The cancer-related protein SSX2 interacts with the human homologue of a Ras-like GTPase interactor, RAB3IP, and a novel nuclear protein, SSX2IP.
The SSX gene family is composed of at least five functional and highly homologous members, SSX1 to SSX5, that are normally expressed in only the testis and thyroid. SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. In addition, the SSX1, SSX2, SSX4, and SSX5 genes were found to be aberrantly expressed in several other malignancies, including melanoma. The SSX proteins are localized in the nucleus and are diffusely distributed. In addition, they may be included in polycomb-group nuclear bodies. Other studies have indicated that the SSX proteins may act as transcriptional repressors. As a first step toward the elucidation of the cellular signaling networks in which the SSX proteins may act, we used the yeast two-hybrid system to identify SSX2-interacting proteins. By doing so, two novel human proteins were detected: RAB3IP, the human homolog of an interactor of the Ras-like GTPase Rab3A; and a novel protein, SSX2IP. RAB3IP did not interact with either SSX1, SSX3, or SSX4 in the yeast two-hybrid system, whereas SSX2IP interacted with SSX3 but not with either SSX1 or SSX4. Further analysis of deletion mutants showed that both RAB3IP and SSX2IP interact with the N-terminal moiety of the SSX2 protein. Immunofluorescence analyses of transfected cells revealed that the RAB3IP protein is normally localized in the cytoplasm. However, coexpression of both RAB3IP and SSX2 led to colocalization of both proteins in the nucleus. Likewise, the SSX2IP protein was found to be colocalizing with SSX2 in the nucleus. By performing glutathione-S-transferase pull-down assays, we found that both RAB3IP and SSX2IP interact directly with SSX2 in vitro. These newly observed protein/protein interactions may have important implications for the mechanisms underlying normal and malignant cellular growth.
de Bruijn
Diederik R H
DR
Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, The Netherlands. d.debruin@antrg.azn.nl
dos Santos
Nuno R
NR
Kater-Baats
Ellen
E
Thijssen
José
J
van den Berk
Lieke
L
Stap
Jiska
J
Balemans
Matthé
M
Schepens
Marga
M
Merkx
Gerard
G
van Kessel
Ad Geurts
AG
D000328:Adult; D002352:Carrier Proteins / Q000378:metabolism*; D002874:Chromosome Mapping / Q000379:methods; D002878:Chromosomes, Human, Pair 1 / Q000235:genetics; D002881:Chromosomes, Human, Pair 12 / Q000235:genetics; D005333:Fetus / Q000737:chemistry / Q000378:metabolism; D015723:Gene Library; D020662:Guanine Nucleotide Exchange Factors; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D020826:rab3A GTP-Binding Protein / Q000378:metabolism*
D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010455:Peptides; C092115:Rab3ip protein, rat; D012097:Repressor Proteins; C093962:synovial sarcoma X breakpoint proteins; D020826:rab3A GTP-Binding Protein