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Identification of a Core Functional and Structural Domain of the V-Ski

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Identification of a Core Functional and Structural Domain of the V-Ski Oncogene (1997) 15, 459 ± 471 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Identi®cation of a core functional and structural domain of the v-Ski oncoprotein responsible for both transformation and myogenesis Guoxing Zheng1, Jerey Teumer2, Clemencia Colmenares3, Craig Richmond4,5 and Edward Stavnezer1 1Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106; 2Organogenesis, Canton, Massachusetts; 3Department of Cancer Biology, Cleveland Clinic Foundation Research Institute, Cleveland, Ohio 44195; 4Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, Ohio, USA The v-ski oncogene promotes cellular transformation and growth. In QEFs but not CEFs, v-ski also induces myogenic dierentiation. In quail embryo ®broblasts the myogenic dierentiation among cells that it has already two properties are displayed simultaneously and terminal transformed (Colmenares and Stavnezer, 1989). This muscle dierentiation occurs only among cells already phenotype involves the expression of myogenic transformed by v-ski. To understand how the two regulatory genes (myoD and myogenin), and the phenotypes are derived from a single gene, we have subsequent expression of terminal dierentiation undertaken to identify functionally important regions in markers and formation of myotubes when the cells v-ski and to test whether these regions can promote one are shifted into low-growth dierentiation medium phenotype without the other. We have generated both (Colmenares and Stavnezer, 1989). The dual action of random and targeted mutations in v-ski and evaluated v-Ski could be due either to its possession of two the eects of these mutations on expression, intracellular separate functional domains (one responsible for each location, transformation, and myogenesis. Among a total of the observed phenotypes), or alternatively to a single of 26 mutants analysed, we have not found complete domain which functions dierently depending on both separation of the myogenic and transforming properties. cell type and external stimuli. Mutations in the region of v-Ski encoded by exon 1 of c- v-ski contains the ®rst ®ve of eight coding exons of ski frequently abolish both its transformation and muscle the proto-oncogene, c-ski (Stavnezer et al., 1989; dierentiation activities, whereas mutations outside of Sutrave and Hughes, 1989; Grimes et al., 1992). this region are always tolerated. When expressed in cells However, oncogenic and myogenic activation of c-ski from a minigene containing only the exon 1 sequence, the has been shown to result from viral over-expression protein displays the transforming and myogenic activities (Colmenares et al., 1991) and not from the truncation similar to v-Ski. These results argue that the amino acid of its coding sequences that occurred upon transduc- sequence encoded by exon 1 contains the core functional tion of v-ski. Both v-ski and c-ski encode nuclear domain of the oncoprotein. To determine whether this proteins (Barkas et al., 1986; Sutrave et al., 1990) and functional domain has a structural counterpart, we have appear to be transcriptional regulators (Colmenares fragmented the v-Ski protein by limited proteolysis and and Stavnezer, 1990; Engert et al., 1995). Primary found a single proteolytically stable domain spanning the sequence analysis indicates that the amino terminal entire exon 1-encoded region. Physical studies of the two-thirds of v-Ski contains several potentially polypeptide encoded by exon 1 con®rms that it folds into important motifs typical of transcription factors a compact, globular protein. The ®nding that both the (Stavnezer et al., 1989). These include a proline-rich transforming and myogenic properties of v-Ski are region, a putative nuclear localization signal, Cys-His- inseparable by mutation and are contained in a single rich metal-binding motifs, and paired amphipathic domain suggests that they are derived from the same alpha-helices (Colmenares and Stavnezer, 1990). This function. region of the protein is derived from the ®rst coding exon of c-ski and is the most highly conserved region Keywords: oncogene; v-Ski; mutagenesis; proteolysis between Ski and the Ski-related gene product, SnoN (Nomura et al., 1989; Boyer et al., 1993). Consistent with the idea that conservation suggests functional importance, chicken SnoN has been shown to have Introduction Ski-like transforming and myogenic activities (Boyer et al., 1993). However, the actual functional regions of The v-ski oncogene of the Sloan-Kettering viruses neither ski nor sno have been de®ned. (SKVs) causes transformation of embryonic ®broblasts The present report describes a mutational analysis of of both chickens (CEFs) and quails (QEFs) (Stavnezer v-ski to identify functional regions and to determine et al., 1981; Colmenares and Stavnezer, 1989). In both whether its myogenic and transforming properties can cell types the transformed phenotype includes morpho- be disassociated. We have made insertion, frameshift, logical transformation and anchorage-independent deletion and substitution mutations to disrupt various regions of the gene and evaluated the eects of these mutations on protein expression, intracellular location, Correspondence: E Stavnezer transformation, and myogenesis. We also present 5 Present address: Department of Microbiology, University of Wisconsin, Madison, Winsconsin, USA results of studies aimed at determining whether the Received 27 December 1995; revised 7 April 1997; accepted 17 April functional regions de®ned by the mutational analysis 1997 constitute distinct protein structural domains. Our Core domain of v-Ski GZhenget al 460 approach was to subject the v-Ski protein to limited activities of these non-defective viruses are indistin- proteolysis and search for regions in the protein that guishable from those of SKV 5.7 (RAV-SKV). Both are relatively resistant to digestion. We chose this viruses induce morphological transformation and method because it has been successfully used to anchorage independent growth of CEFs (3 ± 5% of identify functional domains in several transcription cells form macroscopic colonies in soft agar). In factors. The two approaches have led to the addition, greater than 75% of QEFs infected by these identi®cation of the amino terminal region of v-ski, viruses form multinucleated myotubes within 3 days of which is derived from the ®rst coding exon of c-ski,as transfer into dierentiation medium. Thus neither the a distinct structural and functional unit required for removal of gag sequences from the amino- and both transformation and myogenesis. carboxyl-terminal ends of v-Ski nor the deletion of codons 4 ± 6 or 427 ± 457 from within the gene have any eect on either its transforming or myogenic potential. In both tc-d9S and dHC-d9S, three codons (Ser24, Results Met25, Ser26) that separate two neighboring SstI sites are deleted from the 5' end of v-ski. These three amino Activity of non-defective SKV acids have proven dispensable for Ski function, In the original SKV (SKV 5.7), v-ski is inserted into allowing use of an SstI fragment extending 1.2 kb the MA coding region of the gag gene (Figure 1) downstream of these sites to a site in the exon 4 without deletion of viral sequences at the insertion site sequence as a convenient target for subsequent linker (Stavnezer et al., 1989). Its translational reading frame insertion mutagenesis (see Materials and methods). is in register with gag sequences at both ends of the insertion. The P110 Ski polyprotein encoded by this Frameshift, deletion, and insertion mutations de®ne the virus uses the gag initiation codon and terminates at region of v-ski necessary for transformation the stop codon of the pol gene (Stavnezer et al., 1981). To assess the role of virus-derived sequences in P110 Having established that the deletions in the dHC-d9S function, we have excised v-ski from SKV5.7 along virus do not reduce its activity relative to SKV5.7, we with a small segment of downstream gag sequence, next examined the activities of a series of frameshift provided it with a translation initiation signal, and mutations within v-ski that resulted from oligonucleo- inserted it into avian retroviral vectors (Hughes et al., tide insertions (Table 1). These frameshifts essentially 1987; Foster and Hanafusa, 1983) downstream of an represent a set of progressive deletions from the 3' end acceptor splice site (Figure 1, tC-d9S). To test the role of v-ski, thereby allowing an assessment of the 3' of the remaining gag sequence at the 3' end of v-ski in boundary of the v-ski sequence necessary for full tc-d9S, we have generated the construct, dHC-d9S, in transforming ability (Table 1). Most informative in this which these sequences are deleted along with the 3'- terminal 30 codons of v-ski (Figure 1). The tc-d9S and dHC-d9S plasmids were transfected Table 1 Eect of frameshift and deletion mutations into CEFs and the viruses produced were used to infect fresh CEFs and QEFs to assess their transforming and myogenic abilities, respectively. We ®nd that the Figure 1 Construction of non-defective v-ski proviruses. The v- ski gene of SKV5.7 encodes a 110 kDa viral/ski fusion protein a Morphologies were compared to RAV-SKV infected, and are (P110), as indicated. A fragment containing v-ski (except codons expressed in a scale ranging from normal CEF morphology (±) to 24 ± 26) plus ®ve upstream and 49 downstream codons derived the RAV-SKV transformed morphology (++++). b Colony from gag was provided with a synthetic initiation codon to formation in soft agar is expressed as a percent of the colony generate the tc-d9S gene which encodes a 54 kDa v-Ski protein. forming eciency induced by RAV-SKV (see text), which is averaged Further deletion of the entire downstream gag region plus the c- about 5% of infected cells. c Myogenesis measured as the number of terminal 30 amino acids of v-Ski produces the dHC-d9S gene nuclei contained in myotubes compared to the total number of nuclei which encodes a 46 kDa protein.
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