Interaction of TRAF6 with MAST205 Regulates NF-κB Activation and MAST205 Stability

Abstract

The binding of immune complexes to macrophage Fc␥ receptor results in a subsequent inhibition of lipopolysaccharide-stimulated interleukin-12 synthesis without affecting the induction of tumor necrosis factor-␣. RNA interference targeting MAST205, a 205-kDa serine/ threonine kinase, and transfection of dominant negative MAST205 mutants also mimic this type II macrophage phenotype. Our previous epistasis experiments suggested that the position of MAST205 in the TLR4 signal pathway was proximal to the I B kinase complex. We now report that MAST205 forms a complex with TRAF6, resulting in the inhibition of TRAF6 NF-B activation. We have identified a peptide (residues 218 -233) from the N terminus of MAST205 that, when coupled to a protein transduction domain, inhibits the lipopolysaccharidestimulated activation of NF-B, modulates the size of the MAST205⅐TRAF6 complex, and inhibits ubiquitination of TRAF6. A dominant negative N-terminal MAST205 deletion mutant also inhibits TRAF6 ubiquitination. The domain required for degradation of MAST205 after Fc␥ receptor activation resides within the N-terminal 261 residues, and degradation is triggered by protein kinase C isoform phosphorylation of Ser/Thr residues. These results suggest that MAST205 functions as a scaffolding protein controlling TRAF6 activity and, therefore, plays an important role in regulating inflammatory responses.MAST205 1 is a 205-kDa Ser/Thr kinase that is widely expressed at low levels and highly expressed in spermatids, where MAST205 associates with microtubules. At neuromuscular synapses MAST205 interacts with ␤-2 syntrophin (1, 2). In addition to a well conserved protein kinase C/A kinase domain, MAST205 has a PDZ domain, a protein-protein recognition module (3). The role of MAST205 in cellular physiology, however, has yet to be determined. We have found that MAST205 plays a central role in the regulation of LPS-induced cytokine responses of macrophages (4). In this paper we analyze in greater detail the position of MAST205 in LPS-induced signal pathways and the factors regulating stability of the protein.Macrophages respond to LPS by secreting proinflammatory cytokines such as tumor necrosis factor-␣, IL-1, IL-6, IL-8, and IL-12. IL-12, in particular, is required for the induction of a T helper 1 response that facilitates the clearance of intracellular pathogens. The signal cascade leading to the inflammatory response is complex, reflecting both the importance of controlling the synthesis of potentially harmful cytokines and metabolites, such as reactive oxygen intermediates, and the need to tailor the response depending on the pathogen (5). The inflammatory response is also influenced by previous physiological stimuli to the responding cells, which has lent credence to the concept of macrophage subsets. For example, LPS-stimulated synthesis of IL-12 by macrophages, which is required for the induction of interferon-␥ from NK cells (6), is inhibited by immune complex activation of Fc␥R. As a result, such type II macrophages (7) as antigen present...

Genome-wide expression profiling implicates a MAST3-regulated gene set in colonic mucosal inflammation of ulcerative colitis patients

“…Most members of the MAST family are ubiquitously expressed but their expression is highest in the brain (5). MAST2, the most studied member of this family, is involved in immune reactions, more specifically in the regulation of NF-κB (6). Based on this knowledge, we performed a knockdown of MAST3 in HEK293 cells and tested transcription factor NF-κB activity through reporter assays.…”

Coexpression of MAST205 inhibits the activity of Na+/H+exchanger NHE3

“…The type IIa Na-dependent phosphate transporter in the renal proximal tubule is shown to interact with MAST205 (10). Recent studies suggested a role of MAST205 in interleukin-12 synthesis and NF-B activation via interaction with TRAF6 (32,38). MAST205 also interacts with protocadherin LKC, which induces contact inhibition of cell proliferation (23).…”

MAST205 Competes with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-associated Ligand for Binding to CFTR to Regulate CFTR-mediated Fluid Transport

“…Such a complex can also be a target for therapeutic interventions in diarrheal diseases because down-regulation of MAST205 activates NHE3 and inhibits CFTR channel function. Xiong et al (24) identified a peptide from the N terminus of MAST205 that inhibits the lipopolysaccharide-stimulated activation of NF-B. It is reasonable to speculate that MAST205 peptides or small molecules that inhibit the binding of MAST205 to CFTR have the potential to combat diarrheal diseases.…”

“…MAST205 has a serine/threonine protein kinase domain and a PDZ domain. MAST205 has been shown to interact with several proteins, including ␤2-syntrophin, protocadherin LKC, and the Na ϩ /H ϩ exchanger NHE3 (21)(22)(23)(24)(25). It has been reported that MAST205 forms a complex with TNF receptor-associated factor 6, an E3 ubiquitin ligase, resulting in the inhibition of TNF receptor-associated factor 6 activation.…”