. (j, k) Effect of TRAF6 overexpression on wild type and E2A CRTC2 induced G6pase mRNA amounts (j) and glucose output (k) in primary hepatocytes exposed to glucagon (20 nm) (*P<0.05).