A minimal serine/threonine protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in crassulacean acid metabolism-induced leaves of the common ice plant.
Plant phosphoenolpyruvate carboxylase (PEPc) activity and allosteric properties are regulated by PEPc kinase (PPcK) through reversible phosphorylation of a specific serine (Ser) residue near the N terminus. We report the molecular cloning of PPcK from the facultative Crassulacean acid metabolism (CAM) common ice plant (Mesembryanthemum crystallinum), using a protein-kinase-targeted differential display reverse transcriptase-polymerase chain reaction approach. M. crystallinum PPcK encodes a minimal, Ca(2+)-independent Ser/threonine protein kinase that is most closely related to calcium-dependent protein kinases, yet lacks both the calmodulin-like and auto-inhibitory domains typical of plant calcium-dependent protein kinase. In the common ice plant PPcK belongs to a small gene family containing two members. McPPcK transcript accumulation is controlled by a circadian oscillator in a light-dependent manner. McPPcK encodes a 31.8-kD polypeptide (279 amino acids), making it among the smallest protein kinases characterized to date. Initial biochemical analysis of the purified, recombinant McPPcK gene product documented that this protein kinase specifically phosphorylates PEPc from CAM and C(4) species at a single, N-terminal Ser (threonine) residue but fails to phosphorylate mutated forms of C(4) PEPc in which this specific site has been changed to tyrosine or aspartate. McPPcK activity was specific for PEPc, Ca(2+)-insensitive, and displayed an alkaline pH optimum. Furthermore, recombinant McPPcK was shown to reverse the sensitivity of PEPc activity to L-malate inhibition in CAM-leaf extracts prepared during the day, but not at night, documenting that PPcK contributes to the circadian regulation of photosynthetic carbon flux in CAM plants.
Taybi
T
T
Department of Biochemistry and Molecular Biology, 147 Noble Research Center, Oklahoma State University, Stillwater, Oklahoma 74078-3035, USA.
Patil
S
S
Chollet
R
R
Cushman
J C
JC
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002245:Carbon Dioxide / Q000378:metabolism; D002940:Circadian Rhythm; D003001:Cloning, Molecular; D019684:Magnoliopsida / Q000201:enzymology / Q000235:genetics*; D008293:Malates / Q000378:metabolism; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D008293:Malates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002245:Carbon Dioxide; C030298:malic acid; D017346:Protein Serine-Threonine Kinases; D010730:Phosphoenolpyruvate Carboxylase