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9
Ruvinsky, I., Sharon, N., Lerer, T., Cohen, H., Stolovich-Rain, M., Nir, T., Dor, Y., Zisman,
P. and Meyuhas, O. (2005) Ribosomal protein S6 phosphorylation is a determinant of
cell size and glucose homeostasis. Genes Dev. 19, 2199–2211
Shahbazian, D., Roux, P. P., Mieulet, V., Cohen, M. S., Raught, B., Taunton, J., Hershey, J.
W., Blenis, J., Pende, M. and Sonenberg, N. (2006) The mTOR/PI3K and MAPK pathways
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Wang, X., Li, W., Williams, M., Terada, N., Alessi, D. R. and Proud, C. G. (2001)
Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J. 20,
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Dibble, C. C., Asara, J. M. and Manning, B. D. (2009) Characterization of Rictor
phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1. Mol. Cell.
Biol. 29, 5657–5670
Treins, C., Warne, P. H., Magnuson, M. A., Pende, M. and Downward, J. (2010) Rictor is
a novel target of p70 S6 kinase-1. Oncogene 29, 1003–1016
to determine the specific roles of isoforms of kinases. Thus far
only SKAR (S6K1 Aly/REF-like target) [39] and Rictor [9] have
been shown to be specific S6K1 substrates. No specific substrates
of S6K2 have yet been described. Hence PF-4708671 could help
to elucidate isoform-specific functions of S6K1 and S6K2. For
example, treatment of S6K2−/− mouse embryonic fibroblasts with
PF-4706871 would allow the identification of proteins that are
specifically phosphorylated by S6K1.
Previous studies characterizing S6K1-knockout mice revealed
significant residual phosphorylation of ribosomal S6 protein,
which suggested that S6K2 may also phosphorylate ribosomal S6
protein [3,38]. However, our data demonstrate that concentrations
of PF-4708671 that do not inhibit S6K2, ablate phosphorylation of
ribosomal S6 protein at Ser235, Ser236, Ser240 and Ser244 within
30 min. This suggests that S6K1 is the predominant kinase that
phosphorylates ribosomal S6 protein, at least in HEK-293 cells
that were employed in the present study. It is likely that in the
S6K1-knockout mice compensatory pathways have evolved to
enable ribosomal S6 protein to become phosphorylated. Indeed
mRNA levels of S6K2 were markedly elevated in all tissues of
S6K1-knockout mice [3].
Taken together with the recent advances in the discovery of
novel specific kinase inhibitors such as Akti-1/2 [33], or MK-
2206 [40] (Akt inhibitors) and BI-D1870 (RSK inhibitor) [31],
the identification of PF-4708671 should help to yield further
information on the specific cellular roles of AGC kinases. In
addition the development of this S6K1 inhibitor, together with
the recently solved structure of the inactive conformation of S6K1
[41], could act as a basis upon which to develop improved S6K1
kinase inhibitors, which might one day contribute to the treatment
of human diseases, including cancer and insulin-resistance.
10 Julien, L. A., Carriere, A., Moreau, J. and Roux, P. P. (2010) mTORC1-activated S6K1
phosphorylates Rictor on threonine 1135 and regulates mTORC2 signaling. Mol. Cell.
Biol. 30, 908–921
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Tempst, P. and Sabatini, D. M. (2002) mTOR interacts with raptor to form a
nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175
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AUTHOR CONTRIBUTION
Laura Pearce performed all experiments shown. Gordon Alton, Daniel Richter, John
Kath, Laura Lingardo, Justin Chapman and Catherine Hwang elaborated PF-4708671 and
demonstrated that this drug inhibited S6K1. Laura Pearce, Gordon Alton and Dario Alessi
planned experiments and analysed the data. Laura Pearce and Dario Alessi wrote the paper.
18 Selman, C., Tullet, J. M., Wieser, D., Irvine, E., Lingard, S. J., Choudhury, A. I., Claret,
M., Al-Qassab, H., Carmignac, D., Ramadani, F. et al. (2009) Ribosomal protein S6
kinase 1 signaling regulates mammalian life span. Science 326, 140–144
19 Harrison, D. E., Strong, R., Sharp, Z. D., Nelson, J. F., Astle, C. M., Flurkey, K., Nadon,
N. L., Wilkinson, J. E., Frenkel, K., Carter, C. S. et al. (2009) Rapamycin fed late in life
extends lifespan in genetically heterogeneous mice. Nature 460, 392–395
20 Brunn, G. J., Hudson, C. C., Sekulic, A., Williams, J. M., Hosoi, H., Houghton, P. J.,
Lawrence, Jr, J. C. and Abraham, R. T. (1997) Phosphorylation of the translational
repressor PHAS-I by the mammalian target of rapamycin. Science 277, 99–101
21 Jung, C. H., Jun, C. B., Ro, S. H., Kim, Y. M., Otto, N. M., Cao, J., Kundu, M. and Kim,
D. H. (2009) ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy
machinery. Mol. Biol. Cell 20, 1992–2003
ACKNOWLEDGEMENTS
screen.mrc.ac.uk) for undertaking the kinase specificity screening, the Sequencing Service
(School of Life Sciences, University of Dundee, Dundee, Scotland, U.K.) for DNA
sequencing, and the protein production and antibody purification teams [Division of
Signal Transduction Therapy (DSTT), University of Dundee, Dundee, Scotland, U.K.],
co-ordinated by Hilary McLauchlan and James Hastie, for expression and purification of
antibodies.
22 Hosokawa, N., Hara, T., Kaizuka, T., Kishi, C., Takamura, A., Miura, Y., Iemura, S.,
Natsume, T., Takehana, K., Yamada, N. et al. (2009) Nutrient-dependent mTORC1
association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol.
Cell 20, 1981–1991
23 Ganley, I. G., Lam du, H., Wang, J., Ding, X., Chen, S. and Jiang, X. (2009)
ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy.
J. Biol. Chem. 284, 12297–12305
24 Collins, B. J., Deak, M., Murray-Tait, V., Storey, K. G. and Alessi, D. R. (2005) In vivo
role of the phosphate groove of PDK1 defined by knockin mutation. J. Cell Sci. 118,
5023–5034
25 Durocher, Y., Perret, S. and Kamen, A. (2002) High-level and high-throughput
recombinant protein production by transient transfection of suspension-growing human
293-EBNA1 cells. Nucleic Acids Res. 30, E9
26 Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C. J., McLauchlan, H., Klevernic, I.,
Arthur, J. S., Alessi, D. R. and Cohen, P. (2007) The selectivity of protein kinase
inhibitors: a further update. Biochem. J. 408, 297–315
27 Kuzmic, P., Elrod, K. C., Cregar, L. M., Sideris, S., Rai, R. and Janc, J. W. (2000)
High-throughput screening of enzyme inhibitors: simultaneous determination of
tight-binding inhibition constants and enzyme concentration. Anal. Biochem. 286,
45–50
FUNDING
L.R.P. is funded by a Medical Research Council UK Studentship. We thank the Medical
Research Council, and the pharmaceutical companies supporting the Division of Signal
Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck-
Serono and Pfizer) for financial support.
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The Authors Journal compilation 2010 Biochemical Society