A Heat-Sensitive Arabidopsis thalianaKinase Substitutes for Human p70^s6k Function In Vivo

Abstract

In mammalian cells, mitogen-induced phosphorylation of ribosomal protein S6 by p70 s6k has been implicated in the selective translational upregulation of 5TOP mRNAs. We demonstrate here that the homologous Arabidopsis thaliana protein, AtS6k2, ectopically expressed in human 293 cells or isolated from plant cells, phosphorylates specifically mammalian and plant S6 at 25°C but not at 37°C. When Arabidopsis suspension culture cells are shifted from 25 to 37°C, the kinase becomes rapidly inactivated, consistent with the observation that heat shock abrogates S6 phosphorylation in plants. Treatment with potato acid phosphatase reduced the specific activity of immunoprecipitated AtS6k2 threefold, an effect which was blocked in the presence of 4-nitrophenyl phosphate. In quiescent mammalian cells, AtS6k2 is activated by serum stimulation, a response which is abolished by the fungal metabolite wortmannin but is resistant to rapamycin. Treatment of mammalian cells with rapamycin abolishes in vivo S6 phosphorylation by p70 s6k ; however, ectopic expression of AtS6k2 rescues the rapamycin block. Collectively, the data demonstrate that AtS6k2 is the functional plant homolog of mammalian p70 s6k and identify a new signalling pathway in plants.

High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome

“…The last explanation is probably valid for the ribosomal protein S6, which could not be identified in our ribosome preparation, although it is a sufficiently large protein (28 kDa). This protein is known to be phosphorylated at multiple sites in eukaryotes (Wettenhall and Morgan, 1984), a finding that was recently confirmed for a corresponding S6 protein in A. thaliana (Turck et al, 1998). The second group of proteins identified from the 80S fraction (Table 1) contained 32 protein spots that were associated with the plastid 70S ribosome (MIPS database), whereas the third group (Table 1) comprised 43 protein spots of Number of independent spots identified for each protein family.…”

Stress and mTORture signaling

“…Vries et al (1997) did not observe a decrease in S6K activity upon heat stress despite a decrease in 4EBP-1 phosphorylation, suggesting that the effects on S6K are cell typedependent or that an unknown phosphatase might dephosphorylate 4EBP-1. Furthermore, heat shock in plants inactivates AtS6K and induce dephosphorylation of S6 (Scharf and Nover, 1982;Turck et al, 1998). On the other hand, PKB1 has been reported to become activated within minutes upon heat shock, presumably reflecting the pro-survival effects of Akt/PKB (Konishi et al, 1997).…”

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

“…In this study we identify the A. thaliana S6 kinase AtS6k2 as a potential substrate for AtPDK1. As AtS6k2 can phosphorylate an A. thaliana ribosomal S6 protein [8], our findings suggest that the PDK1-S6K pathway, which regulates S6 and cell growth in animal systems [2], is also present in A. thaliana. Interestingly, in preliminary studies, we could detect a protein kinase able to phosphorylate AtS6k2 in an A. thaliana ribosomal preparation.…”

“…These protein kinases, whose gene expression is enhanced in response to salt and low temperature stress, share 87% amino acid sequence similarity with the human S6K [7]. In addition, three phosphorylation sites that are critical for mammalian S6K activation, Thr-229, Ser-371 and Thr-389 are conserved in AtS6k1 and AtS6k2 [8]. In mammalian cells, the phosphorylation and activation of S6K by PDK1 promotes phosphorylation of the 40S ribosomal protein S6, which in turn controls cell growth and proliferation via the consequential regulation of 5´-terminal oligopyrimidine tract mRNA translation [2].…”

“…In mammalian cells, the phosphorylation and activation of S6K by PDK1 promotes phosphorylation of the 40S ribosomal protein S6, which in turn controls cell growth and proliferation via the consequential regulation of 5´-terminal oligopyrimidine tract mRNA translation [2]. Interestingly, one of the A. thaliana S6K homologues, AtS6k2, has been shown to phosphorylate a plant ribosomal S6 protein in vivo, a phosphorylation sensitive to wortmannin and LY294002, PtdIns 3-kinase inhibitors that suppress mammalian S6K activity [8][9][10]. Also in maize (Zea mays), an S6K homologue, ZmS6K, phosphorylated a ribosomal S6 protein in vivo [11].…”