(Received for publication, July 25, 1996, and in revised form, October 14, 1996)
From the Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280
Phosphorylation of ribosomal protein S6 is requisite for prothoracicotropic hormone (PTTH)-stimulated specific protein synthesis and subsequent ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm, Manduca sexta. To better understand the role of S6 in regulating ecdysteroidogenesis, S6 cDNA was isolated from a Manduca prothoracic gland cDNA library and sequenced. The deduced protein is comprised of 253 amino acids, has a molecular weight of 29,038, and contains four copies of a 10-amino acid motif defining potential DNA-binding sites. This Manduca S6 possesses a consensus recognition sequence for the p70s6k binding domain as well as six seryl residues at the carboxyl-terminal sequence of 17 amino acids. Phosphoamino acid analysis revealed that the phosphorylation of Manduca prothoracic gland S6 is limited exclusively to serine residues. Although alterations in the quantity of S6 mRNA throughout the last larval instar and early pupal-adult development were not well correlated with the hemolymph ecdysteroid titer, developmental expression and phosphorylation of S6 were temporally correlated with PTTH release and the hemolymph ecdysteroid titer. These data provide additional evidence that S6 phosphorylation is a critical element in the transduction pathway leading to PTTH-stimulated ecdysteroidogenesis.
Insect molting and metamorphosis are elicited by a class of steroid hormones, ecdysteroids, originating in the prothoracic gland (1, 2). These glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH),1 that acts via a cascade that includes a Ca2+/calmodulin-dependent increase in intracellular cAMP, activation of a cAMP-dependent protein kinase, and ultimate phosphorylation of S6, a protein of the ribosomal 40 S subunit (1-4).
Previous studies demonstrated that S6 in the prothoracic glands of the tobacco hornworm, Manduca sexta, was phosphorylated at up to five sites under PTTH stimulation (4, 5), a phenomenon typical of S6 phosphorylation in mammals (6). Temporal analysis of PTTH-stimulated S6 phosphorylation showed that phosphorylation and dephosphorylation of S6 closely paralleled the increase and decrease in PTTH-stimulated ecdysteroidogenesis (4). Most importantly, the multiple phosphorylation of S6 was inhibited completely by rapamycin, an inhibitor of S6 phosphorylation (7, 8), resulting in the inhibition of PTTH-stimulated specific protein synthesis and subsequent ecdysteroidogenesis (4). These data indicate that S6 phosphorylation is required for both specific protein synthesis and ecdysteroidogenesis in PTTH-stimulated glands.
Ribosomal protein S6 is the major substrate for several protein kinases
in the eukaryotic ribosome, and it may have an important role in
controlling cell growth and proliferation through the selective
translation of particular classes of mRNA (6). Although steroidogenic tissues such as the insect prothoracic glands do not
respond to hormonal stimulation by cell proliferation, steroidogenesis in some mammalian tissues appears to require the rapid synthesis of
relatively short-lived proteins (9), which in turn aid in the transit
of cholesterol into the mitochondria for the synthesis of steroids (10,
11). In insects, RNA and protein synthesis are required for the full
response of the prothoracic glands to PTTH (12-16). Although the rapid
synthesis of 60-kDa (14), 50-kDa (-tubulin), 70-kDa (Hsp70), and
100-kDa (15, 16) proteins in the prothoracic glands was observed
following exposure to PTTH, calcium ionophore, or cAMP, the specific
macromolecules necessary for the dynamic response of Manduca
prothoracic glands to PTTH have not yet been identified. These
observations suggest that S6 phosphorylation resulting from PTTH
stimulation may be critical in regulating the synthesis of these
proteins that are required for ecdysteroid biosynthesis, and this has
led us to investigate further the relationships between S6
phosphorylation and ecdysteroidogenesis.
M. sexta were reared on an artificial diet at 25 °C, >60% relative humidity under a photoperiod of 18 h light:6 h dark with 2400 h artificial Zeitgeber time (lights off) set at 2200 h Eastern Standard Time. A synchronous population of animals was selected by routinely staging on days zero of the third instar, fifth instar, and pupal stage (3, 4).
ChemicalsStandard and phosphate-free Grace's insect tissue culture medium were obtained from Life Technologies, Inc. Carrier-free [32P]04 (10 mCi/ml) was from Amersham Corp., whereas rapamycin was a gift from Wyeth-Ayerst Research. Other reagents were from Sigma, Bio-Rad, Fisher, and U. S. Biochemical Corp.
cDNA Cloning and SequencingProthoracic glands from day
7, fifth instar (V7) larvae were used to develop a cDNA
library.2 First-strand cDNA was
synthesized with an oligo(dT)12-18 primer using Moloney
murine leukemia virus reverse transcriptase (TimeSaver cDNA
synthesis kit; Pharmacia Biotech, Inc.). Second-strand cDNA was
obtained by nick translation using DNA polymerase I, followed by the
addition of EcoRI/NotI adapter to each end.
cDNA in the 0.5-kilobase pair size range was ligated into the
EcoRI-digested Zap II cloning vector. Phage packaged
in vitro were plated (440,000 primary recombinants) and
screened by standard plaque hybridization techniques using a
full-length Drosophila S6 probe (graciously supplied by Dr.
Kellie Watson). Prehybridization was performed in 3 × standard
saline citrate (SSC)(1 × SSC = 0.15 M NaCl,
0.015 M sodium citrate, pH 7.2) containing 2 × Denhardt's medium, 0.5% SDS, and 50 µg/ml salmon sperm DNA.
Hybridization used the same solution with the 32P-labeled
Drosophila S6 probe, and washes in 0.1 × SSC
containing 0.1% SDS were performed at 50 °C. Selected hybridizing
plaques were taken through secondary and tertiary rounds of
purification, and isolated phage clones were used for the in
vivo rescue of cDNA inserts in the pBluescript
SK
plasmid vector. Preliminary restriction mapping of
cDNA inserts indicated that a single class of inserts was
represented. Partial sequence analysis with T3 and T7 primers from both
ends of nine selected inserts revealed that they were identical. Thus,
a single clone, with 0.9 kilobase pair, was selected for
sequencing.
DNA sequencing was performed on double-stranded plasmid DNA using the chain termination method with the Sequenase enzyme (U. S. Biochemical Corp.). Much of the sequencing was done using ordered deletions across the cDNA insert (17). All regions of both strands were sequenced. Nucleotide sequences were analyzed with the Program Manual Software (Genetics Computer Group, Inc., Madison, WI).
Phosphoamino Acid AnalysisProthoracic glands were dissected from V7 larvae, labeled with 32P for 30 min, and challenged with PTTH for 1 h in the presence or absence of 10 nM rapamycin as described previously (4). Ribosomes (80 S) were prepared using sucrose gradient centrifugation and were subjected to SDS-PAGE (12.5%) electrophoresis and autoradiography (4). The labeled band corresponding to S6 was excised, eluted from the gel using a model 422 Electro-eluter (Bio-Rad), and subjected to partial acid analysis in 6 N HCl for 1 h at 110 °C. The supernatant was lyophilized and resuspended in pH 1.9 electrophoresis buffer containing cold phosphoamino acid standards (0.2 mg/ml each of phosphoserine, phosphothreonine, and phosphotyrosine). The phosphoamino acids were separated by electrophoresis using 10 × 10-cm thin layer cellulose plates at pH 1.9 for 30 min in the first dimension and pH 3.5 for 20 min in the second dimension (18)(HTLE-FS apparatus; CBS Scientific, Del Mar, CA). The plates were stained with ninhydrin following electrophoresis so that the positions of these phosphoamino acid standards could be monitored and subjected to autoradiography.
RNA Preparation and Northern Blot AnalysisProthoracic
glands from ice-chilled larvae were dissected as described previously
(4), immediately frozen on dry ice, and kept at 80 °C until
extracted for RNA. Total RNA was prepared by the rapid RNA extraction
procedure of Jowett (19). RNA was size fractionated on 1%
agarose/formaldehyde gels and transferred to nylon membranes. The
0.9-kilobase pair cDNA insert of the Manduca S6 cDNA
clone was excised from the pBluescript SK
plasmid vector
by SalI + NotI digestion, gel purified, and
labeled by random-primed synthesis with [32P]dCTP to a
specific activity of >109 counts/min/µg. Nylon membranes
were hybridized at 42 °C with a S6 insert probe (2 × 106 counts/min/ml) in a solution consisting of 5 × SSC, 5 × Denhardt's solution, 50% formamide (v/v), 1% SDS, and
100 µg/ml sonicated salmon testis DNA. Following hybridization, blots
were washed in 2 × SSC plus 0.5% SDS at 50 °C, dried briefly
between two sheets of filter paper, and subjected to
autoradiography.
A synthetic peptide corresponding to the C-terminal 21 amino acids of deduced Manduca prothoracic gland S6 protein was generated and conjugated to BSA. Two female New Zealand White rabbits were initially injected with 200 µg of bovine serum albumin-conjugated S6 synthetic peptide mixed with RIBI adjuvant system (RIBI ImmunoChem Research, Inc., Hamilton, MT) at a ratio of 1:1 and boosted once with the same amount of immunogen 4 weeks later. Antiserum was titered by enzyme-linked immunosorbent assay, precipitated with ammonium sulfate, and affinity-purified with a protein A column (Bio-Rad). Antibody specificity was examined by Western blot analysis (20) using both gland lysate and purified 80 S ribosomes from prothoracic glands separated by one-dimensional SDS-PAGE or two-dimensional PAGE (4).
Western Blot Analysis of S6 ExpressionProthoracic glands were dissected from the last larval instar (V1-V9) and animals during early pupal-adult development (P0-P4) as described previously (4), homogenized by sonication (10 s) in 10 mM Tris buffer, pH 7.5, containing 0.15 M NaCl. The homogenate was centrifuged for 10 min at 10,000 × g. Proteins in the supernatant were quantified (21), subjected to SDS-PAGE (10% gel) separation, and transferred onto nitrocellulose membranes. The membranes were then immunostained with the Manduca S6 antibody as described previously (3) and analyzed densitometrically (Molecular Dynamics model 300A densitometer).
S6 PhosphorylationTo investigate whether in vivo the S6 phosphorylation state was correlated with the hemolymph ecdysteroid titer, prothoracic glands were dissected from different developmental stages as indicated and immediately placed on dry ice. 80 S ribosomes were purified, separated by two-dimensional PAGE, and silver stained (4).
The S6 cDNA from
Manduca prothoracic glands includes 33 nucleotides of the 5
noncoding sequence, an open reading frame of 759 bases corresponding to
a polypeptide of 253 amino acids, and 34 residues of the 3
noncoding
sequence exclusive of the poly(A) tract (Fig. 1). Within
the coding region, the nucleotide sequence is 69% identical to the
Drosophila cDNA and 63% identical to the human cDNA
sequence.
Primary Structure of the Manduca S6 Protein
The deduced primary structure of the Manduca ribosomal protein S6 consists of 253 amino acids (Fig. 1), and the molecular weight is 29,038 for the unmodified protein. This is close to the 31 kDa estimated from SDS-PAGE gel analysis (3, 4). This S6 has an excess of basic residues (33 arginyl, 33 lysyl, and 3 histidyl) when compared to the acidic residues (11 aspartyl and 16 glutamyl). The basic amino acids comprise 27.3% of the total, while the acid residues make up 10.7% of the total. The estimated isoelectric point is 11.48. There are three cysteinyl residues at position 12, 83, and 100, but it is not known if they form disulfide bridges. There are a total of 16 seryl residues in the Manduca S6, 6 of which are located within the carboxyl-terminal sequence of 17 amino acids. This includes the serines at positions 237, 239, 243, 245, 246, and 249.
Comparison of the Manduca S6 Sequence to That of Other SpeciesSequence comparison of Manduca S6 to the S6 of
four other species, ranging from yeast to Drosophila, rat,
and human (Fig. 2), revealed that this ribosomal protein
is highly conserved at the amino-terminal region but much less so at
the carboxyl terminus. The deduced Manduca prothoracic gland
S6 polypeptide shares 79% identity (89% similarity) with the
Drosophila ribosomal protein S6 (23-25), 75% identity
(84% similarity) with the rat (26) and human (27, 28) homolog of S6,
and 60% identity (77% similarity) with the yeast S10 homolog of S6
(29). The deduced Manduca S6 protein is 4 amino acids longer
than the S6 of Drosophila, rat, and human and contains a
total of 16 seryl residues, the same number as in Drosophila
S6, whereas there are only 15 seryl residues in rat and human and 11 in
yeast. It should be noted that the positions of all six potentially
phosphorylatable seryl residues located in the carboxyl-terminal region
of the Manduca S6 are not identical to that in other species
(Fig. 2). The Manduca S6 sequence reveals four copies of a
10-amino acid motif that is also common to the other four species whose
consensus sequence includes an initial proline residue and four to six
basic amino acids (Fig. 2). This motif is postulated to be a nuclear
localization signal (26). The S6 sequences of all five species also
reveal a consensus recognition sequence for the mitogen-activated
p70s6k (30, 31). The numbers and positions of the three
cysteine residues are identical among the species compared (Fig.
2).
Phosphoamino Acid Analysis
Although in vertebrates S6
phosphorylation is limited to seryl residues at the carboxyl-terminal
region (32, 33), no analogous information existed for the
Manduca S6 or for the S6 of any other insect. Therefore, a
phosphoamino acid analysis was carried out. Prothoracic glands were
labeled with 32P, treated with rapamycin, and challenged
with PTTH as described previously (4). Ribosomal 80 S proteins were
purified and subjected to SDS-PAGE (12.5%) electrophoresis. The
Coomassie Blue-stained ribosomal 80 S proteins in the absence (Fig.
3a, lane 1) or presence (Fig. 3a, lane
2) of rapamycin were subjected to autoradiography. The labeled
band corresponding to S6 in the absence of rapamycin (Fig. 3a,
lane 3) was excised from the gel and subjected to partial acid
hydrolysis and two-dimensional thin layer electrophoresis. Rapamycin
treatment inhibited PTTH-stimulated S6 phosphorylation completely (Fig.
3a, lane 4; see also Refs. 3 and 4), and this was used as a
control to confirm that the excised band represents S6. Phosphoamino
acid analysis revealed that S6 was phosphorylated exclusively at the
serine residues (Fig. 3b), a result consistent with all past
reports on the subject (34).
S6 Antibody Specificity
The high specificity of the S6
antiserum against the carboxyl-terminal 21 amino acids of the deduced
Manduca S6 was demonstrated by Western blot analysis (Fig.
4). The antibody detected only a single band of 31 kDa
in both prothoracic gland cytosol (Fig. 4b, lanes 1 and
2) and 80 S ribosomes purified from prothoracic glands (Fig.
4b, lanes 3 and 4). The blot containing
equivalent amounts of prothoracic gland cytosol (Fig. 4a, lanes
1 and 2) and 80 S ribosomes (Fig. 4a, lanes
3 and 4) was stained with Amido Black and used as a
reference. Preimmune serum displayed no immunoreactivity to S6 (data
not shown).
To determine if the immunostained band corresponding to S6 in
one-dimensional SDS-PAGE is indeed S6 and whether the antibody recognized all forms of the phosphorylated or unphosphorylated S6,
two-dimensional PAGE was performed. The purified 80 S ribosomal proteins from PTTH-stimulated or control glands were separated by
two-dimensional PAGE (4), transferred onto nitrocellulose membranes,
and immunostained with the purified antibody (Fig. 5).
S6 was phosphorylated at all five sites in PTTH-stimulated glands as
shown by the Ponceau S-stained membrane (Fig. 5b), and only
one site was phosphorylated in control glands (Fig. 5a), a
result consistent with previous studies (4, 5). These same blots were
then destained in water to remove the Ponceau S stain and immunostained
with the antibody. The antibody recognized all forms of the prothoracic
gland S6 protein in both PTTH-stimulated (Fig. 5d) and
control glands (Fig. 5c) with virtually equal affinity to
all forms of S6 when compared to the corresponding Ponceau S-stained
blots (Fig. 5, a and b).
S6 Gene Expression
To investigate the functional relationship
between prothoracic gland S6 expression at both the transcriptional and
translational levels and ecdysteroid biosynthesis, both Northern and
Western blot analyses were performed to monitor the expression of S6
through the last larval instar and during early pupal-adult
development. The developmental Northern blot analysis revealed a
0.9-kilobase transcript expressed throughout the fifth larval instar
and early pupal-adult development (Fig. 6b).
The S6 mRNA is abundant in the prothoracic glands since total RNA
(Fig. 6a) was probed. The densitometric analysis of S6
mRNA expression (Fig. 6c) revealed that the level of
S6-specific mRNA was relatively high at stages V1-V3, started to decrease at V4,
reached its lowest level at V7-V9, recovered
slightly after pupation, and declined again at P1-P4. These results reveal that the peak of
S6 mRNA was inversely correlated with the peaks of the hemolymph
ecdysteroid titer, although the small reprogramming ecdysteroid peak at
V3 occurs at a time of high S6 gene expression. Attempts to
probe the blot with Drosophila rp49 cDNA produced no
visible band, presumably due to the low signal in the total RNA
preparation. It should be noted that each prothoracic gland consists of
about 220 cells, and purification of poly(A) from prothoracic glands
for a developmental assay is technically very difficult and labor
intensive. Therefore, total RNA on the gels was stained with ethidium
bromide before transfer, photographed, and analyzed densitometrically
(Fig. 6a) to insure that the developmental fluctuations in
S6 mRNA expression were not due to sample loading.
S6 Protein Expression
Using the highly specific antibody
generated against the Manduca S6 protein, developmental
changes in the amount of S6 protein in the prothoracic glands were
analyzed by Western blot through the last larval instar and early
pupal-adult development to determine possible correlations with
ecdysteroid biosynthesis. The data reveal that the levels of S6 in the
prothoracic glands were stage-dependent (Fig.
7a). S6 immunoreactivity was relatively
constant until V5, increased rapidly at V6, was
almost double in V7 glands relative to V5, and
decreased rapidly by V8. S6 immunoreactivity remained at a
low level in P0-P2 prothoracic glands but
increased dramatically at P3-P4 in concert
with the ecdysteroid titer. Both of the peaks of S6 immunoreactivity
(Fig. 7) paralleled the large peaks in the hemolymph ecdysteroid
concentration (35, 36). To demonstrate that the fluctuation in S6
immunoreactivity in parallel with the hemolymph ecdysteroid titer was
not simply coincidental, Western blot analysis was conducted for the
fat body and midgut S6 during the same developmental period. The
results from both fat body (Fig. 7b) and midgut (data not
shown) revealed that the pattern of S6 immunoreactivity differed
dramatically from that of the prothoracic gland S6 and was not directly
correlated with the ecdysteroid titer.
In Vivo S6 Phosphorylation
Our previous in vitro
studies showed that S6 phosphorylation is a prerequisite for
PTTH-stimulated protein synthesis and subsequent ecdysteroidogenesis.
However, the in vivo relationships between PTTH release, S6
phosphorylation and the hemolymph ecdysteroid titer remain conjectural.
Fig. 8 reveals that S6 phosphorylation in
vivo was closely correlated with the hemolymph ecdysteroid titer;
S6 remained in a dephosphorylated state in prothoracic glands from
stages V1, V5, V7, V9,
and P1, but was phosphorylated at V3,
V6, and P3. All five sites were phosphorylated
at V6, whereas only one or two sites were phosphorylated at
stages V3 and P3. Phosphorylation of S6 at a
single site is sufficient for the initiation of ecdysteroidogenesis
(4). The phosphorylation of S6 at stages V3,
V6, and P3 occurred in concert with the three
peaks of hemolymph ecdysteroids.
Phosphorylation of the S6 protein in the 40 S ribosomal subunit is a critical event in the initiation of cell growth and proliferation and is well conserved among eukaryotic organisms (6). A full-length cDNA encoding the Manduca prothoracic gland S6 has been cloned and sequenced. Sequence comparisons between Manduca and other species, ranging from yeast to invertebrate to vertebrate, revealed that the key elements required for S6 function are well conserved (Fig. 2). All four copies of the 10-amino acid motif postulated to be a nuclear localization signal (26), a consensus recognition sequence for the mitogen-activated p70s6k, and five phosphorylatable serines in the carboxyl-terminal region (30) are well conserved among eukaryotic species, whereas the yeast S6 has only two serines in that region. The number of basic and acidic residues is almost identical in all species compared, a biochemical characteristic of this very basic ribosomal protein. A striking observation is that both the number (3) and the position (12, 83, and 100) of the cysteine residues are identical in the S6 of the five species listed in Fig. 2. Phosphoamino acid analysis (Fig. 3) revealed that S6 phosphorylation is limited to seryl residues, a result consistent with that reported in mammalian systems (32, 33, 37). The exact positions of the five phosphoseryl residues of Manduca S6 have not yet been identified, but they are presumably located within the carboxyl-terminal 17 amino acids of S6 (32, 33). These results, along with our previous data, demonstrate that PTTH-stimulated ecdysteroidogenesis is associated with the increased phosphorylation of ribosomal S6 at seryl residues.
However, the sequences differ significantly in the carboxyl-terminal half of the molecule and typify a specific group of organisms, i.e. yeast versus invertebrates versus vertebrates. This region of S6 revealed 66.7 and 67.2% of the total amino acid substitutions in the primary structure of the deduced protein between Manduca and Drosophila and between Manduca and human, respectively. Although the amino acid sequence differences between Manduca and yeast are more evenly distributed, 56.2% of the substitutions are located within the carboxyl-terminal half of the protein. In addition, Manduca S6 contains only six serine residues at the carboxyl terminus portion rather than the seven occurring in more evolutionarily advanced species such as the fruit fly, rat, and human S6, but there are four more serines than was found in the S6 of more primitive species, e.g. yeast.
With the cloned S6 cDNA probe (Fig. 1) and specific S6 antibody (Figs. 4 and 5) in hand, we investigated the possible correlation between S6 expression and ecdysteroid biosynthesis, the latter requiring specific protein synthesis (3, 4, 15). The small increase in the larval hemolymph ecdysteroid titer at V3 is responsible for cellular reprogramming, whereas the major surge in the larval hemolymph ecdysteroid titer takes place at about stage V7 and is responsible for initiating the larval-pupal molt (38). The peak at P3 elicits pupal-adult development (36). Thus, the latter two dramatic increases in the hemolymph ecdysteroid titer are responsible for the two metamorphic molts of this insect. Northern blot analysis revealed that transcriptional expression of S6 through larval and early pupal-adult development was not correlated with the ecdysteroid titer, i.e. the S6 mRNA signal was at its lowest level at stages V7-V8 and P3-P4 (Fig. 6). However, Western blot analysis demonstrated that the quantity of S6 gene product peaked at stages V6-V7 and P3-P4 in concert with the peaks in the hemolymph ecdysteroid titer (Fig. 7). The rapid decline in S6 mRNA (Fig. 6) accompanied the rapid increase in the level of S6 protein at the times that the hemolymph ecdysteroid titer peaked, suggesting that the increase in S6 results from increased translation, and that more ribosomal S6 protein is required at these stages, perhaps to facilitate the synthesis of specific proteins (enzymes?) required for ecdysteroidogenesis. Although direct evidence for a specific role of S6 in protein synthesis and ecdysteroidogenesis is not yet available for the Manduca prothoracic gland, in mammalian cells, following activation of the cells by mitogens, mRNAs encoding ribosomal proteins (including S6) and elongation factors are shifted from nonactive ribosomes to the active polysome fraction within 30 min and synthesis of specific proteins begins prior to the general increase in protein synthesis (39).
Our previous in vitro studies showed that high levels of S6
phosphorylation are closely associated with increased rates of protein
and ecdysteroid synthesis and that inhibition of S6 phosphorylation by
rapamycin resulted in the inhibition of specific protein synthesis and
subsequent ecdysteroid synthesis (3, 4). The present data (Fig. 8)
revealed that the multiple phosphorylation of S6 occurred in
vivo as well in concert with the increase in hemolymph ecdysteroidogenesis at all three critical periods of development, i.e. V3, V6, and P3, and
therefore, with the release of endogenous PTTH. The data therefore
indicate that the release of PTTH in vivo initiates S6
phosphorylation, which in turn elicits specific protein synthesis and
subsequent ecdysteroidogenesis. Studies in other biological systems
have also shown a correlation between a high level of S6
phosphorylation and an increase in protein synthesis, one of the
earliest events required for cell growth and proliferation (6). S6
phosphorylation is an event that is believed to facilitate the movement
of inactive 80 S ribosomes into actively translating polysomes (39),
increase translational efficiency (40), selectively translate protein
(41), and regulate a specific class of messages with a polypyrimidine
tract at their 5 cap site (42).
Recent studies also suggest that S6 may play a role in the up-regulation of ribosome biogenesis (6) and in controlling tumor production in Drosophila (23, 25). It is possible that the increased levels of S6 protein expression that correlate with the peaks of hemolymph ecdysteroids may not only facilitate specific protein synthesis required for ecdysteroidogenesis but may also be involved in the feedback regulation of ecdysteroid synthesis. Thus, S6 not only participates in "housekeeping" roles but may be critically involved in the control of insect growth, molting, and metamorphosis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U64795[GenBank].
We are grateful to Virginia Mergner for excellent assistance in cDNA library screening and for use of the Drosophila S6 cDNA probe supplied by Dr. Kellie Watson. We thank Prof. Beverly Errede and Qingyuan Ge for technical help in the phosphoamino acid analysis. We also thank Susan Whitfield for photography and Pat Cabarga for clerical assistance.
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