First published online July 24, 2003; 10.1104/pp.103.022749
Plant Physiology 132:2086-2097 (2003)
© 2003 American Society of Plant Biologists
ENVIRONMENTAL STRESS AND ADAPTATION
Regulated Phosphorylation of 40S Ribosomal Protein S6 in Root Tips of Maize1
Alan J. Williams2,3,
Joanna Werner-Fraczek3,
Ing-Feng Chang3 and
Julia Bailey-Serres*
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]Pi 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.
1 This work was supported by the U.S. Department of Agriculture/National
Research Initiative Competitive Grants Program (grant no.
97–35100–4191 to J.B.S.). A.J.W. was supported by a National
Science Foundation Graduate Research Traineeship (grant no.
DGE–9355042), and I.-F.C. was supported by the Ministry of Education,
Republic of China, Taiwan.
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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