The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function.

Melanie A Sultana,Thomas Ratajczak,Jiake Xu,Nicole Polain,Carmel Cluning,Wai-Sin Kwong,Sarah L Rea,Nathan J Pavlos,John P Walsh,Avaniyapuram Kannan Murugan

doi:10.1371/journal.pone.0259556

Abstract

The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget’s disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget’s disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget’s, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival.

Highlights

Mutations in SQSTM1 affecting the region coding for the UBA domain of SQSTM1/p62 are reported in 25–50% of patients with familial Paget’s disease of bone, with the majority of mutations reported to date causing either an amino acid substitution within the UBA domain or truncation of the translated protein [1,2]
SQSTM1/p62 is a scaffold protein involved in regulation of the transcription factor nuclear factor kappa B (NF-κB) in response to nerve growth factor, interleukin-1, tumour necrosis factor (TNF) α and receptor activator of NF-κB ligand (RANKL) [6], forming signalling complexes that typically contain TNF-receptor associated factor 6 (TRAF6), SQSTM1/p62 and atypical protein kinase C z
Ajuba is an important component of the atypical protein kinase C z (aPKC), TRAF6, SQSTM1/p62 complex involved in NF-κB signalling [7]

Summary

Introduction

Mutations in SQSTM1 affecting the region coding for the UBA domain of SQSTM1/p62 are reported in 25–50% of patients with familial Paget’s disease of bone, with the majority of mutations reported to date causing either an amino acid substitution within the UBA domain or truncation of the translated protein [1,2]. SQSTM1/p62 is a scaffold protein involved in regulation of the transcription factor nuclear factor kappa B (NF-κB) in response to nerve growth factor, interleukin-1, tumour necrosis factor (TNF) α and receptor activator of NF-κB ligand (RANKL) [6], forming signalling complexes that typically contain TNF-receptor associated factor 6 (TRAF6), SQSTM1/p62 and atypical protein kinase C z (aPKC). The related proteins Ajuba and LIMD1 have been identified as additional components of SQSTM1/p62 protein complexes involved in signalling to NF-κB and activator protein-1, respectively. Both Ajuba and LIMD1 can interact with SQSTM1/p62 directly [7,8]. LIM proteins shuttle into the nucleus where they may influence cell lineage fate determination, the precise nuclear and cytoplasmic roles of these proteins is not clear [10]

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