Phospho-Proteomics Identifies D-Group MAP Kinases as Substrates of the Arabidopsis Tyrosine Phosphatase RLPH2

Despite being one of the few bona fide plant tyrosine phosphatases, the Arabidopsis thaliana Rhizobiales-like phosphatase 2 (RLPH2) has no known substrates. Utilizing phospho-proteomics, we identified the activation loop phospho-tyrosine of several A. thaliana D-group mitogen-activated protein kinases (MPKs) as potential RLPH2 substrates. All Arabidopsis D-group MPKs possess a TDY activation loop phosphorylation motif, whereas other MPKs (Groups A, B, and C) contain a TEY motif. Our findings reveal that RLPH2 has a strong preference for aspartate (D) in the TXY motif, providing specificity for RLPH2 to exclusively target and dephosphorylate the D-group MPKs. Additionally, D-group MPKs contain a unique activation loop insertion that conforms to a protein phosphatase one (PP1) binding motif, with findings presented here confirming Arabidopsis PP1 phosphatases dock at this site. Intriguingly, only D-group MPKs among all identified Arabidopsis protein kinases possess this PP1 recruiting motif. Using multiple RLPH2-deficient plant lines, we demonstrate that RLPH2 represses seed dormancy release. Overall, this work highlights the power of phospho-proteomics in identifying substrates of this novel plant tyrosine phosphatase while also revealing new complexities in the interactions between MPK activation loops and multiple phospho-mediated cell signaling events.