PB1 Domain Interaction of p62/Sequestosome 1 and MEKK3 Regulates NF-κB Activation

Kazuhiro Nakamura,Adam J Kimple,David P Siderovski,Gary L Johnson

doi:10.1074/jbc.m109.065102

Kazuhiro Nakamura, Adam J Kimple + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.065102

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Abstract

p62/Sequestosome 1 is a scaffold protein involved in the regulation of autophagy, trafficking of proteins to the proteasome, and activation of NF-kappaB. p62 encodes an N-terminal PB1 domain in addition to the ZZ domain, TRAF6-binding domain, LC3 interaction region, and ubiquitin-associated domain, each critical for the physiological function of p62. PB1 domains have a beta-grasp topology where the front end of one PB1 domain binds the back end of a second PB1 domain. The p62 PB1 domain homodimerizes as well as heterodimerizes with other PB1 domains. The front end of the PB1 domain in p62 binds the PB1 domain of atypical protein kinases C, the MAPK kinase, MEK5, and the NBR1 protein. Other than its role in homodimerization, the rear end acidic cluster region of the p62 PB1 domain had no previous defined binding partners. Herein, we demonstrate that the rear end acidic cluster region of the p62 PB1 domain binds the front end basic region of the MAPK kinase kinase, MEKK3. p62 and MEKK3 co-localize in speckles or aggregates that are centers for organizing TRAF6-regulated NF-kappaB signaling and the assembly of polyubiquinated proteins sorting to sequestosomes and proteasomes. The p62-MEKK3 complex binds TRAF6, which regulates the ubiquitination of the IKK complex and NF-kappaB activation. p62 is required for the association of MEKK3 with TRAF6 and short hairpin RNA knockdown of p62 inhibits IL-1 and MEKK3 activation of NF-kappaB. The rear end acidic cluster of the p62 PB1 domain is used to organize cytosolic aggregates or speckles-associated TRAF6-p62-MEKK3 complex for control of NF-kappaB activation.

Highlights

Plus and minus signs indicate positive and negative binding based on the surface plasmon resonance spectroscopy results of Fig. 2, respectively
The Kd value for specific PB1 domain interactions was calculated according to dose-response measurements with increasing concentrations (5 nM to 5 ␮M) of His-PB1 domains passed over GST-PB1 domain immobilized on the sensor surface coated with anti-GST Ab, as described under “Experimental Procedures” and the legend to Fig. 2
The affinity in p40phox-p67phox binding was calculated based on the equilibrium binding measurements at each different concentration of analyte

Summary

Introduction

The data are shown as an average of relative luciferase activity in triplicate with an error bar of S.D. MEKK3 and p62 PB1 Domains Heterodimerize—In an attempt to define PB1 domain interactions, we have expressed different PB1 domains as either GST fusions or HA-tagged proteins. The analysis shows that a component of expressed wild type MEKK3 and wild type p62 co-localize in aggregates, as shown in the coordinate peaks of intensity in the upper left panel of Fig. 4C.

Results

Conclusion