Characterizing the interaction of protein phosphatase RLPH2 with the D group MPKs from Arabidopsis thaliana

Abstract

Protein phosphorylation is a prevalent post-translational modification that is involved in the regulation of every aspect of cell life. This is achieved through the opposing activities of protein kinases and protein phosphatases, which add or remove a phosphoryl group on proteins, respectively. In Arabidopsis thaliana, there are 942 protein kinases and ~150 protein phosphatases, indicating that protein phosphatases have more adaptive specificity towards its substrates as it can interact with multiple regulatory subunits that directs its enzymatic activity. Novel bacteria like phosphatases have been identified in Arabidopsis thaliana, one of which is Rhizobiales-like phosphatase (RLPH). Using phosphoproteomics to compare the protein phosphorylation profile of WT and rlph2 KO lines, the D group mitogen activated protein kinases (MPK) were identified to be possible substrates of RLPH2, specifically regulating the TxY motif of their activation loop (in this case TDY). From in vitro peptide work, this dephosphorylation activity seemed to be dependent on the aspartic acid residue being present in the TxY motif. This thesis confirmed RLPH2 specificity using in vitro dephosphorylation assays of full length proteins of MPK and the importance of that aspartic acid in recruiting RLPH2 to D-group MPKs. We have also identified a potential region in the C-terminus of the D group MPKs that may be involved in substrate recruitment, which we explored using binding assays and confirmed an interaction. Lastly, we observed hyperphosphorylation of the D group MPKs in rlph2 KO lines specifically between days 30-42, which may indicate that it is involved in some aspect of the senescent phenotype that was also observed during that period. This research is important in finding the regulatory function of RLPH2 on MPK9 and can be used in understanding how that might affect downstream substrates of D group MPKs.