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A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified protein-protein interactions.

Maitra R, Sadofsky MJ - Nucleic Acids Res. (2009)

Bottom Line: We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66.Phylogenetic analysis shows the WW-like module to be highly conserved.The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.

ABSTRACT
More than one-third of the RAG1 protein can be truncated from the N-terminus with only subtle effects on the products of V(D)J recombination in vitro or in a mouse. What, then, is the function of the N-terminal domain? We believe it to be regulatory. We determined, several years ago, that an included RING motif could function as an ubiquitin E3 ligase. Whether this activity is limited to automodification, or may alter other proteins in the cell, remains an open question. We revisited the issue of additional protein-protein interactions between RAG1 and other proteins by means of the yeast two-hybrid assay. We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66. A luciferase reporter assay demonstrates that a protein complex containing RAG proteins and the transcription factor can assemble in cells. Further mapping identified a region within the N-terminal domain resembling a WW motif. Point mutation directed at residues conserved in WW motifs eliminated binding to one of the partners. Phylogenetic analysis shows the WW-like module to be highly conserved. The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

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The effect of site specific mutations within the WWL motif using the mammalian two-hybrid assay. (Top) Mouse RAG1 from 173-206, spanning the WWL motif. Residues in larger font are variously mutated. (Bottom) Mammalian two-hybrid assay revealing interaction between the RAG1 peptide and the three other proteins. Data are raw luciferase units. The interaction with SF3A2 is very sensitive to all mutations.
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Figure 7: The effect of site specific mutations within the WWL motif using the mammalian two-hybrid assay. (Top) Mouse RAG1 from 173-206, spanning the WWL motif. Residues in larger font are variously mutated. (Bottom) Mammalian two-hybrid assay revealing interaction between the RAG1 peptide and the three other proteins. Data are raw luciferase units. The interaction with SF3A2 is very sensitive to all mutations.

Mentions: Given the strong conservation, especially across mammals, we chose to test whether mutation could confirm a functional role of these residues in binding interactions. We prepared several mutated versions of the minimal RAG1 construct used in Figure 4, spanning the region 173–250. We created single alanine substitutions at each of the two tryptophan residues (W179A or W203A), the double mutant containing W203A and P205A, and the clustered replacement of three alanines for the three hydrophobic residues (VYF193AAA). The constructs are presented in Figure 7, along with the luciferase assays reflecting the binding to each of the three interacting proteins using the mammalian two-hybrid system. KPNA2 and GMEB1 each show only a modest reduction in binding (∼2-fold), which may be attributed to subtle effects on protein structure or stability within the RAG1 peptide. In striking contrast, binding to the SF3A2 peptide shows a 20-fold reduction in binding to each of the mutant RAG1 proteins. These data are in complete accord with the prediction that a WW-like domain is binding to a proline-containing peptide. They suggest that the WW-like domain plays a necessary role in stabilizing the interaction with SF3A2 but is not as important for KPNA2 and GMEB1. For these latter proteins, the interaction with the basic region appears to be dominant.Figure 7.


A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified protein-protein interactions.

Maitra R, Sadofsky MJ - Nucleic Acids Res. (2009)

The effect of site specific mutations within the WWL motif using the mammalian two-hybrid assay. (Top) Mouse RAG1 from 173-206, spanning the WWL motif. Residues in larger font are variously mutated. (Bottom) Mammalian two-hybrid assay revealing interaction between the RAG1 peptide and the three other proteins. Data are raw luciferase units. The interaction with SF3A2 is very sensitive to all mutations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691831&req=5

Figure 7: The effect of site specific mutations within the WWL motif using the mammalian two-hybrid assay. (Top) Mouse RAG1 from 173-206, spanning the WWL motif. Residues in larger font are variously mutated. (Bottom) Mammalian two-hybrid assay revealing interaction between the RAG1 peptide and the three other proteins. Data are raw luciferase units. The interaction with SF3A2 is very sensitive to all mutations.
Mentions: Given the strong conservation, especially across mammals, we chose to test whether mutation could confirm a functional role of these residues in binding interactions. We prepared several mutated versions of the minimal RAG1 construct used in Figure 4, spanning the region 173–250. We created single alanine substitutions at each of the two tryptophan residues (W179A or W203A), the double mutant containing W203A and P205A, and the clustered replacement of three alanines for the three hydrophobic residues (VYF193AAA). The constructs are presented in Figure 7, along with the luciferase assays reflecting the binding to each of the three interacting proteins using the mammalian two-hybrid system. KPNA2 and GMEB1 each show only a modest reduction in binding (∼2-fold), which may be attributed to subtle effects on protein structure or stability within the RAG1 peptide. In striking contrast, binding to the SF3A2 peptide shows a 20-fold reduction in binding to each of the mutant RAG1 proteins. These data are in complete accord with the prediction that a WW-like domain is binding to a proline-containing peptide. They suggest that the WW-like domain plays a necessary role in stabilizing the interaction with SF3A2 but is not as important for KPNA2 and GMEB1. For these latter proteins, the interaction with the basic region appears to be dominant.Figure 7.

Bottom Line: We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66.Phylogenetic analysis shows the WW-like module to be highly conserved.The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.

ABSTRACT
More than one-third of the RAG1 protein can be truncated from the N-terminus with only subtle effects on the products of V(D)J recombination in vitro or in a mouse. What, then, is the function of the N-terminal domain? We believe it to be regulatory. We determined, several years ago, that an included RING motif could function as an ubiquitin E3 ligase. Whether this activity is limited to automodification, or may alter other proteins in the cell, remains an open question. We revisited the issue of additional protein-protein interactions between RAG1 and other proteins by means of the yeast two-hybrid assay. We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66. A luciferase reporter assay demonstrates that a protein complex containing RAG proteins and the transcription factor can assemble in cells. Further mapping identified a region within the N-terminal domain resembling a WW motif. Point mutation directed at residues conserved in WW motifs eliminated binding to one of the partners. Phylogenetic analysis shows the WW-like module to be highly conserved. The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

Show MeSH