Biochemical characterization of two group III-members of the Solanum tuberosum calcium dependent protein kinase (CDPK) family

Abstract

As sessile organisms, plants have developed complex signal transduction pathways to cope with the environmental fluctuations to which they are exposed during their life cycle. Calcium is a ubiquitous messenger involved in the signaling of environmental stimuli, whose oscillations are decoded by different sensors. Calcium-dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. In this work, isoforms StCDPK22 and StCDPK24 from the CDPK family of Solanum tuberosum were characterized. Both isoforms belong to Group III, and contain only three EF-hand sites in their calmodulin-like regulatory domain (CLD) instead of four sites like all other members of the family. StCDPK22 encodes a 59.4 kDa protein (524 aa; pI 7.6) while StCDPK24 encodes a 60 kDa protein (532 aa; pI 6.12), both with an N-terminal variable domain (NTV) which presents myristoylation and palmitoylation consensus sites. StCDPK22 gene (circa 4 kb) is localized in chromosome 10, while StCDPK24 gene (circa 5 kb) is localized in chromosome 11. Both isoforms share the eight exons and seven introns structure and have 80% identity and 89% similarity at the protein level. According to RNAseq data from the potato genome StCDPK22 and StCDPK24 expression is ubiquitous, however StCDPK22 transcripts are much more abundant in stolons, leaves, shoots, tubers and roots. The recombinant 6xHis:StCDPK22 and 6xHis:StCDPK24 were obtained and kinase assays were performed. Although both isoforms are active kinases, StCDPK24 activity is ten-fold higher. Kinetic parameters were determined for StCDPK24. We observed that this kinase can use either Mg2+ or Mn2+ as cofactor and full activity was obtained with 1µM calcium, however kinase activity was also observed in the presence of 10 mM EGTA suggesting that calcium may not be a prerequisite for activity. However, in vitro assays indicate that calcium is required for autophosphorylation.