Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains.

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

Phosphorylation of Enabled by the Drosophila Abelson tyrosine kinase regulates the in vivo function and protein-protein interactions of Enabled.

Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.

Can clues to the molecular defects in chronic myelogenous leukemia come from genetic studies on the Abelson tyrosine kinase in fruit flies?

Translocations affecting the structure of the c-abl proto-oncogene are involved in the development or progression of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). Leukemic cells from patients with CML show alterations in adhesive properties that may play a part in the pathology of these diseases. Mutations in the Drosophila Abl homolog are lethal and indicate that Abl may mediate processes involving differential cell adhesion. These observations suggest that Abl may regulate similar adhesive processes in human beings and Drosophila. Genetic analysis of Abl function in Drosophila has identified novel proteins that function in Abl-related processes. Analysis of the functions of these new molecules may provide insight into mechanisms by which oncogenic abl proteins participate in the etiology of CML and ALL.

enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding properties.

Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identified alleles of only one gene, enabled (ena). We report that the ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains, ena is also a substrate for the Abl kinase; tyrosine phosphorylation of ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. We conclude that a critical function of Drosophila Abl is to phosphorylate and negatively regulate ena protein during neural development.

The ovarian tumor protein isoforms of Drosophila melanogaster exhibit differences in function, expression, and localization.

The Drosophila melanogaster ovarian tumor (otu) gene, required for normal proliferation and differentiation of the female germ-line, encodes two cytoplasmic protein isoforms, 98 and 104 kDa. Mutants with defects in this gene are typically grouped into one of three phenotypic classes: quiescent (germ cells do not proliferate), oncogenic or tumorous (germ-line cells proliferate uncontrollably), and differentiated (germ-line cells initiate but do not complete differentiation). Analysis of transformants expressing only one of the otu isoforms showed that the 104-kDa isoform (otu-104) can rescue all classes of otu mutants, whereas only differentiated mutants are rescued to a significant extent by the 98-kDa isoform (otu-98). Western analysis of protein extracts prepared from ovaries of various developmental stages indicated that otu-104 predominates in predifferentiated stages, while otu-98 is prevalent in differentiated egg chambers. Immunolocalization experiments demonstrated that otu protein is present in the cytoplasm of oogonial stem cells that populate third instar larvae and in all germ-line-derived cells until late in oogenesis. In stage 10 egg chambers, otu protein shifts to the subcortical region of nurse cells. This type of analysis also showed that upon formation of a 16-cell syncytium otu-104, but not otu-98, preferentially accumulates in the developing oocyte cytoplasm. The otu mutant protein does not show this pattern of enhanced accumulation, nor does it occur in ovaries of egalitarian and Bicaudal-D mutants, which are defective in oocyte determination. Thus, these studies indicate that the 104-kDa isoform is required for normal proliferation of female germline cells and perhaps for oocyte differentiation. The 98-kDa isoform appears to be dispensable but can provide an otu function needed for the completion of oocyte maturation.

Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression.

We have identified a genomic DNA fragment which restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila melanogaster. Germ-line transformants bearing this fragment express otu mRNA with the same tissue specificity as, and at levels comparable to, the wild-type otu gene. Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region. Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression from the otu gene. A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp.

Specificity of DNA uptake during whole cell transformation of S. cerevisiae.

We have studied the mechanism of DNA transformation of whole yeast cells in Saccharomyces cerevisiae with particular emphasis on the role of the cell wall complex in DNA uptake. Two new aspects of the process have been investigated in order to evaluate its specificity. Such aspects are: (i) effect of monovalent vs. divalent cations during incubation with the transforming DNA and (ii) timing of DNA adsorption and uptake. We found that the specificity for cation requirement is a strain-dependent characteristic influenced by the presence of transforming DNA in the cell suspension. This finding is supported by reports from several laboratories that some yeast strains show mutually exclusive transformability with monovalent vs. divalent cations. While irreversible adsorption of plasmid DNA molecules is induced by both heat shock and polyethylene-glycol (PEG), DNA uptake seems to occur only after the removal of PEG. In the course of this study we have developed a new, alternative method of whole cell DNA transformation with CaCl2 able to transform strains that do not respond to other methods.