12. T. Pfeiffer, S. Schuster, S. Bonhoeffer,
C. V. Dang, G. L. Semenza,
Trends Biochem. Sci.
14. Construction and assay of the S6K1-GST construct
were described previously (
). For the construction
of the GST-
C-S6K1 and D3E,K100Q-S6K1-GST plas-
mids, similar cloning strategies were used.
15. Total RNA was isolated from HEK293 cells as de-
). Deacylation of tRNA was accomplished
by mild alkaline hydrolysis in 0.1 M tris-HCl (pH 8.0)
at 75°C for 5 min. Total RNA was electrophoresed as
), and resolved tRNA was visualized
with ethidium bromide staining before being trans-
ferred to a nylon membrane by electroblotting. The
individual tRNA species were probed with radiola-
beled oligonucleotides as follows: for tRNALeu, 5
; for tRNAHis,
; and for
16. C. J. Lynch, H. L. Fox, T. C. Vary, L. S. Jefferson, S. R.
J. Cell. Biochem.
, 234 (2000).
Transiently transfected, HA-tagged mTOR from
HEK293 cells was extracted after a 16-hour serum
starvation and immunoprecipitated with a monoclo-
nal antibody to HA. The immunocomplex was
washed once with 1 M NaCl in assay buffer [30 mM
Mops (pH 7.5), 5 mM NaF, 20 mM
phate, 1 mM dithioerythritol, 0.1 % Triton X-100,
and 10 % glycerol] and twice with assay buffer alone.
g of a kinase-inactive, puriﬁed, soluble S6K1
substrate (S6K1-D3E,K100Q-GST ) or GST-4E-BP1
was added along with assay buffer containing 10 mM
and 1 mM ATP and incubated for 30 min at
30°C. Quantitation for
measurements was carried
out with scanning densitometry and ImageQuant
software (Molecular Dynamics).
18. F. M. Gribble
J. Biol. Chem.
, 30046 (2000).
19. A. M. Edelman, D. K. Blumenthal, E. G. Krebs,
, 567 (1987).
20. T. Gaal, M. S. Bartlett, W. Ross, C. L. Turnbough Jr.,
R. L. Gourse,
, 2092 (1997).
21. M. Derenzini
, 181 (2000).
22. N. Pullen
, 707 (1998).
23. J. A. Enriquez, G. Attardi,
24. Conﬂuent cultures of HEK293 cells were treated as
), washed with PBS, and drained thor-
oughly. The cells were then scraped into 500
water and sonicated with four 10-s pulses. Cell debris
was removed by centrifugation, and sulfosalicylic
acid was added to the supernatant to a ﬁnal concen-
tration of 2%. The samples were then placed on ice
for 30 min followed by centrifugation to remove
precipitated proteins. The extracts were then ana-
lyzed for amino acid content with a Biochrom 20 plus
amino acid analyzer (Amersham-Pharmacia Biotech).