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Molecular Cloning, Developmental Expression, and Phosphorylation of Ribosomal Protein S6 in the Endocrine Gland Responsible for Insect Molting

1997; Elsevier BV; Volume: 272; Issue: 7 Linguagem: Inglês

10.1074/jbc.272.7.4429

1083-351X

Qisheng Song, Lawrence I. Gilbert,

Insect Resistance and Genetics

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. 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. INTRODUCTIONInsect molting and metamorphosis are elicited by a class of steroid hormones, ecdysteroids, originating in the prothoracic gland (1Gilbert L.I. Combest W.L. Smith W.A. Meller V.H. Rountree D.B. BioEssays. 1988; 8: 153-157Crossref PubMed Scopus (80) Google Scholar, 2Gilbert L.I. Rybczynski R. Tobe S.S. Gilbert L.I. Tata J.R. Atkinson B.G. Metamorphosis. Academic Press, Inc., San Diego1996: 60-98Google Scholar). These glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH), 1The abbreviations used are: PTTHprothoracicotropic hormonePAGEpolyacrylamide gel electrophoresis. 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 (1Gilbert L.I. Combest W.L. Smith W.A. Meller V.H. Rountree D.B. BioEssays. 1988; 8: 153-157Crossref PubMed Scopus (80) Google Scholar, 2Gilbert L.I. Rybczynski R. Tobe S.S. Gilbert L.I. Tata J.R. Atkinson B.G. Metamorphosis. Academic Press, Inc., San Diego1996: 60-98Google Scholar, 3Song Q. Gilbert L.I. Dev. Genet. 1994; 15: 332-338Crossref PubMed Scopus (52) Google Scholar, 4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar).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 (4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar, 5Song Q. Gilbert L.I. Arch. Insect Biochem. Physiol. 1996; 31: 465-480Crossref PubMed Scopus (16) Google Scholar), a phenomenon typical of S6 phosphorylation in mammals (6Stewart M.J. Thomas G. BioEssays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). Temporal analysis of PTTH-stimulated S6 phosphorylation showed that phosphorylation and dephosphorylation of S6 closely paralleled the increase and decrease in PTTH-stimulated ecdysteroidogenesis (4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar). Most importantly, the multiple phosphorylation of S6 was inhibited completely by rapamycin, an inhibitor of S6 phosphorylation (7Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (585) Google Scholar, 8Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (561) Google Scholar), resulting in the inhibition of PTTH-stimulated specific protein synthesis and subsequent ecdysteroidogenesis (4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar). 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 (6Stewart M.J. Thomas G. BioEssays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). 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 (9Garren L.D. Ney R.L. Davis W.W. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 1443-1450Crossref PubMed Scopus (328) Google Scholar), which in turn aid in the transit of cholesterol into the mitochondria for the synthesis of steroids (10Orme-Johnson N.R. Biochim. Biophys. Acta. 1990; 1020: 213-231Crossref PubMed Scopus (67) Google Scholar, 11Whitehouse B.J. J. Endocrinol. 1992; 134: 1-3Crossref PubMed Scopus (11) Google Scholar). In insects, RNA and protein synthesis are required for the full response of the prothoracic glands to PTTH (12Smith W.A. Rountree D.B. Bollenbacher W.E. Gilbert L.I. Borkovec A. Gelman D. Progress in Insect Neurochemistry and Neurophysiology. Humana Press, Clifton, NJ1987: 319-322Google Scholar, 13Smith W.A. Gilbert L.I. J. Exp. Zool. 1989; 252: 262-270Crossref Scopus (22) Google Scholar, 14Keightley D.A. Lou K.J. Smith W.A. Mol. Cell. Endocrinol. 1990; 74: 229-237Crossref PubMed Scopus (52) Google Scholar, 15Rybczynski R. Gilbert L.I. Insect Biochem. Mol. Biol. 1994; 24: 175-189Crossref PubMed Scopus (63) Google Scholar, 16Rybczynski R. Gilbert L.I. Mol. Cell. Endocrinol. 1995; 115: 73-85Crossref PubMed Scopus (26) Google Scholar). Although the rapid synthesis of 60-kDa (14Keightley D.A. Lou K.J. Smith W.A. Mol. Cell. Endocrinol. 1990; 74: 229-237Crossref PubMed Scopus (52) Google Scholar), 50-kDa (β-tubulin), 70-kDa (Hsp70), and 100-kDa (15Rybczynski R. Gilbert L.I. Insect Biochem. Mol. Biol. 1994; 24: 175-189Crossref PubMed Scopus (63) Google Scholar, 16Rybczynski R. Gilbert L.I. Mol. Cell. Endocrinol. 1995; 115: 73-85Crossref PubMed Scopus (26) Google Scholar) 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.DISCUSSIONPhosphorylation 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 (6Stewart M.J. Thomas G. BioEssays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). 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 (26Chan Y.-L. Wool I.G. J. Biol. Chem. 1988; 263: 2891-2896Abstract Full Text PDF PubMed Google Scholar), a consensus recognition sequence for the mitogen-activated p70s6k, and five phosphorylatable serines in the carboxyl-terminal region (30Ferrari S. Bandi H.R. Hofsteenge J. Bussian B.M. Thomas G. J. Biol. Chem. 1991; 266: 22770-22775Abstract Full Text PDF PubMed Google Scholar) 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 (3Song Q. Gilbert L.I. Dev. Genet. 1994; 15: 332-338Crossref PubMed Scopus (52) Google Scholar) 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 (32Krieg J. Hofsteenge J. Thomas G. J. Biol. Chem. 1988; 263: 11473-11477Abstract Full Text PDF PubMed Google Scholar, 33Bandi H.R. Ferrari S. Krieg J. Meyer H.E. Thomas G. J. Biol. Chem. 1993; 268: 4530-4533Abstract Full Text PDF PubMed Google Scholar, 37Wettenhall R.E.H. Morgan F.J. J. Biol. Chem. 1984; 259: 2084-2091Abstract Full Text PDF PubMed Google Scholar). 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 (32Krieg J. Hofsteenge J. Thomas G. J. Biol. Chem. 1988; 263: 11473-11477Abstract Full Text PDF PubMed Google Scholar, 33Bandi H.R. Ferrari S. Krieg J. Meyer H.E. Thomas G. J. Biol. Chem. 1993; 268: 4530-4533Abstract Full Text PDF PubMed Google Scholar). 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 (Fig. 4, Fig. 5) in hand, we investigated the possible correlation between S6 expression and ecdysteroid biosynthesis, the latter requiring specific protein synthesis (3Song Q. Gilbert L.I. Dev. Genet. 1994; 15: 332-338Crossref PubMed Scopus (52) Google Scholar, 4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar, 15Rybczynski R. Gilbert L.I. Insect Biochem. Mol. Biol. 1994; 24: 175-189Crossref PubMed Scopus (63) Google Scholar). 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 (38Bollenbacher W.E. Vedeckis W.V. Gilbert L.I. O'Connor J.D. Dev. Biol. 1975; 44: 46-53Crossref PubMed Scopus (222) Google Scholar). The peak at P3 elicits pupal-adult development (36Warren J.T. Gilbert L.I. Insect Biochem. 1986; 16: 65-82Crossref Scopus (223) Google Scholar). 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 (39Nielsen P.J. Duncan R. McConkey E.H. Eur. J. Biochem. 1981; 120: 523-527Crossref PubMed Scopus (37) Google Scholar).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 (3Song Q. Gilbert L.I. Dev. Genet. 1994; 15: 332-338Crossref PubMed Scopus (52) Google Scholar, 4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar). 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 (6Stewart M.J. Thomas G. BioEssays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). S6 phosphorylation is an event that is believed to facilitate the movement of inactive 80 S ribosomes into actively translating polysomes (39Nielsen P.J. Duncan R. McConkey E.H. Eur. J. Biochem. 1981; 120: 523-527Crossref PubMed Scopus (37) Google Scholar), increase translational efficiency (40Burkhard S.J. Traugh J.A. J. Biol. Chem. 1983; 258: 14003-14008Abstract Full Text PDF PubMed Google Scholar), selectively translate protein (41Thomas G. Thomas G. Luther H. Proc. Natl Acad. Sci. U. S. A. 1981; 78: 5712-5716Crossref PubMed Scopus (98) Google Scholar), and regulate a specific class of messages with a polypyrimidine tract at their 5′ cap site (42Jefferies H.B.J. Thomas G. Thomas G. J. Biol. Chem. 1994; 269: 4367-4372Abstract Full Text PDF PubMed Google Scholar).Recent studies also suggest that S6 may play a role in the up-regulation of ribosome biogenesis (6Stewart M.J. Thomas G. BioEssays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar) and in controlling tumor production in Drosophila (23Stewart M.J. Denell R. Mol. Biol. Evol. 1993; 10: 1041-1047PubMed Google Scholar, 25Watson K.L. Konrad K.D. Woods D.F. Bryant P.J. Proc. Natl Acad. Sci. U. S. A. 1992; 89: 11302-11306Crossref PubMed Scopus (156) Google Scholar). 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. INTRODUCTIONInsect molting and metamorphosis are elicited by a class of steroid hormones, ecdysteroids, originating in the prothoracic gland (1Gilbert L.I. Combest W.L. Smith W.A. Meller V.H. Rountree D.B. BioEssays. 1988; 8: 153-157Crossref PubMed Scopus (80) Google Scholar, 2Gilbert L.I. Rybczynski R. Tobe S.S. Gilbert L.I. Tata J.R. Atkinson B.G. Metamorphosis. Academic Press, Inc., San Diego1996: 60-98Google Scholar). These glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH), 1The abbreviations used are: PTTHprothoracicotropic hormonePAGEpolyacrylamide gel electrophoresis. 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 (1Gilbert L.I. Combest W.L. Smith W.A. Meller V.H. Rountree D.B. BioEssays. 1988; 8: 153-157Crossref PubMed Scopus (80) Google Scholar, 2Gilbert L.I. Rybczynski R. Tobe S.S. Gilbert L.I. Tata J.R. Atkinson B.G. Metamorphosis. Academic Press, Inc., San Diego1996: 60-98Google Scholar, 3Song Q. Gilbert L.I. Dev. Genet. 1994; 15: 332-338Crossref PubMed Scopus (52) Google Scholar, 4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar).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 (4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar, 5Song Q. Gilbert L.I. Arch. Insect Biochem. Physiol. 1996; 31: 465-480Crossref PubMed Scopus (16) Google Scholar), a phenomenon typical of S6 phosphorylation in mammals (6Stewart M.J. Thomas G. BioEssays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). Temporal analysis of PTTH-stimulated S6 phosphorylation showed that phosphorylation and dephosphorylation of S6 closely paralleled the increase and decrease in PTTH-stimulated ecdysteroidogenesis (4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar). Most importantly, the multiple phosphorylation of S6 was inhibited completely by rapamycin, an inhibitor of S6 phosphorylation (7Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (585) Google Scholar, 8Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (561) Google Scholar), resulting in the inhibition of PTTH-stimulated specific protein synthesis and subsequent ecdysteroidogenesis (4Song Q. Gilbert L.I. Insect Biochem. Mol. Biol. 1995; 25: 591-602Crossref PubMed Scopus (54) Google Scholar). 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 (6Stewart M.J. Thomas G. BioEssays. 1994; 16: 809-815Crossref PubMed Scopus (69) Google Scholar). 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 (9Garren L.D. Ney R.L. Davis W.W. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 1443-1450Crossref PubMed Scopus (328) Google Scholar), which in turn aid in the transit of cholesterol into the mitochondria for the synthesis of steroids (10Orme-Johnson N.R. Biochim. Biophys. Acta. 1990; 1020: 213-231Crossref PubMed Scopus (67) Google Scholar, 11Whitehouse B.J. J. Endocrinol. 1992; 134: 1-3Crossref PubMed Scopus (11) Google Scholar). In insects, RNA and protein synthesis are required for the full response of the prothoracic glands to PTTH (12Smith W.A. Rountree D.B. Bollenbacher W.E. Gilbert L.I. Borkovec A. Gelman D. Progress in Insect Neurochemistry and Neurophysiology. Humana Press, Clifton, NJ1987: 319-322Google Scholar, 13Smith W.A. Gilbert L.I. J. Exp. Zool. 1989; 252: 262-270Crossref Scopus (22) Google Scholar, 14Keightley D.A. Lou K.J. Smith W.A. Mol. Cell. Endocrinol. 1990; 74: 229-237Crossref PubMed Scopus (52) Google Scholar, 15Rybczynski R. Gilbert L.I. Insect Biochem. Mol. Biol. 1994; 24: 175-189Crossref PubMed Scopus (63) Google Scholar, 16Rybczynski R. Gilbert L.I. Mol. Cell. Endocrinol. 1995; 115: 73-85Crossref PubMed Scopus (26) Google Scholar). Although the rapid synthesis of 60-kDa (14Keightley D.A. Lou K.J. Smith W.A. Mol. Cell. Endocrinol. 1990; 74: 229-237Crossref PubMed Scopus (52) Google Scholar), 50-kDa (β-tubulin), 70-kDa (Hsp70), and 100-kDa (15Rybczynski R. Gilbert L.I. Insect Biochem. Mol. Biol. 1994; 24: 175-189Crossref PubMed Scopus (63) Google Scholar, 16Rybczynski R. Gilbert L.I. Mol. Cell. Endocrinol. 1995; 115: 73-85Crossref PubMed Scopus (26) Google Scholar) 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.