In animals such as Drosophila and mouse, growth hormone-insulin and TGFb signal pathways are known to control body size and organ size (Conlon and Raff, 1999). In C. elegans, mutants in the TGFb/DBL-1 pathway are all small indicating that this pathway is important in body size control (Patterson and Padgett, 2000). We previously isolated many big mutants, and showed that several of them are mutants in the
egl-4 gene encoding a cGMP-dependent protein kinase (G kinase). Genetic interaction studies suggest that EGL-4 inhibits activity of DBL-1/TGFb pathway for body size control (Hirose et al. 2003). Although DBL-1 and SMA-2 could be substrates of EGL-4, both relation between EGL-4 and the TGFb pathway and mechanisms of body size control by the TGFb pathway remain poorly understood. To determine these issues, we screened for proteins that bind to EGL-4 using yeast two hybrid system, and identified six proteins: Ras homolog (DRN-1), seven transmembrane protein (C52B9.4), CDP diglyceride synthetase ortholog (C33H5.18), collagen related protein (COL-167), transport protein Sec61a subunit homolog (Y57G11C.15) and an unknown protein (Y43F8C.11).
egl-4 gene is known to produce two alternatively spliced variants called PKGa and PKGb, which differ in the promoters and the first exon. We found that the above proteins specifically bind in yeast to the PKGa N-terminal region containing a glycine-rich domain encoded by the PKGa first exon, suggesting an important role of this domain to allow access by the binding proteins. We also found that mutants in the three binding proteins,
drn-1(
ok400), C52B9.4
(tm2431) and
col-167(
tm3691) were slightly smaller than wild type, which suggest that these proteins play a role in body size control. At present, we are trying to purify tagged EGL-4 synthesized in a cell-free protein synthesis system for substrate identification. We are also examining direct interaction in vitro between EGL-4 and the proteins found by the yeast two-hybrid screening. In the process of these works, when we grew worms on E. coli HB101, adult worms were found to be larger in volume than those fed on OP50 by about 50%. Although it was known that HB101 food speeds up the growth to adulthood (Shtonda and Avery 2006), our result shows that it also affects the maximum body volume. The worms fed on HB101 have enhanced body size difference depending on the genetic background, resulting in easier identification of body size phenotypes.