Poster: Signaling, cell-to-cell

Abs # 439: Identification of phosphatidylinositol phosphate kinase interacting proteins

Presenter:	Davis, Amanda J., ajspell@unity.ncsu.edu
Authors	Davis, Amanda J. (A)   Perera, Imara Y. (A)   Boss, Wendy F. (A)
Affiliations:	(A): North Carolina State University

Phosphatidylinositol phosphate kinases are a family of enzymes that phosphorylate phosphatidylinositol 4-phosphate (PtdInsP) to form phosphatidylinositol 4,5-bisphosphate (PtdInsP₂). Changes in PtdInsP₂ and inositol 1,4,5 trisphosphate (InsP₃) occur in plants in response to many environmental stimuli, including gravity and osmotic stress (Stevenson, et al., Trends Plant Sci. 5:252-258, 2000). Our goals are to identify and characterize PtdInsP kinase interacting proteins and to determine how these PtdInsP kinase binding proteins regulate the PtdInsP kinases. Interactions with Rho-type G-proteins have been shown to increase Arabidopsis PtdInsP kinase activity (Kost, et al., J Cell Biol. 145:317-330. 1999) and posttranslational modification of the PtdInsP kinases by phosphorylation decreases kinase activity (Westergren et al., Biochem J. 359:583-589, 2001). We chose to characterize two PtdInsP kinases, AtPIPK1 (At1g21980) and AtPIPK11 (At4g01190), representatives of the two subfamilies of PtdInsP kinases in Arabidopsis. The cDNAs were used create GST-fusion constructs of the PtdInsP kinases. Recombinant proteins, GST-AtPIPK1 and GST-AtPIPK11, were produced in E. coli and purified on glutathione-Sepharose beads. Solubilized Arabidopsis microsomes isolated from suspension culture cells were incubated with purified recombinant GST-fusion proteins. In addition, protein binding studies were done in the presence and absence of GTP and ATP to identify selectively interacting proteins. Peptides that selectively bound GST-AtPIPK1 and GST-AtPIPK11 in the presence of GTP or ATP were recovered and sequenced by mass spectroscopy. The interactions between the recombinant proteins and the sequenced peptides are being characterized by Western blotting with isoform specific antibodies.