Identification and characterization of an SH2 -ANK repeat tyrosine kinase in Drosophila melanogaster

Abstract

Tyrosine kinases play critical roles in the processing of developmental information. In an attempt to more clearly understand how tyrosine kinases function in these processes a molecular search for tyrosine kinases was undertaken. Using a polymerase chain reaction technique, we attempted to identify tyrosine kinases in Drosophila melanogaster that would permit both biochemical and genetic analysis. The primary objective was the cloning of a homologue of the mammalian receptor tyrosine kinase for the hematopoietic growth factor CSF-1. No such clone was identified among the six novel kinases cloned. However, a non-receptor tyrosine kinase was cloned which defined a new family of these enzymes. This tyrosine kinase like the mammalian Syk and ZAP-70 kinases possessed two SH2 domains and no SH3 domains. Strikingly located between the SH2 domains were a series of five ankyrin (ANK) repeats. This is the first kinase to be identified which possess ANK repeats. In addition the protein contained a stretch of 50 amino acids that are proline rich and very basic. The predicted structure of the protein led us to name it, SH2, ANK repeat kinase or SHARK.;In an attempt to understand the function of this protein we determined its mRNA expression pattern. Flies express SHARK at low but detectable levels in most stages but significantly higher levels in early embryos and imaginal disks. The protein expression pattern confirmed that the post gastrulation embryos produced the highest levels. Embryos limited expression of SHARK to ectodermally derived epithelial structures in a staining pattern almost identical to the product of the crumbs gene. Crumbs, an EGF repeat containing protein, is necessary for proper maintenance of epithelial character of cells that express it. Furthermore, the SHARK protein, like Crumbs localizes to the apical surface of expressing cells. Characterization of its genomic organization indicated that SHARK resides within two kilobases of another distinct transcriptional unit. Localization of SHARK by in situ hybridization to polytene chromosomes places the gene at 53A, an area believed to contain a haploinsufficient locus. These two facts have hampered isolation of mutants in SHARK.