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

Abstract

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 [ 32 P]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 (RRS 238 KLS 241 AAAKAS 247 AAT 250 S 251 A-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.Ribosomal protein S6 (RPS6) assembles onto the 45S rRNA precursor in the nucleolus and is located at the small head region of the cytosolic 40S ribosomal subunit (Nygärd and Nilsson, 1990). RPS6 can be directly cross-linked to mRNA, tRNA, and initiation factors, confirming its location in a region involved in the initiation of translation. RPS6, a 30-kD protein, is the major phosphoprotein of eukaryotic ribosomes. In mammals and fruitfly (Drosophila melanogaster), it is sequentially phosphorylated at five carboxyterminal Ser residues (Krieg et al., 1988;Fumagalli and Thomas, 2000;Radimerski et al., 2000). RPS6 phosphorylation in the nucleolus occurs during active ribosome biogenesis, whereas RPS6 phosphorylation in the cytoplasm is thought to promote the selective translation of a subset of transcripts, primarily ribosomal protein and elongation factor mRNAs with a 5Ј-terminal oligopyrimidine tract (5ЈTOP; Reinhard et al., 1994;Jefferies et al., 1997;Fumagalli and Thomas, 2000). Ribosomes with the highest degree of RPS6 phosphorylation appear to have a selective advantage in mobilization into polysomes (Jefferies and Meyuhas and Hornstein, 2000). RPS6 phosphorylation might not be sufficient to re...

Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation

“…Phosphorylation of ribosomal protein S6 and p70 S6 kinase has also been reported in maize (Williams et al, 2003;de la Cruz et al, 2004). For ribosomal protein S6, phosphorylated isoforms in Ser-238 and Ser-241 have been described (Williams et al, 2003), while for p70 S6 kinase, residue Thr-389 was found to be phosphorylated in correlation with its activation (de la Cruz et al, 2004).…”

“…Phosphorylation of S6 was described previously in plant ribosomes. In response to several stress treatments, hyperphosphorylated isoforms of S6 accumulate in maize root tips (Williams et al, 2003). Of particular interest are the Ser-238 and Ser-241 residues, because these are differentially phosphorylated after specific environmental changes.…”

“…A total of 0.5 mg of purified ribosomes, quantified as described in Williams et al (2003), were incubated overnight at 37°C with RNase T1 and RNase V1 (4,000 units mL 21 each; Ambion, Austin, TX) plus RNase A (4,000 mg mL 21 ; Qiagen, Valencia, CA) in a buffer containing 10 mM Tris-HCl, pH 8.1, 0.5 mM EDTA, and 8 M UREA, and Complete protease inhibitor cocktail (Roche, Indianapolis). The next day, 8,000 units mL 21 each of RNase T1 and V1 plus 8,000 mg of mL 21 RNase A were added followed by 2 h incubation.…”

“…Furthermore, Nomura et al (2001) confirmed that UV-B induces activation of ribosomal p70 S6 kinase in cultured mouse epidermal cells. Recently, the accumulation of hyper-phosphorylated isoforms of S6 were described in root tips of maize; four Ser and one Thr residue can be phosphorylated, and the extent of modification is modulated by environmental conditions (Williams et al, 2003). Consequently, a similar pathway as well as other mechanisms could exist in plant cells that mediate selective translation of ribosomal proteins and translation factors after UV-B damage.…”

RNA Interference-Mediated Repression of S6 Kinase 1 Impairs Root Nodule Development in Soybean

“…A carboxy-terminal region of RPS6 has been shown to be phosphorylated by S6K1 in animal cells (Krieg et al, 1988;Radimerski et al, 2000) as well as in plant (Williams et al, 2003). In addition, the GFP-S6K1 was shown to be able to phosphorylate a carboxy-terminal region of Arabidopsis RPS6 (a.a. 150-249; AtRPS6-CT) (Mahfouz et al, 2006).…”