Abstract 1639 Chloroplast protein phosphatase SLP1 counters protein kinase, casein kinase 2

The serine/threonine protein phosphatase SLP1 is targeted to the higher plant chloroplast and has no known substrates. We have performed quantitative phosphoproteomics on wildtype, SLP1 knockout and over-expressor lines to uncover substrates of the enzyme. Analysis of Arabidopsis photosynthetic tissue identified over 18,000 phosphorylation sites on proteins found in multiple cellular compartments. We identified >200 phosphorylation sites on ∼100 proteins whose phospho-status increased up to 112-fold in the absence of the protein phosphatase SLP1 and remarkably all are known (or predicted) to localize to the chloroplast where SLP1 resides. Motif analysis of putative substrates revealed a dominant acidic motif surrounding phospho-sites that matched casein kinase 2 (CK2) substrates, of which a single isoform, CK2a4, resides in the chloroplast. Dephosphorylation assays with substrate phospho-peptides confirmed SLP1s preference for an acidic motif supporting the idea that SLP1 counters CK2 in the chloroplast. SLP1 and CK2 substrates include proteins involved in the Calvin cycle, the photosynthetic apparatus, starch, fatty acid and RNA metabolism and membrane transporters linked with carbon and fatty acid export. This is the first study utilizing quantitative mass spectrometry to identify plant protein phosphatase substrates and demonstrates the power of this approach. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).