Plant S6 kinases do not require hydrophobic motif phosphorylation for activity in yeast lacking Ypk3

Abstract

The ribosomal protein S6 kinases (S6K) are among the major substrates and crucial effectors of the target of rapamycin (TOR) kinase, which is an evolutionarily conserved regulator of cell growth and proliferation. Recent research indicates that yeast Ypk3 is an ortholog of mammalian S6Ks. Here, we find that plant S6Ks restore ribosomal protein S6 phosphorylation in a rapamycin-sensitive manner in yeast cells lacking Ypk3. However, phosphorylation of a hydrophobic motif, which is mediated through TOR signaling and essential for mammalian S6K activity, is not detected in plant S6Ks. Furthermore, deletion of the N-terminal region of rice S6Ks shows phosphorylation of the hydrophobic motif and reduced rapamycin sensitivity. Our findings suggest a mechanism of plant S6K activation distinct from that of mammalian S6Ks.

The activation mechanism of plant S6 kinase (S6K), a substrate of TOR kinase, is different from that of mammalian S6K

“…B), we tested the activity of OsS6K with double mutation at the T‐loop and HM [OsS6K1 T‐loop/HM (S307A/S465E)] or TM and HM [OsS6K1 TM/HM (S449A/S465E)] sites. The activity of OsS6K1 was lost when the T‐loop or TM site was replaced by Ala . However, unexpectedly, when the HM site was replaced by Glu, the mutation at the T‐loop or TM site did not reduce the activity (Fig.…”

“…The observation that the HM site in the full‐length plant S6Ks was not phosphorylated, except in SlS6K (Fig. ), and that plant S6K activation did not require HM phosphorylation in ypk3Δ cells raised a question as to whether the mammalian S6Ks are also activated without HM site phosphorylation in ypk3Δ cells. Therefore, we examined whether the HM site in the full‐length mammalian S6Ks was phosphorylated in ypk3Δ cells.…”

“…Furthermore, sitedirected mutagenesis at the HM site of OsS6K1 did not affect its activity. Overall, we concluded that the phosphorylation of the HM site is not required for the activity of plant S6Ks [32]. However, the HM site was shown to be phosphorylated through TOR signaling in plant cells and it is considered that TOR signaling activates S6Ks in plants as it does in animal cells [6,7].…”

“…1A). Interestingly, HM phosphorylation was detected in ypk3D cells expressing full-length SlS6K, although it was not detected in cells expressing the fulllength OsS6Ks, AtS6K1 [32], and GmS6K (Fig. 1A).…”

“…In a previous study, we analyzed the mechanism of activation of plant S6Ks using yeast cells lacking Ypk3, which was recently identified to be a functional ortholog of S6Ks . We showed that the HM sites of S6Ks from rice (OsS6K1 and OsS6K2) and Arabidopsis (AtS6K1 and AtS6K2) were not phosphorylated in the ypk3Δ cells although, except for AtS6K2, they phosphorylated Rps6.…”