Investigating the role of Ricinus communis Ca2+-dependent protein kinase-1 (RcCDPK1) autophosphorylation and the regulatory networks of RcCDPK1 and its Arabidopsis ortholog AtCPK4

Abstract

Castor (Ricinus communis) oil seeds (COS) have become an important model for studies of oil seed metabolism owing to their unusually high accumulation of storage triacylglycerides. R. communis Ca2+-dependent protein kinase-1 (RcCDPK1) catalyzes in vivo inhibitory phosphorylation of bacterial-type phosphoenolpyruvate carboxylase (PEPC) at Ser451 in developing COS. Bacterial-type PEPC (BTPC) functions as a regulatory and catalytic subunit of an unusual, mitochondrial surface-associated Class-2 PEPC complex, hypothesized to play crucial roles in controlling anaplerotic photosynthate partitioning to storage oils while recycling respiratory CO2 in developing COS. This thesis aimed to address how autophosphorylation influences RcCDPK1’s transphosphorylation of BTPC at Ser451, as well as to expand the kinase’s regulatory networks in conjunction with its close Arabidopsis ortholog AtCPK4. LC-MS/MS of fully autophosphorylated RcCDPK1 detected 42 phosphorylated Ser, Thr, or Tyr residues. Prior autophosphorylation markedly attenuated the ability of RcCDPK1 to transphosphorylate its BTPC substrate at Ser451. Tyr30 was identified as an autophosphorylation site of particular interest as it occurs at the junction of RcCDPK1’s N-terminal variable and catalytic domains and is widely conserved in plant and protist CDPKs. Analysis of a RcCDPK1Y30F mutant indicated that Tyr30 autophosphorylation primes RcCDPK1 for transphosphorylating BTPC at Ser451. To expand RcCDPK1’s and AtCPK4’s known regulatory networks a combination of biochemical and functional genetic approaches were integrated. RcCDPK1 and AtCPK4 effectively phosphorylated Arabidopsis abscisic acid (ABA) transcription factor-4 (AtABF4) and its closest castor ortholog, RcTRAB1. LC-MS/MS revealed several conserved AtABF4 and RcTRAB1 Ser or Thr residues were targeted by both CDPK orthologs. A medium-throughput screen known as a Kinase-Client (KiC) assay was also used to identify additional RcCDPK1/AtCPK4 substrates. These included Ras-group-related leucine-rich repeat protein-9 (AtPIRL9), as well as the E3-ubiquitin ligase AtATL6. The overall results of this thesis link RcCDPK1/AtCPK4 to novel regulatory pathways involved in ABA and immune signaling, and carbon/nitrogen interactions, while creating interesting testable hypotheses for future research. Collectively, this project uncovered novel aspects of RcCDPK1 and AtCPK4 regulation that shed light on autophosphorylation in CDPKs, as well as identifying putative conserved substrates that may be regulated by these critical kinases.