Poster: Intracellular Signaling

Abs # 827: SPIRL1, a Glutamate Receptor homologue of unknown function from Samanea motor cells, is phosphorylated in vitro by PKA

Presenter:	Yu, Ling , yuling@agri.huji.ac.il
Authors	Yu, Ling  (A)   Levi, Hadas  (A)   Becker, Dirk  (B)   Moshelion, Menachem  (A)   Bienner, Eva  (C)   Moran, Nava  (A)
Affiliations:	(A): Inst. Plant Sci. Gen. Agric.,The Hebrew University of Jerusalem, Rehovot 76100, Israel (B): Julius von Sachs Inst., University of Wuerzburg, Wuerzburg, Germany (C): Inst. of Biochem. Food Sci., The Hebrew University of Jerusalem, Rehovot 76100, Israel

The ionotropic glutamate receptor channels (iGluRs), extensively sudied in animals, are non-selective glutamate-gated cation channels. While dozens of iGluR-homologues were identified in plants and one even in bacteria, the biochemistry and physiology of the protein products of these plant genes, are still unknown. Spirl1 is an iGluR homologue from leaf motor organs, pulvini, which are responsible for the rhythmic leaf movements of Samanea saman, a leguminous tree of the Mimosa family. We cloned Spirl1 using a probe based on the conserved region of a K channel. The full length of the predicted protein sequence is 903 amino acids and its size is ~100 kDa. This sequence is ~60 % identical to several of the Arabidopsis GLR2 gene family. When over-expressed in the insect SF9 cell line, SPIRL1 was located in the plasma membrane and inner membrane systems, as visualized with confocal microscope imaging and immuno-staining using antibody purified from anti-SPIRL1 antiserum, raised against a synthetic peptide consisting of 18-amino-acids in the C-terminus of SPIRL1. In western blots SPIRL1 yielded a strong band of ~105 kDa and a weaker band of ~85 kDA. Since SPIRL1 possesses a few predicted consensus sites for phosphorylation by cyclic AMP-dependent Protein Kinase (PKA), we tested it for susceptibility to phosphorylation in vitro: SPIRL1 was solubilized from the microsomal fraction of SF9 and immunoprecipited using the purified antibody. Subsequently, SPIRL1 was incubated with 32P-g-ATP and the catalytic subunit of PKA. Both bands of SPIRL1 were phosphorylated, and this phosphorylation was inhibited by PKI (the PKA-specific inhibitory peptide. Supported by grants from BSF (2000-191-1) and ISF (550/01-1) to NM and by a 2002 Short-term EMBO fellowship to LY.