MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF StCDPK2

GIAMMARIA V; GRANDELLIS C; VILLASUSO AL; ULLOA RM

St. Louis, MO, USA

Congreso; 9th IPMB Meeting; 2009

IPMB

MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF StCDPK2, Giammaria V., Grandellis C., Ulloa RM.  Instituto de ingenier¨ªa Gen¨¦tica y Biolog¨ªa Molecular, Buenos Aires, Argentina.  rulloa@dna.uba.ar Calcium plays an essential role in plant cell signalling. Calcium dependent protein kinases (CDPKs) are the best-characterized calcium sensors in plants. These Ser/Thr protein kinases have an N-terminal kinase domain joined to a C-terminal CaM-like domain via a junction region that serves to stabilize and maintain the kinase in an auto-inhibited state. CDPKs regulate many aspects of plant growth and development as well as plant adaptation to biotic and abiotic stresses. StCDPK2 is a single copy gene with eight exons and seven introns that is localized in chromosome 7 of the potato genome. StCDPK2 encodes a CDPK isoform expressed in potato leaves and tuber sprouts. Its expression is positively modulated by light and GA. The recombinant StCDPK2::x6His protein is an active kinase, its kinetic parameters (Km and Vmax) for Syntide-2, the best substrate acceptor (1.75 mM and 0.4 ¦Ìmol/min.mg) and for ATP (18 mM and 0.3 ¦Ìmol/min.mg) were determined. Calcium dependent autophosphorylation of StCDPK2x6His was observed. Maximal activity was detected in the presence of 2mM calcium, however Phosphatidic Acid (PA8 and PA18:1) and DGPP further enhanced kinase activity, while both chlorpromazine and staurosporine inhibited it. Specific antibodies against the recombinant protein were able to recognize a 57 kDa protein in soluble and plasma membrane enriched fractions from leaves of potato plants. StCDPK2::x6His displayed the same chromatographic behavior in DEAE and Phenyl-sepharose columns than the CDPK activity present in leaf extracts from potato plants suggesting that StCDPK2 could be responsible for it. This work was supported by FONCYT, CONICET and UBACYT.