Ping Gong et al. (2002) Mid-west Worm Meeting "UNC-44 Protein Interaction"

Jerald Perschall, Ping Gong, Dina Darwis, Anthony Otsuka

[Mid-west Worm Meeting, 2002]

The correct connection of neurons is a critical part of neural development. The growth of neurons to their targets has been shown to require dozens of genes in the nematode, Caenorhabditis elegans. One of those genes is uncoordinated-44 (unc-44) that encodes a series of ankyrin proteins. Previous studies showed that the large AO13 ankyrin isoform, rather than the other isoforms is required for proper axonal guidance. To determine which molecules interact with UNC-44 and whether they form a signal transduction complex, the yeast two-hybrid system has been used. One bait, pDD5, was constructed by inserting the DNA coding for the carboxyl domain of AO13 ankyrin into plasmid pAS2-1. Transformation of pDD5 into yeast cells containing C. elegans library plasmids yielded positive colonies that were picked and DNA sequenced. Four of ten clones analyzed encode W08G11.4 which is related to the B56 or B' regulatory subunit of phosphatase 2A (PP2A). PP2A has been shown to be involved in many cellular processes by combining a limited number of catalytic (C) and adapter (A) subunits with a variety of regulatory (B, B', B'', and B''') subunits. The B-type subunits of PP2A determine the substrate specificity of PP2A phosphatase. PP2A containing the B56 regulatory subunit has been reported to be involved in axonal guidance in Drosophila.

Ping Gong et al. (2001) International C. elegans Meeting "Two-hybrid Screen for UNC-44 AO13 Interacting Proteins"

Dina Darwis, Jorge Munera, Anthony J Otsuka, Janna Lindemulder, Jerry Perschall, Brent Bill, Ping Gong

[International C. elegans Meeting, 2001]

Mutations in unc-44 result in axon outgrowth and guidance defects. The cloning of unc-44 demonstrated that it encodes a series of ankyrins, including a novel ankyrin called AO13 ankyrin [Otsuka, A. J. et al. J. Cell Biol. 129 , 1081-1092 (1995)]. AO13 ankyrin is unusual in that it is much larger than other ankyrins (6994 aa compared to 4377 aa for the largest vertebrate ankyrin, ankyrin G ). AO13 ankyrin is also unusual because the carboxyl end of the protein contains 7 potential transmembrane domains and Ser/Thr/Glu/Pro-rich (STEP) repeats. We hypothesize that UNC-44 AO13 ankyrin may be part of a signal transduction complex involved in axon guidance. To test this hypothesis, two-hybrid screens [Fields, S. and Song, O. Nature 340 , 245-247 (1989)] were performed with AO13-specific baits. Baits were constructed from a STEP repeat region (pDD3) and from the carboxyl terminus of AO13 ankyrin (pDD4 and pDD5). These baits were introduced into either yeast strain Y190 ( HIS3 and lacZ reporters) or AH109 ( HIS3, lacZ , and ADE2 reporters). These strains were transformed with R. Barstead's C. elegans cDNA clone libraries. A number of positive clones were obtained and are currently being analyzed. In addition to the two-hybrid approach, the UNC-44 ankyrins are being examined for changes in phosphorylation. Finally, dsRNAi is being used in an attempt to knock out AO13 ankyrin expression.