Dosage-sensitive, reciprocal genetic interactions between the Abl tyrosine kinase and the putative GEF trio reveal trio's role in axon pathfinding.

The Abelson tyrosine kinase (Abl) is integrated into signal transduction networks regulating axon outgrowth. We have identified the Drosophila trio gene through a mutation that exacerbates the Abl mutant phenotype. Drosophila Trio is an ortholog of mammalian Trio, a protein that contains multiple spectrin-like repeats and two Dbl homology (DH) domains that affect actin cytoskeletal dynamics via the small GTPases Rho and Rac. Phenotypic analysis demonstrates that trio and Abl cooperate in regulating axon outgrowth in the embryonic central nervous system (CNS). Dosage-sensitive interactions between trio and Abl, failed axon connections (fax), and enabled (ena) indicate that Trio is integrated into common signaling networks with these gene products. These observations suggest a mechanism by which Abl-mediated signaling networks influence the actin cytoskeleton in neuronal growth cones.

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.

Reactivation of host-dependent src kinase activity by co-expression with a heterologous tyrosine kinase.

XD4 is a host range deletion mutant (delta 77-225) of the v-src transforming gene. This mutant transforms chicken embryo fibroblasts (CEF) but not Rat-2 cells. It encodes a product (XD4-src) that is phosphorylated at tyrosine and active as a tyrosine kinase in CEF, but is neither phosphorylated at tyrosine nor active as a kinase in Rat-2 cells. We report here that the XD4-src kinase activity in Rat-2 cells can be restored by co-expression with the tyrosine kinase encoded by v-fps, but not by co-expression with activated Ha-ras. Mutation of the major tyrosine autophosphorylation site (Y416) in XD4-src reduced but did not abolish the reactivation of XD4 tyrosine phosphorylation and kinase activity by v-fps. These results suggest that deletion of the SH2 and SH3 domains renders v-src kinase activity dependent on tyrosine phosphorylation at Y416 and other sites. The reactivation of XD4-src may result either from direct phosphorylation by the v-fps gene product or may be an indirect consequence of v-fps expression.

Intracellular targeting of pp60src expression: localization of v-src to adhesion plaques is sufficient to transform chicken embryo fibroblasts.

To define the effects of pp60v-src activity at different intracellular sites, we have constructed chimeric molecules which target the pp60v-src kinase to specific intracellular locations. pp60v-src was targeted to the nucleus by insertion of the SV40 large T antigen nuclear localization signal. Nuclear pp60v-src was active as a tyrosine kinase and phosphorylated nuclear proteins at tyrosine. However, cells expressing the nuclear pp60v-src were phenotypically normal by a number of criteria, and nuclear src kinase did not induce the expression of an mRNA (CEF-4) whose induction is characteristic of transformation by wild-type v-src. pp60v-src was targeted to perinuclear membranes by fusion to rat growth hormone and vesicular stomatitis G protein sequences. Cells expressing this chimeric molecule were phenotypically normal by most criteria. However the perinuclear src protein did induce elevated levels of CEF-4 mRNA, indicating that the v-src kinase expressed at this site induces partial transformation. The v-src and activated c-src kinases were targeted to adhesion plaques by fusion to the talin-binding sequence of vinculin. Cells expressing these fusion proteins were transformed by morphological, physiological and biochemical criteria, although the foci induced by these viruses were distinct from those induced by wild-type v-src. A chimeric protein which targeted c-src to adhesion plaques was not transforming. Thus targeting pp60src to adhesion plaques, although not sufficient to activate the transforming capacity of c-src, is sufficient to allow transformation by v-src.

Host range mutants of v-src: alterations in kinase activity and substrate interactions.

Host range mutants of Schmidt-Ruppin v-src that transform chicken embryo fibroblasts (CEF) but not Rat-2 cells were generated previously by linker insertion-deletion mutagenesis (J. E. DeClue and G. S. Martin, J. Virol. 63:542-554, 1989). One of these mutants, SRX5, in which Tyr-416 is substituted by the sequence Ser Arg Asp, retained high levels of kinase activity in vitro and in vivo, both in CEF and in Rat-2 cells. Phosphorylation of p36 (the calpactin I heavy chain) was drastically reduced in cells expressing SRX5 src, suggesting that the phenotype of SRX5 results from an alteration in substrate recognition by the src kinase. Three mutants, SPX1, SHX13, and XD6, containing linker insertions or small deletions within the src homology 2 (SH2) region, induced reduced levels of kinase activity in both CEF and Rat-2 cells. However, the residual levels of kinase activity in Rat-2 cells were above the threshold at which wild-type pp60v-src transforms Rat-2 cells, indicating that the reduction in kinase activity was not sufficient to account for the failure to transform. Cells infected by these mutants exhibited reduced levels of phosphorylation of 120- and 62-kDa proteins. We have reported elsewhere (M. F. Moran, C. A. Koch, D. Anderson, C. Ellis, L. England, G. S. Martin, and T. Pawson, Proc. Natl. Acad. Sci. USA 87:8622-8626, 1990) that ras GTPase-activating protein GAP and associated protein p62 are not tyrosine phosphorylated in Rat-2 cells expressing SHX13 or XD6. The transformation defect in Rat-2 cells may result from the failure to phosphorylate those proteins. The fifth mutant, XD4, contains a deletion which removes all of the src homology 3 (SH3) and most of the SH2 sequences of src. The protein encoded by XD4 is active as a kinase when expressed in CEF, indicating that in CEF the SH2 and SH3 regions of v-src are not necessary for kinase activity and transformation. The XD4 src product is not tyrosine phosphorylated and is inactive as a kinase when expressed in Rat-2 cells. Thus, host cell factors can affect the tyrosine phosphorylation and activity of the v-src kinase in the absence of the SH2 and SH3 regions. These results indicate that the host-dependent transformation phenotype results from alterations in src kinase activity and substrate specificity.

12-O-tetradecanoylphorbol-13-acetate activates the synthesis of phosphatidylethanol in animal cells exposed to ethanol.

Tumor-promoting phorbol esters acutely activate a pathway in lymphocytes leading to the synthesis and accumulation of phosphatidylethanol, using exogenous ethanol as a precursor. This product is a representative of a unique class of acidic glycerophospholipids in which the head group is a primary alcohol. The formation of this lipid, in response to different phorbol ester derivatives, correlates with their activity as tumor promoters and inducers of growth changes in a variety of animal cells. Since phosphatidylethanol represents an unusual metabolite of ethanol, it is proposed that studies of its synthesis and biological functions may also provide new perspectives on the biology of alcohol addiction as well as the role of this biological pathway in tumor promotion.