MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF StCDPK2

GIAMMARIA V; GRANDELLIS C; VILLASUSO AL; ULLOA RM

St. Louis, MO – USA

Congreso; International Plant Molecular Biology (IPMB); 2009

IPMB

Abstract Body 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 and are involved in hormone signalling. StCDPK2 is a single copy gene with eight exons and seven introns localized in chromosome 7 of the potato genome. StCDPK2 encodes a 57 kDa CDPK isoform expressed in potato leaves and tuber sprouts. Antibodies against StCDPK2::x6His recombinant protein revealed a 57 kDa protein in soluble and plasma membrane enriched fractions from potato leaves. StCDPK2::x6His displayed the same chromatographic behavior in DEAE and Phenyl-sepharose columns than CDPK activity/ies present in leaves´ extracts. The recombinant protein had a Km of 1.75 µM for syntide-2 and of 18 µM for ATP and a Vmax of 0.4 µmol/min.mg for both. Maximal activity was detected in the presence of 2 µM calcium, however PA and DGPP further enhanced kinase activity, while both chlorpromazine and staurosporine inhibited it. Calcium dependent autophosphorylation of StCDPK2::x6His was detected. StCDPK2 expression is positively modulated by light and GA. GA also modified the activity of lipid kinases in potato plants grown in vitro resulting in increased levels of PIP2, PA and DGPP. We propose that phospholipids could participate in GA signaling modulating StCDPK2 activity.