PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2

J Biol Chem. 2018 Sep 7;293(36):13897-13909. doi: 10.1074/jbc.RA118.002176. Epub 2018 Jul 11.

Abstract

PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.

Keywords: Caenorhabditis elegans (C. elegans); E3 ubiquitin ligase; F-box; FBXO45; FSN-1; MYCBP2; NMNAT; PAM; PHR protein; RPM-1; axon; axon degeneration; fluorescence resonance energy transfer (FRET); neurodegeneration; neuron; proteasome; ubiquitin ligase; ubiquitination.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amidine-Lyases / chemistry*
  • Animals
  • Caenorhabditis elegans
  • F-Box Proteins / metabolism
  • Humans
  • Mixed Function Oxygenases / chemistry*
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / physiology
  • Nicotinamide-Nucleotide Adenylyltransferase / chemistry*
  • Nicotinamide-Nucleotide Adenylyltransferase / metabolism
  • Protein Binding
  • S-Phase Kinase-Associated Proteins
  • SKP Cullin F-Box Protein Ligases / chemistry*
  • SKP Cullin F-Box Protein Ligases / physiology
  • Ubiquitin-Protein Ligases
  • Ubiquitination*

Substances

  • Adaptor Proteins, Signal Transducing
  • F-Box Proteins
  • FBXO45 protein, human
  • Multiprotein Complexes
  • S-Phase Kinase-Associated Proteins
  • SKP1 protein, human
  • Mixed Function Oxygenases
  • PAM protein, human
  • MYCBP2 protein, human
  • SKP Cullin F-Box Protein Ligases
  • Ubiquitin-Protein Ligases
  • NMNAT2 protein, human
  • Nicotinamide-Nucleotide Adenylyltransferase
  • Amidine-Lyases