F. Michael Hoffmann

Genetic analysis of a drosophila neural cell adhesion molecule: Interaction of fasciclin I and abelson tyrosine kinase mutations

Drosophila fasciclin I is a homophilic cell adhesion molecule expressed in the developing embryo on the surface of a subset of fasciculating CNS axons, all PNS axons, and some nonneuronal cells. We have identified protein-null mutations in the fasciclin I (fas I) gene, and show that these mutants are viable and do not display gross defects in nervous system morphogenesis. The Drosophila Abelson (abl) proto-oncogene homolog encodes a cytoplasmic tyrosine kinase that is expressed during embryogenesis primarily in developing CNS axons; abl mutants show no gross defects in CNS morphogenesis. However, embryos doubly mutant for fas I and abl display major defects in CNS axon pathways, particularly in the commissural tracts where expression of these two proteins normally overlaps. The double mutant shows a clear defect in growth cone guidance; for example, the RP1 growth cone (normally fas I positive) does not follow its normal path across the commissure.

Drosophila Dpp signaling is mediated by the punt gene product: A dual ligand-binding type II receptor of the TGFβ receptor family

Signaling by TGF beta-related factors requires ligand-induced association between type I and type II transmembrane serine/threonine kinases. In Drosophila, the saxophone (sax) and thick veins (tkv) genes encode type I receptors that mediate signaling by decapentaplegic (dpp), a member of the bone morphogenetic protein (BMP) subgroup of TGF beta-type factors. In this report, we demonstrate that the Drosophila punt gene encodes atr-II, a previously described type II receptor that on its own is able to bind activin but not BMP2, a vertebrate ortholog of dpp. Mutations in punt produce phenotypes similar to those exhibited by tkv, sax, and dpp mutants. Furthermore, punt will bind BMP2 in concert with tkv or sax, forming complexes with these receptors. We suggest that punt functions as a type II receptors for dpp and propose that BMP signaling in vertebrates may also involve sharing of type II receptors by diverse ligands.

Identification of two bone morphogenetic protein type I receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor

Drosophila sequences at chromosomal positions 25D (Brk25D) and 43E (Brk43E) are similar to the TGF beta type I receptor serine/threonine kinases and are expressed broadly during embryogenesis. Brk25D binds dpp protein and bone morphogenetic protein 2 with high affinity. Mutations affecting Brk25D map to the gene thick veins and block the expression of two decapentaplegic-responsive (dpp-responsive) genes, dpp and labial, in the embryonic midgut. Defects in Brk25D receptor function combined with reduced expression of dpp ligand produce mutant phenotypes in the embryo and adult. Brk43E is the product of the gene saxophone, which also interacts with dpp. We conclude that dpp signaling in vivo is mediated by at least two receptors, Brk25D and Brk43E.

Drosophila abl tyrosine kinase in embryonic CNS axons: a role in axonogenesis is revealed through dosage-sensitive interactions with disabled.

During Drosophila embryogenesis, the Abelson tyrosine kinase (abl) is localized in the axons of the central nervous system (CNS). Mutations in abl have no detectable effect on the morphology of the embryonic CNS, and the mutant animals survive to the pupal and adult stages. In the absence of abl function, however, heterozygous mutations or deletions of disabled (dab) exert dominant effects, disrupting axonal organization and shifting the lethal phase of the animals to embryonic and early larval stages. Embryos that are homozygous mutant for both abl and dab fail to develop any axon bundles in the CNS, although the peripheral nervous system and the larval cuticle appear normal. The genetic interaction between these two genes begins to define a process in which both the abl tyrosine kinase and the dab gene product participate in establishing axonal connections in the embryonic CNS of Drosophila.

MAGE-A, mMage-b, and MAGE-C Proteins Form Complexes with KAP1 and Suppress p53-Dependent Apoptosis in MAGE-Positive Cell Lines

The MAGE-A, MAGE-B, and MAGE-C protein families comprise the class-I MAGE/cancer testes antigens, a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing sperm. Aberrant expression of class I MAGE proteins occurs in melanomas and many other malignancies, and MAGE proteins have long been recognized as tumor-specific targets; however, their functions have largely been unknown. Here, we show that suppression of class I MAGE proteins induces apoptosis in the Hs-294T, A375, and S91 MAGE-positive melanoma cell lines and that members of all three families of MAGE class I proteins form complexes with KAP1, a scaffolding protein that is known as a corepressor of p53 expression and function. In addition to inducing apoptosis, MAGE suppression decreases KAP1 complexing with p53, increases immunoreactive and acetylated p53, and activates a p53 responsive reporter gene. Suppression of class I MAGE proteins also induces apoptosis in MAGE-A-positive, p53wt/wt parental HCT 116 colon cancer cells but not in a MAGE-A-positive HCT 116 p53-/- variant, indicating that MAGE suppression of apoptosis requires p53. Finally, treatment with MAGE-specific small interfering RNA suppresses S91 melanoma growth in vivo, in syngenic DBA2 mice. Thus, class I MAGE protein expression may suppress apoptosis by suppressing p53 and may actively contribute to the development of malignancies and by promoting tumor survival. Because the expression of class I MAGE proteins is limited in normal tissues, inhibition of MAGE antigen expression or function represents a novel and specific treatment for melanoma and diverse malignancies.

A novel tyrosine kinase-independent function of Drosophila abl correlates with proper subcellular localization

Abstract The axonal localization of the Drosophila abl protein and its genetic interactions with the disabled and fasciclin I genes implicate this cytoplasmic tyrosine kinase in the process of axonal pathfinding. Several changes at the amino terminus of abl permitted proper function and localization of the altered proteins. In contrast, the presence of human c-abl type 1a amino-terminal sequences or the murine c-abl carboxy-terminal domain interfered with function and axonal locallzation. Rescue of phenotypes caused by mutations in abl alone did not require tyrosine kinase activity, indicating a novel kinase-independent function for the properly localizaed abl protein. However, abl kinase activity was required to rescue the mutant phenotypes in genetic backgrounds also mutant for disabled .

The Drosophila abelson proto-oncogene homolog: Identification of mutant alleles that have pleiotropic effects late in development

The Abelson gene in Drosophila (abl) consists of ten exons extending over 26 kb of genomic DNA. The DNA sequence encodes a protein of 1520 amino acids with sequence homology to the human c-abl proto-oncogene product, beginning at the amino terminus and extending 656 amino acids through the region essential for tyrosine kinase activity. Mutant lesions in the abl gene were identified first by their failure to complement chromosomal deletions that overlap the abl DNA sequence and then by rescue of the mutant phenotypes with an abl minigene in transgenic flies. Elimination of abl zygotic function by mutations produces some recessive lethality at the pharate adult pupal stage, and mutant adults with reduced longevity, reduced fecundity, and an irregular pattern of retinal cells.

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

BackgroundEpithelial to Mesenchymal Transition (EMT) induced by Transforming Growth Factor-β (TGF-β) is an important cellular event in organogenesis, cancer, and organ fibrosis. The process to reverse EMT is not well established. Our purpose is to define signaling pathways and transcription factors that maintain the TGF-β-induced mesenchymal state.ResultsInhibitors of five kinases implicated in EMT, TGF-β Type I receptor kinase (TβRI), p38 mitogen-activated protein kinase (p38 MAPK), MAP kinase kinase/extracellular signal-regulated kinase activator kinase (MEK1), c-Jun NH-terminal kinase (JNK), and Rho kinase (ROCK), were evaluated for reversal of the mesenchymal state induced in renal tubular epithelial cells. Single agents did not fully reverse EMT as determined by cellular morphology and gene expression. However, exposure to the TβRI inhibitor SB431542, combined with the ROCK inhibitor Y27632, eliminated detectable actin stress fibers and mesenchymal gene expression while restoring epithelial E-cadherin and Kidney-specific cadherin (Ksp-cadherin) expression. A second combination, the TβRI inhibitor SB431542 together with the p38 MAPK inhibitor SB203580, was partially effective in reversing EMT. Furthermore, JNK inhibitor SP600125 inhibits the effectiveness of the TβRI inhibitor SB431542 to reverse EMT. To explore the molecular basis underlying EMT reversal, we also targeted the transcriptional repressors ZEB1 and ZEB2/SIP1. Decreasing ZEB1 and ZEB2 expression in mouse mammary gland cells with shRNAs was sufficient to up-regulate expression of epithelial proteins such as E-cadherin and to re-establish epithelial features. However, complete restoration of cortical F-actin required incubation with the ROCK inhibitor Y27632 in combination with ZEB1/2 knockdown.ConclusionsWe demonstrate that reversal of EMT requires re-establishing both epithelial transcription and structural components by sustained and independent signaling through TβRI and ROCK. These findings indicate that combination small molecule therapy targeting multiple kinases may be necessary to reverse disease conditions.

Drosophila abl and genetic redundancy in signal transduction

Genetic studies on Drosophila Abl and, more recently, on mouse c-Abl and c-Src indicate that the functions of these non-receptor tyrosine kinases may duplicate activities of other molecules within signal transduction pathways. In Drosophila, second-site mutations have been recovered that disrupt the redundant functions so that the Abl tyrosine kinase is essential to the formation of axonal connections in the embryonic central nervous system and for attachment of embryonic muscles to the body wall. Molecular isolation and analysis of the genes identified by these second-site mutations should define the molecular basis for the genetic redundancy.

Lactimidomycin, Iso-migrastatin and Related Glutarimide-containing 12-membered Macrolides are Extremely Potent Inhibitors of Cell Migration

Migrastatin (1), iso-migrastatin (5) and lactimidomycin (7) are all glutarimide-containing polyketides known for their unique structures and cytotoxic activities against human cancer cell lines. Migrastatin, a strong inhibitor of tumor cell migration, has been an important lead in the development of antimetastatic agents. Yet studies of the related 12-membered macrolides iso-migrastatin, lactimidomycin, and related analogues have been hampered by their limited availability. We report here the production, isolation, structural characterization, and biological activities of iso-migrastatin, lactimidomycin, and 23 related congeners. Our studies showed that, as a family, the glutarimide-containing 12-membered macrolides are extremely potent cell migration inhibitors with some members displaying activity on par or superior to that of migrastatin as exemplified by compounds 5, 7, and 9-12. On the basis of these findings, the structures and activity of this family of compounds as cell migration inhibitors are discussed.