Worm Breeder's Gazette 14(1): 82 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Inst. Zoology, University of Fribourg, CH-1700 Fribourg.|
|2||Inst. Biochemistry, University of Fribourg, CH-1700 Fribourg. E-mail: MatthiasPaul.Wymann@UniFR.ch, Fax: ++41 37 29 86 55|
Phosphatidylinositol (3,4,5)P3 is considered to be a novel 2nd messenger and has been found to accumulate subsequently to cell surface receptor stimulation. A family of enzymes catalysing the phosphorylation of phosphoinositides at the D-3 position has been identified and cloned. Catalytic subunits of the p110[[alpha]] and [[beta]] -subtypes have been found in tight association with regulatory p85 subunits containing an SH3, two SH2 and a BCR domain. Activation of this heterodimeric PtdIns 3-kinase occurs during its translocation to autophosphorylated growth factor receptors, where the SH2 domains of p85 specifically recognise phosphorylated YXXM motives. The p110[[gamma]], on the other hand, is activated by [[beta]][[gamma]]-subunits of G-proteins and seems to transduce signals from serpentine receptors. While the p110[[alpha]]-[[gamma]] accept PtdIns, PtdIns 4-P and PtdIns(4,5)P2 as substrates, homologs of the yeast Vps34 gene product utilise solely PtdIns to produce PtdIns 3-P . Vps34p has been found to be indispensable for the sorting of various proteins to the yeast vacuole and is associated with the Vps15p serine/threonine kinase1.
Although many PtdIns 3-kinases have been identified from various species, very little is known about their downstream targets and their role in developmental processes. Since the yeast system does not allow the study of multicellular organisation and the mammalian excludes genetics, we decided to search for PtdIns 3-kinase genes in C. elegans.
Nested PCR with degenerated primers - derived from conserved sequences within the catalytic domain of the PtdIns 3-kinase family - yielded among others a product with the expected length and homology. This probe was used to screen a Barstead [[lambda]]-ZAP cDNA library and lead to the isolation of a full length cDNA clone (P51Y) of 2900 bp encoding an ORF of 901 amino acids. The 3' end was found to correspond to the sequence tag of yk2e6.3, a clone identified in the cDNA project from Yuji Koharas lab. Northern blot analysis of mixed stage polyA+ mRNA with a 3' probe from P51Y, accommodating the conserved regions of the PtdIns 3-kinase family, produced a single hybridization signal at 2.9 kb. The putative translation product of P51Y is homologous to the Vps34 family with high degrees of identity within the C-terminal conserved regions and significant similarities throughout the entire protein up to the N-terminus.
The cDNA hybridized to cosmid F53D11, which localizes the gene to chromosome I, to a region where A.Rose (Vancouver) has mapped 3 let-mutants. Rescuing of these 3 mutant candidates, cellular localization studies of the gene product and lipid kinase assays are currently carried out in our labs.
1. Stack, J. H. et al., J. Cell Biol. 129 (1995), 321-334.
2. Volina, S. et al. EMBO J. 14 (1995), 3339-3348.