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ENVIRONMENTAL STRESS AND ADAPTATION

Regulated Phosphorylation of 40S Ribosomal Protein S6 in Root Tips of Maize¹

Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, California 92521–0124

Ribosomal protein S6 (RPS6) is located in the mRNA binding site of the 40S subunit of cytosolic ribosomes. Two maize (Zea mays) rps6 genes were identified that encode polypeptides (30 kD, 11.4 pI) with strong primary amino acid sequence and predicted secondary structure similarity to RPS6 of other eukaryotes. Maize RPS6 was analyzed by the use of two-dimensional gel electrophoresis systems, in vivo labeling with [³²P]P_i and immunological detection. Nine RPS6 isoforms were resolved in a two-dimensional basic-urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry performed on trypsin-digested isoforms identified four serine (Ser) and one threonine (Thr) residue in the carboxy-terminal region as phosphorylation sites (RRS₂₃₈KLS₂₄₁AAAKAS₂₄₇AAT₂₅₀S₂₅₁A-COOH). Heterogeneity in RPS6 phosphorylation was a consequence of the presence of zero to five phosphorylated residues. Phosphorylated isoforms fell into two groups characterized by (a) sequential phosphorylation of Ser-238 and Ser-241 and (b) the absence of phospho-Ser-238 and presence of phospho-Ser-241. The accumulation of hyper-phosphorylated isoforms with phospho-Ser-238 was reduced in response to oxygen deprivation and heat shock, whereas accumulation of these isoforms was elevated by cold stress. Salt and osmotic stress had no reproducible effect on RPS6 phosphorylation. The reduction in hyper-phosphorylated isoforms under oxygen deprivation was blocked by okadaic acid, a Ser/Thr phosphatase inhibitor. By contrast, the recovery of hyper-phosphorylated isoforms upon re-oxygenation was blocked by LY-294002, an inhibitor of phosphatidylinositol 3-kinases. Thus, differential activity of phosphatase(s) and kinase(s) determine complex heterogeneity in RPS6 phosphorylation.

² Present address: Affymetrix Inc., 6550 Vallejo Street, Suite 100, Emeryville, CA 95608.

³ These authors contributed equally to this paper.

^* Corresponding author; e-mail serres{at}mail.ucr.edu; fax 909–787–4437.

Received March 3, 2003; returned for revision March 27, 2003; accepted April 2, 2003.

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