A functional screen for regulators of the c-Abl protein tyrosine kinase.
c-Abl protein tyrosine kinase activity is tightly regulated in vertebrate cells. Several mutations can activate Abl and convert it into an oncogene. In man, chromosomal translocations result in fusion proteins associated with chronic myelogenous leukemias and some acute lymphocytic leukemias. In viral forms of abl, gag sequences are fused to Abl portions resulting in a deletion of N-terminal sequences. To study c-Abl activity in a cellular environment likely to lack specific regulators, we have expressed human c-Abl in Schizosaccharomyces pombe in an inducible fashion. c-Abl causes growth arrest followed by death of the cells. Mutations in the SH2 domain or in the autophosphorylation site dramatically reduce the ability of Abl to confer the growth arrest phenotype and to phosphorylate endogenous proteins, suggesting a fundamental role of these structures in the activity of the enzyme. An SH3 domain deletion mutant of Abl is as active as c-Abl in yeast indicating that there is no intrinsic regulation of c-Abl occurring via the SH3 domain and suggesting that the inhibitory effect of the SH3 domain observed in cells of vertebrate origin is mediated by a factor that is absent in fission yeast. We have used this assay to functionally screen a human cDNA library for molecules able to counteract the lethal effect of c-Abl expression. We are currently in the process of characterising the isolated clones. We hope to identify among them the molecule(s) responsible for regulating c-Abl activity in human cells.
Moro
M
M
European Molecular Biology Laboratory, Heidelberg, Germany.
Walkenhorst
J
J
Goga
A
A
Witte
O N
ON
Superti-Furga
G
G
D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015723:Gene Library; D016313:Genes, abl; D006801:Humans; D016296:Mutagenesis; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development; D017384:Sequence Deletion; D014714:Vertebrates
D018076:DNA, Complementary; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl