Mitogen-activated kinase phosphatase Mkp-1 is an essential negative regulator of innate immune responses. In enterocytes, Mkp-1, which is transiently expressed in response to Toll-like receptor (TLR) ligands, plays a role as an initial step in establishing tolerance to bacteria and their pro-inflammatory molecules. Mkp-1 has a very short half-life (about 30 min) in IEC-6 enterocytes. Here we examined the mechanism of Mkp-1 rapid degradation in IEC-6 cells. Immunoprecipitation and proteolysis inhibitor analysis indicated that Mkp-1 is monoubiquitinated and degraded by proteasome. Dominant negative ubiquitin increased Mkp-1 half-life, indicating involvement of ubiquitination in Mkp-1 degradation. Stability of Mkp-1 is not affected by LPS treatment, consistent with constitutive degradation. U0126, a potent and selective inhibitor of extracellular response kinase (ERK) had negligible effect on the stability of both intrinsic and ectopically expressed Mkp-1, therefore enterocytes, unlike other cell types, do not regulate Mkp-1 degradation via ERK-dependent phosphorylation. C-Truncations of the C-terminal non-catalytic domain (at amino acids 306, 331, and 357), the 306-330 deletion obliterating the potential PEST domains, as well as S296A mutation, but not S323A, S358A, or S363A mutations in potential ERK phosphorylation sites dramatically stabilized Mkp-1. C-terminal fusion of the non-catalytic C-terminal domain of Mkp-1 conferred accelerated degradation on the intrinsically stable green fluorescent protein. According to these results, rapid degradation of Mkp-1 in IEC-6 enterocytes is ubiquitin-proteasome-dependent and ERK-independent. Multiple elements of the C-terminal non-catalytic domain play essential roles in the formation of the complex rapid degradation signal.
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