The Ski oncoprotein interacts with Skip, the human homolog of Drosophila Bx42.

The v-Ski avian retroviral oncogene is postulated to actas a transcription factor. Since protein–protein interac-tions have been shown to play an important role in thetranscription process, we attempted to identify Skiprotein partners with the yeast two-hybrid system. Usingv-Ski sequence as bait, the human gene skip (SkiInteracting Protein) was identiﬁed as encoding a proteinwhich interacts with both the cellular and viral forms ofSki in the two-hybrid system. Skip is highly homologousto the Drosophila melanogaster protein Bx42 which isfound associated with chromatin in transcriptionallyactive pu•s of salivary glands. The Ski-Skip interactionis potentially important in Ski’s transforming activitysince Skip was demonstrated to interact with a highlyconserved region of Ski required for transformingactivity. Like Ski, Skip is expressed in multiple tissuetypes and is localized to the cell nucleus.Keywords: Ski oncogeneIntroductionThe v-Ski oncogene was originally identiﬁed as thetransforming protein of the SKV avian retroviruseswhich were isolated by their ability to transform avianﬁbroblasts in vitro (Stavnezer et al., 1981). V-Ski istruncated at the amino and carboxyl termini comparedto the cellular proto-oncogene c-Ski. Ski has beencloned from several species including human, chicken,and Xenopus (Nomura et al., 1989; Sleeman andLaskey, 1993; Sutrave et al., 1990a); and is thefounding member of a family of proteins whichincludes itself and the Ski related protein Sno (Boyeret al., 1993).For an oncogene Ski has the unusual property ofbeing able to a•ect cellular proliferation, and alsoinitiate di•erentiation. Quail embryo ﬁbroblasts infectedwith a retrovirus expressing Ski di•erentiate intomyoblasts capable of forming myotubes in culture(Colmenares and Stavnezer, 1989), and transgenic miceoverexpressing a truncated c-Ski protein displayenlarged skeletal muscle mass compared to their normallitter mates (Sutrave et al., 1990b). Despite evidence fora role in muscle development, c-Ski is only expressed atvery low levels in normal skeletal muscle tissue, similarto its expression levels detected in other adult tissues(Colmenares and Stavnezer, 1990; Nomura et al., 1989).Little is known about how Ski functions. Both thecellular and the viral genes share transforming andmuscle inducing ability. Ski localizes to the nucleus andis observed to associate with chromatin (Barkas et al.,1986; Sutrave et al., 1990a). Because of its nuclearlocalization and its ability to induce expression ofmuscle speciﬁc genes in quail cells (Colmenares andStavnezer, 1989), Ski has been assumed to be atranscription factor. Consistent with this assumption,c-Ski has been shown to bind DNA in vitro with thehelp of an unknown protein factor (Nagase et al.,1990), and has been recently shown to enhance MyoDdependent transactivation of a myosin light chainenhancer linked reporter gene in muscle cells (Engertet al., 1995).Protein-protein interactions are an establishedmechanism of transcriptional regulation. As discussedabove, Ski has been proposed to be involved intranscriptional regulation and presumably achievesthis regulation by binding to DNA following interac-tion with an unknown protein(s). Thus in order tounderstand the mechanism of action of the Ski proteinit will be necessary to identify these putative bindingpartners. In this report the yeast two-hybrid systemwas used to identify the human gene skip encoding aSki interacting protein which is highly homologous tothe Drosophila melanogaster nuclear protein Bx42.ResultsIdentiﬁcation of a Ski binding partnerTo identify putative Ski binding proteins the yeast two-hybrid system was utilized. We chose to use Skisequence derived from v-Ski as bait since all identiﬁedbiological activity is shared between c-Ski and thesmaller v-Ski (Colmenares et al., 1991a; Sutrave et al.,1990a). V-Ski sequence was cloned into the two-hybridGAL4 DNA binding vector pGBT9 and the resultantplasmid was used to screen a previously constructedtwo-hybrid library made from cDNA of EBVtransformed human lymphoid cells (Durfee et al.,1993).The yeast strain Y153 was used for the screeningsince it allows for double selection using two GAL4regulatable reporter genes: lacZ and HIS3. Coloniespositive for an interaction will result in a phenotypethat allows Y153 to grow on His