Sequestosome 1/p62--more than just a scaffold

Abstract

The interaction of proteins with ubiquitin receptors is key to solving the mystery that surrounds the functional role ubiquitin chains play in directing traffic. The specificity of these interactions is largely mediated by UbL/UBA domains. Sequestosome 1/p62 is a protein that is gaining attention as it is intimately involved in cell signaling, receptor internalization, and protein turnover. Herein we review recent advances in the field.

Fig. 1

Functional properties of p62 UbL and UBA domains. a) PB1 domains can be one of three types: Type A, Type B, or Type A/B. p62 interacts with aPKC and can also dimerize with another p62 molecule via back-to-back binding of the basic cluster and the OPCA motif within the PB1 domain. b) The “MGF” binding pocket of p62’s UBA domain accommodates polyubiquitin binding. Location of p62 UBA mutations, F406V and L413V. c) UBA domain mutants were tested for their ability to bind polyubiquitin chains in a GST-UBA pulldown and Western blotting for ubiquitin (Ub), upper panel. p62 turnover was assessed by transiently transfecting HEK cells with myc-tagged p62 UBA domain mutants. Following overnight incubation, cells were treated with cyclohexamide for 24 hours. Lysates were prepared immediately after treatment 0, or after 3 hours the expression of p62 was monitored by Western blotting for myc. d) The UBA domain of p62 bind both K48 and K63 polyubiquitin chains [19]. GST-tagged p62 UBA domain was used in a pulldown assay along with increasing concentrations of either K48 or K63 polyubiquitin chains (kindly provided by Cecile Pickart).

Fig. 2

Models of p62 function. a) p62 can act as a scaffold for the K63 polyubiquitination of TRAF6 specific substrates. In a native form, the UbL domain of p62 may interact with its UBA domain, prior to interaction with ubiquitin. Next, the protein may “open” allowing access to multiple protein binding domains. Following p62 dimerization through the PB1 domain, dimerized TRAF6 binds to p62 at the TRAF6-binding site allowing the transfer of K63 polyubiquitin chains generated from TRAF6 to specific substrates. K63-tagged substrates can interact with the UBA domain of p62. b) p62 may reside at the intersection of two degradation pathways. Once the polyubiquitin chain of a substrate protein binds to the UBA domain of p62, the substrate can be trafficked to the proteasome for degradation. An example of this is the p62-dependent turnover of tau which requires p62 for proteasomal degradation [21]. p62 may also be involved the autophagic/lysosomal pathway by sequestering aggregated proteins prior to their inclusion in autophagosomes.

Fig. 3

Interaction of polyubiquitin with p62 UBA is necessary for substrate ubiquitination. PC12 cells were transfected with HA-TrkA along with Myc-tagged wild-type (WT) p62, Myc-p62 ΔUBA (a construct lacking the UBA domain), Myc-p62 UBA mutant 1: L398V (no effect on polyubiquitin binding), Myc-p62 UBA mutant 2: F406V (inhibits polyubiquitin binding); or Myc-p62 UBA mutant 4: L417V (inhibits polyubiquitin binding). Complete characterization of the mutants and the effects on binding polyubiquitin has been previously described [9]. Thirty-six hours after transfection the cells were treated with NGF (50 ng/ml) for 15 min, lysed and immunoprecipitated with HA antibody [23], followed by Western blotting for either ubiquitin or TrkA as shown.