Budding in Saccharomyces cerevisiae follows a genetically programmed pattern of cell division which can be regulated by external signals. On the basis of the known functional conservation between a number of mammalian oncogenes and antioncogenes with genes in the yeast budding pathway, we used enhancement of pseudohyphal budding in S. cerevisiae by human proteins expressed from a HeLa cDNA library as a morphological screen to identify candidate genes that coordinate cellular signaling and morphology. In this report, we describe the isolation and characterization of human enhancer of filamentation 1 (HEF1), an SH3-domaincontaining protein that is similar in structure to p130 cas , a recently identified docking protein that is a substrate for phosphorylation by a number of oncogenic tyrosine kinases. In contrast to p130 cas , the expression of HEF1 appears to be tissue specific. Further, whereas p130 cas is localized predominantly at focal adhesions, immunofluorescence indicates that HEF1 localizes to both the cell periphery and the cell nucleus and is differently localized in fibroblasts and epithelial cells, suggesting a more complex role in cell signalling. Through immunoprecipitation and two-hybrid analysis, we demonstrate a direct physical interaction between HEF1 and p130 cas , as well as an interaction of the SH3 domain of HEF1 with two discrete proline-rich regions of focal adhesion kinase. Finally, we demonstrate that as with p130 cas , transformation with the oncogene v-abl results in an increase in tyrosine phosphorylation on HEF1, mediated by a direct association between HEF1 and v-Abl. We anticipate that HEF1 may prove to be an important linking element between extracellular signalling and regulation of the cytoskeleton.

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The product of proto-oncogene c-abl and its related gene, arg, encode nonreceptor tyrosine kinases that are ubiquitously expressed in mouse and human cells (1-3). The two proteins are 95% identical in the N-terminal SH3, SH2, and tyrosine kinase domains. The C-terminal regions that constitute more than one-half of both proteins exhibit an overall identity of only 29% (2). This large C-terminal region that distinguishes c-Abl from the other family of nonreceptor kinases is required for the proper biological function of c-Abl. Truncation of the C terminus causes neonatal lethality in mice, a phenotype similar to the one observed with mice carrying homozygous null mutation for c-abl (4, 5). Several functional domains have been identified in the C-terminal region of c-Abl, including three nuclear localization signals (6), a DNA binding domain composed of three high mobility group-like boxes (7, 8), and binding domains for G-and F-actin (9, 10). Two physiological substrates for c-Abl have been identified for which substrate binding sites have also been identified in the C-terminal region. They are the CRK family of SH2/SH3 adapter proteins (11, 12) and RNA polymerase II (13-15).The largest subunit of RNA polymerase contains a unique C-terminal domain that is composed of a seven-amino acid repeat with the consensus sequence YSPTSPS (16, 17). The heptad sequence is repeated 52 times in mammals, 44 times in Drosophila melanogaster, and 26 times in Saccharomyces cerevisiae (17). The CTD 1 of RNAP is essential for cell growth because truncation of more than half of the repeats in yeast causes cold sensitivity and inability to induce specific gene expression such as INO 1 and GAL 10 (18). In mouse, a similar truncation in an ␣-amanitin-resistant RNA polymerase caused inability of the polymerase to confer ␣-amanitin resistance (19).Because the CTD is rich in serine, threonine, and tyrosine, it serves as a substrate for both serine (threonine) and tyrosine kinases. Several CTD kinases have been identified in yeast and mammals. In yeast, the cyclin-dependent kinase Kin 28, a component of the holo-TFIIH, has been shown to phosphorylate the CTD (20, 21). The mammalian homologue of TFIIH-associated CTD kinase has been shown to be the cdk7/cyclin H kinase pair (22). Another yeast CTD kinase, SRB10/11 kinase-cyclin pair, has been identified as the mammalian cdk8/cyclin C (23). Thus far, the only kinase known to phosphorylate RNAP-CTD on tyrosines is c-Abl (13-15). c-Abl can phosphorylate the CTD to high stoichiometry with the incorporation of Ͼ30 mol of phosphate/mol of CTD. Such a high stoichiometric phosphorylation by Abl requires binding of the SH2 domain of Abl to partially tyrosine-phosphorylated CTD (13). Furthermore, CTD phosphorylation by Abl both in vivo and in vitro requires a CTD-interacting domain (CTD-ID) present at the C terminus of Abl (15).The strongest evidence that c-Abl phosphorylates RNA polymerase II in vivo came from the study of cellular response to DNA damaging agents such as methyl methanesulfonate (MMS). ...

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spoOJ and to make wild-type sporulation catabolite resistant suggested that spoOJ had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spoOJ locus (spoOJ::Tn917fQHU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spoOJ::Tn917Q1HU261 to sporulate in medium with glucose. Sequencing the spoOJ locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spoOJ mutations. Analysis of the deduced amino acid sequence of the spoOJ locus indicated that the spoOJ gene product contains an ot-helix-turn-a-helix unit similar to the motif found in Cro-like DNA-binding proteins.Bacillus subtilis sporulation is a model system that is used to study procaryotic gene expression and cellular differentiation as responses to environmental stimuli. Sporulation is subject to catabolite repression; i.e., the presence of glucose or other readily metabolized carbon sources inhibits sporulation by wild-type cells (33). Initiation of sporulation is controlled by at least seven genes, spoOA, spoOB, spoOE, spoOF, spoOH, spoOJ, and spoOK (23). Glucose represses transcription of spoOA and spoOF (3, 43). However, it is not known how availability of nutrients regulates initiation of sporulation. Several spoO genes have been sequenced (5,9,14,15,30,41). It is evident from this work that the spoOH gene codes for a sigma subunit of RNA polymerase and that some spoO genes are responsible for sensing environmental conditions. The spoOA and spoOF gene products are homologous to the effector molecules of the two-component response regulator systems that have been described for a variety of bacteria (15,41 PMB12 and SP10 are also able to suppress the Spo-and oligosporogenic phenotypes of various spoOJ mutations (6,20,34). As a result, both PMB12-infected and SP10-infected spoOJ mutants sporulate at a significantly higher frequency than uninfected spoOJ mutants. The observations that PMB12-and SP10-infected bacteria display catabolite-resistant sporulation and that these bacteriophages suppress spoOJ mutations suggest that spoOJ has a role in catabolite repression of sporulation.The spoOJ locus was originally thought to be represented by two mutations, spoOJ87 and spoOJ93 (17). However, spoIIA and spaIID are expressed in a strain with spoOJ87, whereas spoOJ93 blocks expression of these genes (7, 13). The wild-type alleles of both mutations have been cloned into bacteriophage 0105 vectors (10,12 spoOJ93 (45).The data presented in this report provide additional evidence for the involvement of spoOJ in catabolite repressi...

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