| Summary: | Programmed cell death (PCD) is a fundamentally important process delicately coordinated throughout an organism’s life cycle. In plants, PCD is an integral part of development, reproduction, and pathogenesis. Numerous types of proteins are involved in regulation of PCD in plants, like phosphatases, metacaspases, and protein kinases. In tomato resistance to the pathogen Pseudomonas syringae pv. tomato (Pst), a Ser/Thr protein kinase, Adi3 (AvrPto-dependent Pto-interacting protein 3), interacts with the pathogen’s avirulence protein AvrPto and the tomato resistance protein Pto. Adi3 is a member of the AGC protein kinase family, a group known to transmit signals via the secondary messengers cAMP, cGMP, and phospholipids. In an unchallenged system, the master regulator of AGC kinases, Pdk1, activates Adi3. Activation of Adi3 enables nuclear localization and cell death suppression – all of which is prevented when challenged by Pst.
A BLAST_p search of the Arabidopsis thaliania genome with the amino acid sequence of Adi3 identified a 67% identical match, AGC1-3. Like Adi3, AGC1-3 at its activation-loop serine and another site, by Pdk1. With N-terminal deletions of AGC1-3, Pdk1 was found to phosphorylate AGC1-3 at two serines – one serine conserved among all Arabidopsis AGC kinases, the other a serine on the N-terminus of the kinase domain. The non-activation loop serine in AGC1-3, Ser269, is conserved at Ser212, in Adi3. Phosphorylation at Ser212 does not impact auto-catalytic activity of Adi3. However it does enhance trans-catalytic activity. Analysis of AGC1-4 and AGC1-7, two proteins closely related to AGC1-3, reveals that phosphorylation of non-activation loop residues by Pdk1 is not restricted to AGC1-3 and Adi3. Functional analysis of AGC1-3 in Arabidopsis protoplasts revealed that like Adi3, nuclear localization and activation-loop phosphorylation are essential for cell viability.
In an effort to elucidate a signaling network controlled by AGC1-3, the KiC (Kinase-client) assay was employed. In the KiC assay, a 2,100-member peptide library was assayed against AGC1-3 and the constitutively active mutant AGC1-3^(S596D). By MS analysis, AGC1-3 and AGC1-3(S596D) phosphorylated 26 and 19 substrate peptides, respectively. Substrate peptides were mapped to proteins involved in central metabolism, transcription, and protein metabolism.
The work presented in this dissertation provides conclusive evidence that Pdk1 phosphorylates AGC1-3 and Adi3 at a non-activation loop residue. The work also supports AGC1-3 as the Arabidopsis homologue of Adi3 and presents novel phosphorylation data of potential AGC1-3 substrates.
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