Wendell L. Combest

A calmodulin-sensitive adenylate cyclase in the prothoracic glands of the tobacco hornworm, Manduca sexta

The Ca2+/calmodulin (CaM) dependence of adenylate cyclase activity in Manduca sexta prothoracic glands was investigated. Membrane fractions from two developmental stages were used, day 3 of the last larval instar and day 0 of the pupal stage, both of which respond to the neuropeptide prothoracicotropic hormone (PTTH) with increased cAMP production dependent on extracellular Ca2+. The data revealed that both larval and pupal prothoracic gland membrane fractions have a Ca2+/CaM-dependent adenylate cyclase which is inhibited by CaM antagonists and EGTA. The larval adenylate cyclase shows a multiphasic response to Ca2+/CaM, with a 2-fold stimulation between 0.02 and 0.01 microM, a further increase in adenylate cyclase activity at concentrations greater than 2 microM and a potentiation of NaF-stimulated activity at doses greater than 0.1 microM Ca2+/CaM. Pupal prothoracic gland membrane fractions exhibit only the second phase of stimulation. Stimulation by the GTP analogs GTP-gamma-S and Gpp(NH)p is dependent on CaM in larval, but not in pupal membrane fractions, suggesting a role for CaM in Gs protein-mediated regulation of adenylate cyclase. However, adenylate cyclase activity in glands from both stages is dependent on CaM, supporting our initial premise that Ca2+ is required for cAMP synthesis in the prothoracic glands.

Involvement of cyclic AMP-dependent protein kinase in prothoracicotropic hormone-stimulated ecdysone synthesis

Prothoracicotropic hormone (PTTH) is a brain neuropeptide that stimulates the prothoracic glands to synthesize ecdysone, an event that leads to insect molting. Both cyclic AMP (cAMP) and calcium have been implicated in PTTH action, with current evidence favoring cAMP as the messenger directly regulating ecdysone synthesis. To further define the role of cAMP in PTTH action, the activity of cAMP-dependent protein kinase (cAMP-PK) was examined in prothoracic glands from two developmental stages of the tobacco hornworm, Manduca sexta (day 3 fifth instar larvae and day 0 pupae). Prothoracic glands at each of these stages of development possess two forms of cAMP-PK which resemble the vertebrate type I and type II isozymes, with the latter being the predominant form (greater than 90%). Marked developmental differences exist in the degree of activation of soluble cAMP-PK following in vitro exposure of the prothoracic glands to PTTH. In larval glands, soluble cAMP-PK is activated within 3-10 min of initial exposure to doses of PTTH that stimulate ecdysone synthesis. By contrast, activation of soluble cAMP-PK in pupal glands occurs only when PTTH is administered in the presence of a phosphodiesterase inhibitor. Developmental differences in the activation of cAMP-PK by PTTH were qualitatively identical to previously observed differences in PTTH-stimulated accumulation of intracellular cAMP. The results suggest an involvement of soluble cAMP-PK in the response of day 3 fifth instar larval prothoracic glands to PTTH, but indicate a difference in the nature, intracellular location, or time course of activation, of hormone-sensitive protein kinase in day 0 pupal glands.

Protein phosphorylation in the prothoracic glands as a cellular model for juvenile hormone-prothoracicotropic hormone interactions

Abstract Prothoracicotropic hormone (PTTH) is a brain peptide that initiates the molting process by acting directly at the cell membrane of the prothoracic glands to increase the intracellular levels of free Ca 2+ and cyclic AMP (cAMP). This, in turn, leads to enhanced cAMP-dependent protein kinase activity resulting in the phosphorylation of a specific protein (M r 34,000), and ultimately to a stimulation of ecdysone synthesis. When prothoracic glands are incubated in the presence of juvenile hormone (JH I) or (7 S ) hydroprene and then challenged with PTTH, the phosphorylation of the 34 kDa protein is decreased in a dose-dependent manner. The morphogenetically inactive methyl farnesoate is ineffective in preventing this downstream effect of PTTH. The JH effect does not appear to be stage specific, as early last larval, late last larval and pupal Manduca sexta prothoracic glands are similarly affected. The mechanism by which JH may prevent this PTTH-stimulated phosphorylation is discussed in terms of inhibition of phosphorylation via stimulation of an ATPase and stimulation of dephosphorylation by activation of a phosphoprotein phosphatase.

Polyamines modulate multiple protein phosphorylation pathways in the insect prothoracic gland

Multiple endogenous substrates phosphorylated by four distinct protein kinases were identified in particulate and cytosolic fractions from the larval prothoracic gland of the tobacco hornworm, Manduca sexta. Three prominent particulate-associated phosphoprotein substrates (19, 21, and 34 kDa) were of particular interest. The in vitro phosphorylation of the 19 and 21 kDa peptides was markedly enhanced by cAMP, Ca2+/calmodulin, as well as Ca2+/phospholipids, presumably via cAMP-dependent protein kinase (cAMP-PK), Ca2+/calmodulin-dependent protein kinase (Ca2+/CaM-PK), and protein kinase C (PKC), respectively. The polyamine spermine markedly inhibits both PKC- and cAMP-PK-mediated phosphorylation of the 19 and 21 kDa peptides but had no effect on the Ca2+/CaMP-PK-mediated phosphorylation. Spermine also inhibits the phosphorylation of the 34 kDa peptide via cAMP-PK but does not affect PKC-promoted phosphorylation. In contrast to this differential inhibition of phosphorylation by a polyamine, four cytosolic and three particulate-associated peptides from the prothoracic glands undergo enhanced phosphorylation in the presence of spermine, presumably by stimulating casein kinase II activity. Therefore, polyamines appear to have multiple effects on protein phosphorylation pathways in this important endocrine gland, perhaps representing an important new regulatory control mechanism.

The brain adenylate cyclase system of the tobacco hornworm, Manduca sexta

The basic characteristics of adenylate cyclase from the brain of Manduca sexta larvae are similar to those described for central nervous system preparations from other insects. The properties investigated include the apparent Km for ATP in the presence and absence of NaF, pH optimum, and effects of divalent cations. In addition, since adenylate cyclase can be activated by peptide hormones and neurotransmitters, several biogenic amines were tested on the Manduca system. The brain adenylate cyclase system is stimulated by octopamine (Ka = 1.0 μM) and dopamine (Ka = 0.3 μM) in the presence of guanine nucleotide, but not by serotonin. With a washed, particulate fraction, amine stimulation depends upon the addition of guanine nucleotide. Adenylate cyclase is stimulated 20–30-fold by NaF, the non-hydrolyzable GTP analog Gpp(NH)p, and the diterpene forskolin. Enzyme activity is dependent upon Mg2+ or Mn2+, with the latter cation being 3–6-fold more effective. Calmodulin activates the brain adenylate cyclase approx. 2-fold and this effect is Ca2+-dependent. When the enzyme is solubilized with Lubrol-PX, it retains its normal responsiveness to NaF but octopamine is no longer effective. The solubilized enzyme subjected to sucrose density centrifugation has a sedimentation coefficient of 8.9 S.

Polyamine Regulation of Protein Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta

Abstract: An analysis of the effects of polyamines on protein phosphorylation in cytosolic fractions of the pupal brain of Manduca sexta showed that spermine elicited an increase in casein phosphorylation in a dose‐dependent manner (maximum three‐ to fourfold at 2.0 mM), whereas spermidine was less effective and putrescine was without effect. In contrast, with phosvitin as the exogenous substrate, higher doses of polyamines, especially spermine, inhibited phosphorylation. High salt conditions abolished the polyamine response. Cytosol protein kinase activity eluted from DEAE‐cellulose at 0.2–0.3 M NaCl. This activity was enhanced in the presence of spermine, and inhibited in the presence of heparin (IC50∼ 30 ng/ml). The enzyme was characterized by a sedimentation coefficient of 6.5S, and a Stokes radius of 49 Å, consistent with a Mr of 130,000. Both GTP (Km, 55 μM) and ATP (Km, 34 μM) were utilized as phosphoryl donors (Vmax for ATP being four‐fold higher than that observed for GTP). These results indicate the presence in the insect brain of an enzyme very similar to vertebrate casein kinase II. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography demonstrated that low concentrations of spermine (100 μM) strongly enhanced the phosphorylation of three high‐molecular‐weight cytosolic proteins (305,000, 340,000, and 360,000) localized in the insect nervous system.

Polyamines Differentially Inhibit Cyclic AMP‐Dependent Protein Kinase‐Mediated Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta

Abstract: The effects of the naturally occurring polyamines spermine and spermidine on phosphorylation promoted by cyclic AMP (cAMP)‐dependent protein kinase (PK) (cAMP‐PK; EC 2.7.1.37) were studied using the brain of the tobacco hornworm, Manduca sexta. Four particulate‐associated peptides (280, 34, 21, and 19 kilodaltons) in day 1 pupal brains are endogenous substrates for a particulate type II cAMP‐PK. These phosphoproteins are present in brain synaptosomal, as well as microsomal, particulate fractions but are not present in the cytosol. They are distributed throughout the CNS and PNS and are present in several nonneuronal tissues as well. Phosphorylation of these proteins via cAMP‐PK was inhibited markedly by micromolar concentrations of spermine and spermidine. Other particulate‐associated peptides phosphorylated via a Ca2+/calmodulin‐PK or Ca2+ and cAMP‐independent PKs were unaffected by polyamines, whereas the phosphorylation of a 260–kilodalton peptide was markedly enhanced. Spermine did not exert its inhibitory effect indirectly by enhancement of cAMP or ATP hydrolysis or via proteolysis, but its action appears to involve a substrate‐directed inhibition of cAMP‐PK‐promoted phosphorylation as well as enhanced dephosphorylation. Although addition of spermine resulted in marked ribosome aggregation in synaptosomal and microsomal particulate fractions, this phenomenon was not involved in the inhibition of cAMP‐PK‐promoted phosphorylation.

Characterization of cyclic AMP-dependent protein kinase activity in the larval brain of Manduca sexta

Soluble cyclic AMP-dependent protein kinase (cAMP-PK) activity in the brains of day 5 last instar larvae of Manduca sexta was quantified and the cAMP-PK characterized. Histones f2b and f2a were the preferred exogenous substrates for the enzyme whereas protamine and bovine serum albumin were poorly phosphorylated. Maximal enzyme activity was obtained in the presence of 5–10 mM Mg2+ while Ca2+ and NaCl were inhibitory with IC50 values of 1.0 mM and 0.15 M, respectively. The apparent Km for ATP was not altered in the presence of maximally stimulatory concentrations of cAMP. The protein inhibitor of cAMP-PK from bovine heart was an effective inhibitor of the larval brain cAMP-PK (IC50 = 7.7 μg). Two peaks of cAMP-PK activity were separated by DEAE-cellulose chromatography, corresponding to mammalian type I and II cAMP-PK. Type II predominates in the larval brain comprising 93% of the total cAMP-PK activity in the soluble fraction. Both types are stimulated 3–5-fold and inhibited (>85%) by the protein kinase inhibitor. The percentage of total cAMP-PK present in the soluble fraction depends on the ionic strength of the homogenization buffer with approx. 80% of the cAMP-PK activity recovered in the 12,000 g supernatant fraction in the presence of 0.5 M NaCl. Particulate associated cAMP-PK can be solubilized completely by treatment with Triton X-100.

Characterization of a calcium and diacylglycerol-activated and phospholipid-dependent protein kinase in the pupal brain of the tobacco hornworm, Manduca sexta: The cytosolic enzyme

Abstract A calcium and diacylglycerol-activated and phospholipid-dependent protein kinase (protein kinase C; PKC) in the cytosol of the pupal brain of the tobacco hornworm Manduca sexta has been characterized. Two peaks of PKC activity were separated by DEAE-cellulose chromatography, both of which were dependent upon Ca 2+ , phosphatidylserine and 1,2 diolein. Maximal enzyme activity was obtained in the presence of 0.7 mM Ca 2+ and 200 μg/ml of phosphatidylserine. Diacylglycerol (1,2 diolein; 50 μg/ml) enhanced PKC activity and calcium sensitivity markedly in the presence of phospholipids. The phorbol ester 12- O -tetradecanoyl phorbol-13-acetate substituted for diacylglycerol in the activation of PKC. By utilizing the differential inhibition of PKC and cyclic AMP-dependent protein kinase by trifluoperazine and protein kinase inhibitor, both enzymes were measured accurately in dilute, crude cytosol preparations using the common substrate histone H1.

Phosphorylation of endogenous substrates by the protein kinases of the larval brain of Manduca sexta

Abstract The larval brain of Manduca sexta yields three prominent proteins ( M r = 52,000, 42,000 and 25,000 ), the in vitro phosphorylation of which are markedly stimulated by cAMP ( K a = 3−5 × 10 −7 M). No measurable differences in the rate of phosphorylation of these proteins by protein kinase was noted through 7 days of the fifth larval instar. The protein kinase inhibitor specific for the catalytic subunit of the cAMP-dependent protein kinase inhibits the phosphorylation of these three proteins. The major phosphoprotein detected in brain homogenates is a 62 kDa polypeptide and its phosphorylation is apparently independent of cyclic nucleotides and Ca 2+ /calmodulin. All of the protein kinase substrates are dependent upon Mg 2+ for maximal phosphorylation. The phosphorylation of the 42 and 25 kDa substrates was stimulated by exogenous Ca 2+ and calmodulin, and was blocked by both the calmodulin antagonist trifluoperazine and the Ca 2+ chelating agent, EGTA.