Poster: Intracellular Signaling

Abs # 817: SPICK2, an AKT2/3 channel homologue 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)   Moshelion, Menachem  (A)   Becker, Dirk  (B)   Naveh, Leah  (A)   Libal,  Yael  (A)   Sekler, Israel  (C)   Moran, Arie  (C)   Lotan, Ilana  (D)   Harmon, Alice  (E)   Moran, Nava  (A)
Affiliations:	(A): Inst. Plant Sci. Gen. Agric., The Hebrew University of Jerusalem, Rehovot 76100,Israel nava.moran@huji.ac.il (B): Julius-von-Sachs-Inst.,University of Wuerzburg, Wuerzburg, Germany (C): Dept. Physiol .,Ben-Gurion University, Beer Sheva, Israel (D): Dept. Physiol. Pharmacol., Tel Aviv Univ., Tel Aviv, Israel (E): Dept. Botany, University of Florida, Gainesville, FL, USA
Web Site:	http://departments.agri.huji.ac.il/botany/moran.html

Recently, we cloned an Akt2/3 homologue , Spick2, from the pulvini (motor organs responsible for rhythmic leaf movements) of the legume Samanea saman. SPICK2 is a putative channel presumed to conduct K⁺-influx into Samanea motor cells and to constitute a necessary component of the leaf-moving “machine”.Spick2 mRNA was regulated in Planta by light and the biological clock⁽¹⁾. Since SPICK2 possesses several consensus sites for phosphorylation by cyclic AMP-dependent Protein Kinase (PKA) as well as for calcium-dependent Protein Kinase (CDPK), we tested SPICK2 susceptibility to phosphorylation in vitro: SPICK2 protein was expressed in an insect cell line, SF9, solubilized from the microsomal fraction of SF9 and immunoprecipited by anti-SPICK2 antiserum, raised against a synthetic peptide consisting of 22-amino-acids in the N-terminus of the predicted protein sequence of SPICK2. Subsequently, SPICK2 was incubated with 32P-g-ATP and either soy-bean a-CDPK or the catalytic subunit of PKA. a-CDPK did not seem to phosphorylate SPICK2 (although it did phosphorylate histone III in control test tubes). In contrast, PKA phosphorylated SPICK2, and PKI (the PKA-specific inhibitory peptide) inhibited the phosphorylation. These results support the hypothesis that SPICK2 may be regulated by phosphorylation in the process of phototransduction and/or signalling from the biological clock. ⁽¹⁾ Moshelion et al., 2002, Plant Physiol.128:634-642. ⁽²⁾ 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. ^# Both authors contributed equally to this work.