Identification of a novel signaling complex that controls axon guidance in Drosophila.

Abstract

Dreadlocks (Dock) is a Src Homology (SH) 3 and SH2 domain containing adapter protein that has been proposed to play a role in the conveyance of guidance signals to the cytoskeleton. Drosophila <italic>dock</italic> mutants display connectivity defects in photoreceptor axons as well as subsets of neurons in the central and peripheral nervous system. The yeast two-hybrid system indicated that the protein tyrosine phosphatase (PTP) DPTP6 IF interacts with Dock. Immunoprecipitation (IP) experiments confirm that DPTP61F and Dock associate in Drosophila Schneider 2 (S2) cell culture and in whole embryos. <italic> In vitro</italic> binding experiments revealed that the second SH3 domain of Dock interacted directly with the first of five proline motifs present in COOH-terminal half of DPTP61F. This suggests that DPTP61F may also be involved in axon guidance since the function of the second SH3 domain of Dock is required for proper axon guidance. Since the DPTP61F/Dock complex contains an SH2 domain and a PTP domain, tyrosine phosphorylation is likely important in the DPTP61F/Dock signaling pathway. IP of Dock from S2 cells coprecipitates two tyrosine phosphorylated proteins that potentially interact with the Dock SH2 domain. A 6xHis tagged version of the Dock SH2 domain was expressed in S2 cells and found to enhance the levels of tyrosine phosphorylation of the two proteins present in Dock IPs as well as three other endogenous proteins. These proteins copurified with the Dock SH2 domain on a Ni-agarose column and were further purified using a 4G10 anti-phosphotyrosine column. Large-scale purification resulted in enough material for microsequencing and cloning of the five associated proteins. Their identities suggest that they form a signaling complex involved in axon guidance. This complex contains the Drosophila homologs of the Down Syndrome Cell Adhesion Molecule (DSCAM), Activated Cdc42 Kinase (Ack), and the actin organizing protein Lasp-1. Also co-purifying are SnxW, a molecule that resembles mammalian sorting nexins, and Ryd1, a putative R&barbelow;GG and ty&barbelow;rosine rich d&barbelow;omain containing protein. dsRNA-mediated interference of protein expression (RNAi) demonstrated that the proteins within this complex play critical roles in axon guidance in Drosophila embryos. This work is the first characterization of a cytoplasmic machine that translates extracellular signals into growth cone motility.