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

Abstract

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 p70 s6k binding domain as well as six seryl residues at the carboxylterminal 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 Ca 2ϩ /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.

A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis

“…Another effect of PTTH is the phosphorylation of ribosomal protein S6 in the PGs of Manduca (Song and Gilbert, 1997). In agreement with this report, phosphorylation of S6 was induced 2.7-fold after 1 h exposure of PGs to PTTH.…”

20E-regulated USP expression and phosphorylation in Drosophila melanogaster

“…Protein samples were prepared according to a protocol modified from Song and Gilbert (1997). In brief, Drosophila tissues were dissected from the indicated stages at room temperature under the Ringers' solution and homogenized on ice in phosphate-buffered saline (PBS) (136 mM NaCl, 1.1 mM K 2 HPO 4 , 2.7 mM KCl, 8.0 mM Na 2 HPO 4 , pH 7.4) using a disposable pestle in an Eppendorf tube.…”

TOR signaling is involved in PTTH-stimulated ecdysteroidogenesis by prothoracic glands in the silkworm, Bombyx mori

“…Previous studies indicated that PTTH-stimulated cAMP and Ca 2þ are intracellular second messengers in both Manduca sexta (Smith et al, 1984(Smith et al, , 1985Fellner et al, 2005) and Bombyx mori (Gu et al, 1996(Gu et al, , 1998. In M. sexta, it was reported that p70S6 kinase (S6K) and ribosomal protein S6 are related to PTTHstimulated ecdysteroidogenesis (Song and Gilbert, 1994, 1997. In addition, extracellular signal-regulated kinase (ERK) phosphorylation was found to be involved in PTTH's stimulation of ecdysteroidogenesis (Rybczynski et al, 2001;Lin and Gu, 2007;Gu et al, 2010).…”