The Caenorhabditis elegans Aurora A kinase, AIR-1, is associated with mitotic centrosomes and is required for chromosome segregation. Using AIR-1 as the bait in a yeast two-hybrid screen, we cloned a novel member of the Ste20/PAK group of kinases (T19A5.2) which we have designated as germinal center kinase (
gck-1). The Ste20p group of kinases has been divided into two families (PAKs and GCKs) based on the location of the kinase domain. The absence of a PAK homology domain as well as the presence of an N-terminal kinase domain and a unique C-terminus classifies GCK-1 as a member of the germinal center kinase (GCK) family. This family is comprised of eight subfamilies, several of which have been shown to activate the MAP kinase pathway.
gck-1 falls into the third GCK (GCK-III) subfamily; however, the GCK-III subfamily proteins have not been previously shown to activate any of the MAP kinase pathways. To determine GCK-1 localization, a rabbit polyclonal antibody was raised against recombinant GCK-1 fused with maltose binding protein (MBP). GCK-1 is associated with P-granules which localize to the germ line precursor cells of the developing C. elegans embryo. Additionally, GCK-1 localizes to the mitotic cleavage furrow in embryonic somatic cells. To determine the role of GCK-1 in C. elegans development, we performed RNA-mediated interference (RNAi).
gck-1(RNAi) results in sterility due to improper meiotic prophase progression. A wild-type C. elegans gonad consists of two U-shaped tubes with mitotic nuclei at the distal end. As the nuclei move proximally within a common cytoplasm they enter pachytene of meiotic prophase I where they remain for a period of time before entering into diakinesis. Linearly arranged diakinetic nuclei become large cellularized oocytes as they progress proximally towards the spermatheca. The bivalents in the most proximal oocytes can be visualized with a phosphohistone H3 antibody. The oocytes are fertilized in the spermatheca, and then pass into the uterus where the maternal nucleus meiotic divisions I and II are followed by several mitotic cell cycles before the embryos are extruded into the environment. Alpha-tubulin and DAPI staining of C. elegans
gck-1(RNAi) gonads reveals what appear to be multiple rows of small cellularized diakinetic oocytes in the proximal end of the gonad. However, only some of the small cellularized bodies have phosphohistone H3 positive bivalents and these cells are randomly distributed in the proximal gonad rather restricted to the most proximal end, as in wild type. The cell cycle stage of the non-staining small cellularized bodies is presently undefined. The
gck-1(RNAi) germ line progression defects are similar to the phenotype resulting from over-activation of
let-60(Ras) in the MAP kinase pathway. The MAP kinase pathway is required for pachytene exit in theC. elegans gonad. Since GCK-1 shares some homology with other protein kinases that are known to be involved in the MAP kinase pathway, genetic epistatic studies are underway to determine the relationship between GCK-1 and the MAP kinase pathway. Additional studies are being conducted to further define the relationship of
gck-1 with other mutants that have germ line progression defects. Future experiments will determine the interaction between GCK-1, the MAP kinase pathway, and the AIR-1 kinase.