Two Solanum tuberosum CDPK isoforms display different kinetic parameters

GIAMMARIA V; GRANDELLIS C; ULLOA RM

Tucumán

Congreso; XLV reunión de SAIB; 2009

SAIB

Two Solanum tuberosum CDPK isoforms display different kinetic parameters Giammaria Verónica, Grandellis Carolina, Ulloa Rita María Instituto de Ingeniería Genética y Biología Molecular (INGEBI-CONICET-UBA)     Changes in calcium cytosolic concentrations [Ca2+]i precede plant cells responses during growth, development and stress conditions. Distinctive calcium signatures are generated and the way Ca2+ signals are encrypted and decoded provides signaling specificity. Calcium-dependent protein kinases, CDPKs, are key calcium sensor in plants. In potato, five isoforms have been characterized. Isoforms StCDPK2 and StCDPK3 share 80% identity in amino acid sequence. Expression analysis revealed that both are expressed in leaves, stems, stolons and roots of potato plants but while StCDPK2 mRNA is high in leaves and is induced in response to osmotic stress, StCDPK3 expression is low in all tissues and is induced in early stolons.  The recombinant StCDPK2::His6x and StCDPK3::His6x proteins were obtained and their kinetic parameters analyzed. Both isoforms have a similar Km for ATP (13-18 mM) and syntide-2 was the preferred substrate, but their Km and Vmax for syntide differed: StCDPK2 (1,75 mM; 400 nmoles.min-1.mg-1), StCDPK3 (35mM; 9.5 nmoles.min-1.mg-1). In addition, StCDPK2 reached maximal activity at 2mM Ca2+ while StCDPK3 needed 50mM. Autophosphorylation of StCDPK2 was dependent on the presence of Ca2+ while StCDPK3 could autophosphorylate in the presence of 5 mM EGTA. Our results support the hypothesis that each isoform may have a distinct role in calcium signal transduction.