Developmental changes in cyclic AMP-dependent protein kinase associated with increased secretory capacity of Manduca sexta prothoracic glands (original) (raw)
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Insect Biochemistry and Molecular Biology, 1996
Prothoraeicotropic hormone (PTTH), a peptide produced by the insect brain, stimulates the prothoracic glands to secrete ecdysteroids. The big form of this peptide (25.5 kDa) has been postulated to act through cyclic AMP in larval Manduca sexta, but the role of the cyclic nucleotide in the action of PTTH in pupal glands has been less clear. Results of the present study indicate that PTTH-stimulated ecdysteroid secretion and protein phosphorylation by glands removed from pupal Manduca sexta are blocked by two inhibitors of cAMP-dependent protein kinase: Rp-cAMPS, an antagonist of cAMP binding to the regulatory subunit of the kinase, and H-89, an inhibitor of the catalytic subunit of the kinase. Further, PTTH stimulates significant accumulation of cAMP in pupal glands, although less than that previously seen in PTTH-stimulated larval glands. Cyclic AMP-dependent protein kinase is found in cytoplasmic and membrane-associated glandular subfractions, as measured by incorporation of [32P]8-N3cAMP into the regulatory subunit of the kinase. PTTH enhances cytoplasmic cAMP content and appears to increase the amount of cAMP bound to a cytoplasmic type II regulatory subunit of cAMP-dependent protein kinase. The results indicate that cAMP plays a requisite role in PTTH action in pupal glands, thus arguing in favor of a uniform mechanism of action for the peptide during Manduca development.
Second Messengers and the Action of Prothoracicotropic Hormone inManduca sexta
American zoologist, 1993
SYNOPSIS. The large (26 kDa) prothoracicotropic hormone of Manduca sexta stimulates ecdysteroid secretion by the prothoracic glands through the action of cyclic AMP (cAMP). Adenylate cyclase in the prothoracic glands is sensitive to calcium/calmodulin, and enhancement of intracellular calcium levels may be the means by which PTTH stimulates cAMP synthesis. The cyclic nucleotide in turn activates cAMP-dependent protein kinase and protein phosphorylation, most notably of a 34 kDa membraneassociated protein. It does not appear that protein kinase C plays a role in the acute action of PTTH, nor has the hormone been found to stimulate formation of inositol trisphosphate undercurrent assay conditions. PTTH rapidly increases protein synthesis by the prothoracic glands, and translation inhibitors block PTTH-stimulated ecdysteroid secretion. Connections between protein phosphorylation, protein synthesis, and ecdysone secretion remain to be clarified.
Stimulation of ecdysteroidogenesis by small prothoracicotropic hormone: Role of cyclic AMP
Molecular and Cellular Endocrinology, 1993
hormones (PTTHs) stimulate synthesis and secretion of ecdysteroids by insect prothoracic glands. In Manduca sex@ PTTH exists as two size variants, small and big PTTH. Experiments were performed to assess the possible role of cyclic AMP in small PTTH signal transduction. CAMP analogs, or agents that increase intracellular CAMP, stimulated ecdysteroidogenesis.
Insect Biochemistry and Molecular Biology, 1995
Prothoracicotropic hormone (PTTH)-stimulated protein phosphorylation leads to ecdysteroidogenesis (molting hormone biosynthesis) in the prothoracic glands of the tobacco hornworm, Munduca sexta. The phosphorylation of 34 and 50 kDa peptides (~34 and ~50) paralleled the increase in ecdysteroidogenesis, and the dephosphorylation of p34 and p50 preceded a decrease in ecdysteroidogenesis. Inhibition by rapamycin of ~34, but not ~50, phosphorylation prevented PTTH-stimulated ecdysteroidogenesis in a dose-dependent manner, suggesting that p34 phosphorylation is requisite for PTTH-stimulated ecdysteroidogenesis. Two proteins whose synthesis was rapidly stimulated by PTTH were p50 and ~70. The time-course of PTTH-stimulated synthesis of p50 paralleled that of p34 phosphorylation and that of ecdysteroidogenesis. Rapamycin inhibited PTTH-stimulated synthesis of p50 and ~70, suggesting that specific protein synthesis is also required for PTTH-stimulated ecdysteroidogenesis, confirming the results of Rybczynski and Gilbert [(1994) Insect Biochem. Molec. Biol. 24, 175-1891, and that p34 phosphorylation may regulate the downstream synthesis of p50 and ~70, possible key regulatory proteins leading to ecdysteroidogenesis. Results from twodimensional (ZD)-PAGE analysis of the ribosomal proteins purified from prothoracic glands, demonstrated that p34 is indeed ribosomal S6, and is pbosphorylated at up to five sites (P,,) upon PTTH stimulation. The multiple phosphorylation of S6 was inhibited completely by rapamycin as shown in 2D gel maps, further confirming that p34 is ribosomal protein S6. Temporal analysis of PTTH-stimulated S6 phosphorylation by ZD-PAGE revealed that phosphorylation of S6 at the P, site was temporally correlated with the initiation of ecdysteroidogenesis, and that multiple phosphorylation at all five sites (P,_5) was correlated with the maximal synthesis of ecdysteroids. Dephosphorylation of S6 was accompanied by a decrease in ecdysteroidogenesis. These data demonstrate that p34 is ribosomal protein S6 and that both the phosphorylation of S6 and specific protein synthesis are required for PTTH-stimulated ecdysteroidogenesis in the prothoracic gland. Ecdysteroidogenesis Prothoracicotropic hormone S6 phosphorylation Rapamycin Protein synthesis ECDYSTEROID BIOSYNTHESIS IN MANDUCA SEXTA 593
Evidence of a stimulatory effect of cyclic AMP on corpus allatum activity in Manduca sexta
Molecular and Cellular Endocrinology, 1994
Injection of dibutyryl-cAMP prevents cuticular melanization of black Manduca sexta larvae, whose pigmentation is related to a defect in the control of the corpus allatum. The cAMP analog has no effect in allatectomized black larvae. Significant stimulation of corpus allatum activity was obtained in vitro with compounds which elicit or mimic elevated intracellular cAMP levels (dibutyryl-, 8bromo-, N 6 benzoyl-, and 8-thiomethyl-cAMP, 3-isobutyl-l-methylxanthine), but not with dibutyryl-cGMP. Relatively inactive glands, such as those on day 4 of last larval stadium or from black mutant larvae, were more sensitive to these compounds than glands actively synthesizing JH/JH acid. JH acid synthesis by corpora allata taken after pupal commitment in the last larval stadium (days 6 and 8) was not stimulated by either dibutyryl-cAMP or 3-isobutyl-l-methylxanthine, but day 8 glands appeared to be inhibited by dibutyryl-cAMP. The results indicate that a cAMP second messenger system is involved in the transduction of signals which stimulate JH/JH acid synthesis by Manduca corpora allata prior to pupal commitment and suggest that it may be involved in the inhibition of JH acid synthesis after commitment. They also imply that the proposed hemolymph factor to which the black mutant corpora allata are differentially sensitive interfaces with the cAMP system.
Cellular changes in the prothoracic glands of diapausing pupae of Manduca sexta
Journal of Experimental Biology, 1986
Prothoracic glands from diapausing pupae of the tobacco hornworm, Manduca sexta, synthesize markedly less ecdysone in vitro in response to prothoracicotropic hormone (PTTH) than do glands from non-diapausing pupae. Impaired steroidogenesis is also observed in glands from diapausing animals exposed to agents that enhance ecdysone synthesis in non-diapausing pupal glands by increasing intracellular levels of cAMP (1-methyl-3-isobutylxanthine, dibutyryl cAMP, and the calcium ionophore A23187). In contrast, prothoracic glands from diapausing pupae synthesize significantly more cAMP in response to PTTH and A23187 than do those from non-diapausing pupae. These observations indicate that the PTTH-refractoriness characteristic of prothoracic glands during diapause results from a lesion in the steroidogenic pathway occurring beyond the level of the PTTH receptor-adenylate cyclase system. The diapause condition of the prothoracic glands (reduced ecdysone synthesis accompanied by enhanced cAMP...
Archives of Insect Biochemistry and Physiology, 1996
The multiple phosphorylation of ribosornal protein S6 appears to be required for prothoracicotropic hormone (PITH)-stimulated protein synthesis and ecdysteroidogenesis by the prothoracic glands of Manduca sexta. The present study investigated the role of protein phosphatase in these phenomena by analyzing the effects of pretreatment of prothoracic glands with the phosphatase inhibitors okadaic acid and calyculin A in both basal and PITH-stimulated glands. Okadaic acid or calyculin A treatment enhanced ribosomal S6 phosphorylation in control glands to a level similar to that observed with PTTHstimulated glands. This treatment also prevented S6 dephosphorylation but had no apparent synergistic effect on S6 phosphorylation in PITH-stimulated glands. Most importantly, okadaic acid or calyculin A treatment inhibited, rather than augmented, ecdysteroidogenesis in both PTTt i-stimulated and non-stimulated glands. The composite data suggest that protein phosphatase activity sensitive to okadaic acid or calyculin A is required for PnH-stimulated ecdysteroidogenesis.
Insect Biochemistry and Molecular Biology, 2000
Multiple assays were conducted in order to determine if the recently available recombinant prothoracicotropic hormone (rPTTH) from Manduca sexta is identical, or similar, to the natural hormone and if results from its use in a variety of assays confirm, or are inconsistent with, previous studies over the past 20 years on PTTH action using brain extract. Brain extracts and rPTTH showed similar, if not identical, effects on the cell biology of Manduca prothoracic gland cells with the following results: increased levels of cAMP (adenosine 3Ј:5Ј cyclic monophosphate) synthesis; requirement for extracellular Ca 2+ in in vitro studies; ecdysteroidogenesis stimulation in vitro; stimulation of general and specific protein synthesis; immunocytochemical identification of the two lateral cells in each brain hemisphere as the source of PTTH (the prothoracicotropes); the ability of antibodies to rPTTH to inhibit ecdysteroidogenesis stimulation in vitro; and the multiple phosphorylation of the ribosomal protein S6. The data revealed that brain extract and rPTTH show equivalent effects in all of the assays, indicating that this rPTTH is the natural PTTH of Manduca and that the data generated with brain extracts over the past two decades are indeed relevant.
Stimulation of ecdysteroidogenesis by small prothoracicotropic hormone: role of calcium
Molecular and Cellular Endocrinology, 1995
Insect prothoracic glands are regulated by neuropeptide prothoracicotropic hormones (PTTH). In Manduca sexta PTTH exists as two size variants, big PTTH (∼25.5 kDa) and small PTTH (∼7 kDa). Previous studies indicate that both size variants employ cAMP as a second messenger and that stimulation of ecdysteroid secretion by big PTTH is Ca2+-dependent. in the present study, experiments were performed to assess the role of Ca2+ in small PTTH-stimulated ecdysteroid secretion by prothoracic glands from fifth instar larvae. Basal ecdysteroid secretion was not affected by Ca2+ channel blockers (verapamil or lanthanum) or by omission of Ca2+ from the incubation medium. Treatment of glands with a Ca2+ ionophore (A23187 or ionomycin) produced a concentration-dependent stimulation of ecdysteroid secretion. Stimulation of ecdysteroid secretion by small PTTH was suppressed (1) by Ca2+ channel blockers and (2) in Ca2+-free medium. A cAMP analog (Sp-cAMPS) stimulated ecdysteroid secretion in the presence of a Ca2+ channel blocker (verapamil) and in Ca2+-free incubation medium, and ionophore-induced ecdysteroid secretion appeared to be suppressed by a cAMP antagonist (Rp-cAMPS). The combined results indicate that basal ecdysteroid secretion is not dependent on external Ca2+, and suggest that small PTTH-stimulated ecdysteroid secretion is mediated by an influx of Ca2+ that precedes cAMP formation.
Biochemical and Biophysical Research Communications, 1991
Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.