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Mining the TRAF6/p62 interactome for a selective ubiquitination motif.

Jadhav TS, Wooten MW, Wooten MC - BMC Proc (2011)

Bottom Line: NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination.Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination.The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cellular and Molecular Biosciences, Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL, 36849, USA. wootemc@auburn.edu.

ABSTRACT
A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [-(hydrophobic)-k-(hydrophobic)-x-x-(hydrophobic)- (polar)-(hydrophobic)-(polar)-(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

No MeSH data available.


Model illustrating the interaction between the ligase and scaffold in directing substrate ubiquitination. A: Schematic representation of the means by which the E3 ligase, TRAF6, interacts with the scaffold, p62, and selects a specific Lysine for ubiquitination. B: The sequence of the consensus motif identified in TRAF6/p62 substrates.
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Figure 1: Model illustrating the interaction between the ligase and scaffold in directing substrate ubiquitination. A: Schematic representation of the means by which the E3 ligase, TRAF6, interacts with the scaffold, p62, and selects a specific Lysine for ubiquitination. B: The sequence of the consensus motif identified in TRAF6/p62 substrates.

Mentions: Two TRAF6/p62 substrates, tyrosine receptor kinase A (TrkA) [3] and neurotrophin receptor interacting factor (NRIF) [4] (Figure 1A) were identified by us. Mutagenesis studies verified that both of these proteins are K63- polyubiquitinated at specific Lysine residues, K485 in TrkA and K19 in NRIF. The RING finger domain of TRAF6 ligase is known to be responsible for its catalytic E3 ligase activity [5] and also is responsible for binding of the substrate [6], which then mediates polyubiquitination of target proteins. The modular scaffold protein p62 provides the platform for the transfer reaction to occur [3]. Other studies have demonstrated TRAF6-mediated polyubiquitination including TRAF6 auto-ubiquitination, NEMO [5], TAB2 and TAB3 [7]. These reactions, however, have not been shown to require p62 to mediate the modification. Moreover, like TRAF6, there are many reported E3 Ub ligases whose potential pool of biological targets is unknown.


Mining the TRAF6/p62 interactome for a selective ubiquitination motif.

Jadhav TS, Wooten MW, Wooten MC - BMC Proc (2011)

Model illustrating the interaction between the ligase and scaffold in directing substrate ubiquitination. A: Schematic representation of the means by which the E3 ligase, TRAF6, interacts with the scaffold, p62, and selects a specific Lysine for ubiquitination. B: The sequence of the consensus motif identified in TRAF6/p62 substrates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3090762&req=5

Figure 1: Model illustrating the interaction between the ligase and scaffold in directing substrate ubiquitination. A: Schematic representation of the means by which the E3 ligase, TRAF6, interacts with the scaffold, p62, and selects a specific Lysine for ubiquitination. B: The sequence of the consensus motif identified in TRAF6/p62 substrates.
Mentions: Two TRAF6/p62 substrates, tyrosine receptor kinase A (TrkA) [3] and neurotrophin receptor interacting factor (NRIF) [4] (Figure 1A) were identified by us. Mutagenesis studies verified that both of these proteins are K63- polyubiquitinated at specific Lysine residues, K485 in TrkA and K19 in NRIF. The RING finger domain of TRAF6 ligase is known to be responsible for its catalytic E3 ligase activity [5] and also is responsible for binding of the substrate [6], which then mediates polyubiquitination of target proteins. The modular scaffold protein p62 provides the platform for the transfer reaction to occur [3]. Other studies have demonstrated TRAF6-mediated polyubiquitination including TRAF6 auto-ubiquitination, NEMO [5], TAB2 and TAB3 [7]. These reactions, however, have not been shown to require p62 to mediate the modification. Moreover, like TRAF6, there are many reported E3 Ub ligases whose potential pool of biological targets is unknown.

Bottom Line: NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination.Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination.The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cellular and Molecular Biosciences, Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL, 36849, USA. wootemc@auburn.edu.

ABSTRACT
A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [-(hydrophobic)-k-(hydrophobic)-x-x-(hydrophobic)- (polar)-(hydrophobic)-(polar)-(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

No MeSH data available.