| A salinity- and dehydration-stress responsive calcium-dependent protein kinase (CPK) was isolated from the common ice plant, Mesembryanthemum crystallinum (McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane localization as determined by transient expression of green fluorescent protein (GFP) fusions in microprojectile bombarded cells. Complete removal of the N-terminal domain (AA1-70) was required to completely abolish plasma membrane localization suggesting that both acylation and the N-terminal domain itself play important roles in membrane association of the kinase. The recombinant, E. coli expressed, full-length McCPKI protein was catalytically active in a calcium-dependent manner (Vmax 0.5 activity at 0.15 μM). Auto-phosphorylation of recombinant McCPK1 was observed on two different serine residues identified as Ser-62 and Ser-420. Mutagenesis of each of these sites reduced autophosphorylation activity 21% and 70%, respectively, relative to the wildtype kinase. Lack of autophosphorylation at Ser-420 decreases kinase activity against McCSP1, a bone fide McCPK1 substrate, by 20.5% relative to McCPK1 suggesting that auto phosphorylation at this site is important for enzyme activity. A potential role in salinity and dehydration stress responses is corroborated by the dynamic and reversible change in subcellular localization that McCPK1 undergoes from the plasma membrane to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton as determined by transient expression of green fluorescent protein (GFP) fusions in microprojectile bombarded cells and confirmed by subcellular fractionation and western blot analysis of 6xHis-tagged McCPK1. To better understand the functional role of McCPK1 we used yeast-two hybrid interaction screening to identify substrates and McCPK1-interacting proteins. This collection of interacting proteins includes substrates and proteins that likely mediate McCPK1 subcellular location changes observed during environmental stress responses. One putative interactor, McCAP2 (M. crystallinum CPK1 Adapter Protein 2), interacts with McCPK1 in the yeast two-hybrid system and in vitro, but does not appear to be a substrate. (Abstract shortened by UMI.)... |
