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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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(A) Yeast two-hybrid assay. Positive interaction between the RAG1 N-terminal domain and any member of the library of fusion proteins allows growth under histidine-restrictive conditions (plates 1–3). In contrast, all cells grow on control double drop-out plates (plate 4). (B) Three proteins were isolated, including the previously known Karyopherin α2. (C) The isolated fragment of SF3A2 is shown. It is composed largely of imperfect repeats of a proline-rich heptad PPAPGVH.
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Figure 2: (A) Yeast two-hybrid assay. Positive interaction between the RAG1 N-terminal domain and any member of the library of fusion proteins allows growth under histidine-restrictive conditions (plates 1–3). In contrast, all cells grow on control double drop-out plates (plate 4). (B) Three proteins were isolated, including the previously known Karyopherin α2. (C) The isolated fragment of SF3A2 is shown. It is composed largely of imperfect repeats of a proline-rich heptad PPAPGVH.

Mentions: Eight isolates emerged from the screen, representing three target proteins. Characterization of the initial plasmids is portrayed (in part) in Figure 2A. Each of the candidate target proteins is coexpressed with the empty DNA-BD vector (sectors labeled 177) or with the RAG1-NTD fusion (sectors labeled 179). All grow, as expected, on the control double-drop out plates lacking leucine and tryptophan (panel A, plate 4) but only candidates that are capable of interaction with the NTD survive on plates additionally lacking histidine, in the presence of 7.5 mM 3-AT (panel A, plates 1–3). One selected target is the protein Karyopherin α2 (also known as KPNA2/RCH1/SRP1α/IPOA1/QIP2), previously recognized by others using two-hybrid selection with full-length RAG1 as bait (17–19). This interaction served as a control in the analysis that follows. The remaining two target proteins have not been reported. These are splicing factor 3A2 (SF3A2) and glucocorticoid modulatory element binding protein 1 (GMEB1). The selected plasmids represented (almost) the full-length GMEB1 protein. In contrast, the several independent isolates of SF3A2 each were composed of the identical C-terminal 145 residues of that protein (Figure 2C). It is possible that this particular clone was over-represented in the library. We note that the residues selected are extraordinarily proline rich, being composed largely of imperfect repeats of the heptad PPAPGVH. The full SF3A2 protein is 475 amino acids long, of which the C-terminal 269 is 43% proline and is largely composed of this same proline-rich repeat (see supplement 1 in Supplementary Data). Potential physiologic relevance of these interactions will be discussed later.Figure 2.


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)

(A) Yeast two-hybrid assay. Positive interaction between the RAG1 N-terminal domain and any member of the library of fusion proteins allows growth under histidine-restrictive conditions (plates 1–3). In contrast, all cells grow on control double drop-out plates (plate 4). (B) Three proteins were isolated, including the previously known Karyopherin α2. (C) The isolated fragment of SF3A2 is shown. It is composed largely of imperfect repeats of a proline-rich heptad PPAPGVH.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: (A) Yeast two-hybrid assay. Positive interaction between the RAG1 N-terminal domain and any member of the library of fusion proteins allows growth under histidine-restrictive conditions (plates 1–3). In contrast, all cells grow on control double drop-out plates (plate 4). (B) Three proteins were isolated, including the previously known Karyopherin α2. (C) The isolated fragment of SF3A2 is shown. It is composed largely of imperfect repeats of a proline-rich heptad PPAPGVH.
Mentions: Eight isolates emerged from the screen, representing three target proteins. Characterization of the initial plasmids is portrayed (in part) in Figure 2A. Each of the candidate target proteins is coexpressed with the empty DNA-BD vector (sectors labeled 177) or with the RAG1-NTD fusion (sectors labeled 179). All grow, as expected, on the control double-drop out plates lacking leucine and tryptophan (panel A, plate 4) but only candidates that are capable of interaction with the NTD survive on plates additionally lacking histidine, in the presence of 7.5 mM 3-AT (panel A, plates 1–3). One selected target is the protein Karyopherin α2 (also known as KPNA2/RCH1/SRP1α/IPOA1/QIP2), previously recognized by others using two-hybrid selection with full-length RAG1 as bait (17–19). This interaction served as a control in the analysis that follows. The remaining two target proteins have not been reported. These are splicing factor 3A2 (SF3A2) and glucocorticoid modulatory element binding protein 1 (GMEB1). The selected plasmids represented (almost) the full-length GMEB1 protein. In contrast, the several independent isolates of SF3A2 each were composed of the identical C-terminal 145 residues of that protein (Figure 2C). It is possible that this particular clone was over-represented in the library. We note that the residues selected are extraordinarily proline rich, being composed largely of imperfect repeats of the heptad PPAPGVH. The full SF3A2 protein is 475 amino acids long, of which the C-terminal 269 is 43% proline and is largely composed of this same proline-rich repeat (see supplement 1 in Supplementary Data). Potential physiologic relevance of these interactions will be discussed later.Figure 2.

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