Autophosphorylation of the Dual-Specificity Calcium-Dependent Protein Kinase RcCDPK1 From Castor Oil Seeds
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Phosphoenolpyruvate carboxylase (PEPC) is a tightly-regulated enzyme that plays essential roles in central plant metabolism, particularly in the replenishment of tricarboxylic acid cycle intermediates withdrawn for biosynthesis or N-assimilation. In developing castor oil seeds (COS) an enigmatic ‘bacterial-type’ PEPC (BTPC) isozyme is highly expressed as a catalytic and regulatory subunit of a novel Class-2 PEPC heteromeric complex. During COS development BTPC is subject to in vivo inhibitory phosphorylation at Ser451, a highly conserved target residue occurring within an intrinsically-disordered domain. This phosphorylation event is catalyzed by RcCDPK1, a member of the castor Ca2+-dependent protein kinase (CDPK) family. Ca2+-dependent autokinase activity of CDPKs has been reported and may influence CDPK transphosphorylation activity and substrate accessibility. However, the functions and in vivo occurrence of CDPK autophosphorylation remain poorly understood. A major objective of this thesis was to test the hypothesis that autophosphorylation influences RcCDPK1’s ability to transphosphorylate its BTPC substrate at Ser451. Ca2+-stimulated autokinase activity of heterologously expressed RcCDPK1WT was readily detected by pIMAGOTM phosphoprotein reagent, and multiple pSer, pThr, and pTyr residues were mapped via LC-MS/MS. These included Tyr30 which: (i) occurs at the interface of RcCDPK1’s N-terminal variable and catalytic domains, and (ii) is conserved and also autophosphorylated in RcCDPK1 orthologs AtCPK4 and GmCDPKβ. The influence of Tyr30 autophosphorylation was examined by generating a phosphoablative Y30F mutant (RcCDPK1Y30F). In vitro dephosphorylated RcCDPK1WT exhibited an ~9-fold increase in its rate of Ca2+-dependent BTPC transphosphorylation at Ser451 relative to autophosphorylated RcCDPK1WT. Conversely, RcCDPK1Y30F showed no obvious difference relative to RcCDPK1 WT with respect to its autokinase or BTPC Ser451 transphosphorylation activity. Although global autophosphorylation of RcCDPK1 is highly inhibitory, Tyr30 autophosphorylation appears to have little impact on its kinase activity. Preliminary attempts to detect in vivo RcCDPK1 phosphorylation in developing COS, as well as following via transient expression in Nicotiana benthamiana leaves were conducted by probing immunoblots of clarified extracts with anti-pTyr30 antibodies. Collectively, results of this thesis provide insight into the links between plant carbon metabolism, Ca2+-dependent signaling, and the biological significance of CDPK autophosphorylation.
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