Autophosphorylation and subcellular localization dynamics of a salt- and water deficit-induced calcium-dependent protein kinase from ice plant.
A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) 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 (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K0.5 = 0.15 microm). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6x His-tagged McCPK1.
Chehab
E Wassim
EW
Department of Biochemistry/MS200, University of Nevada, Reno, Nevada 89557-0014, USA.
Patharkar
O Rahul
OR
Hegeman
Adrian D
AD
Taybi
Tahar
T
Cushman
John C
JC
D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D003890:Desiccation; D019684:Magnoliopsida / Q000201:enzymology*; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010766:Phosphorylation; D012965:Sodium Chloride / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D014162:Transfection
D017931:DNA Primers; D012965:Sodium Chloride; D017868:Cyclic AMP-Dependent Protein Kinases