1Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine Hershey; 2Department of Biology, Lebanon Valley College, Annville; and 3Division of Allergy and Immunology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania
Submitted 30 December 2004 ; accepted in final form 5 May 2005
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ABSTRACT |
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amino acids; gastrocnemius; protein kinase C isoforms; ribosomal protein S6 kinase-1; eukaryotic initiation factor 4E-binding protein-1
Four lines of evidence suggest that amino acids are probably not augmenting protein synthesis merely by increasing substrate availability. First, the tRNAs involved in protein synthesis are essentially fully charged at the intracellular amino acid concentrations found in skeletal muscle. Thus increasing the plasma amino acid concentration above that found in controls would not further charge the tRNA. Second, increasing substrate availability should accelerate protein synthesis by enhancing peptide-chain elongation. However, we (1, 2, 21, 40) and others (28) have provided evidence that amino acids accelerate peptide chain initiation to a greater extent than elongation. Third, removal of leucine can prevent the overall stimulation of protein synthesis by amino acids, yet removal of methionine is without effect (40, 42). Finally, norleucine, a structural analog of leucine, stimulates protein synthesis (32, 33) even though norleucine cannot be incorporated into skeletal muscle proteins. Thus amino acids affect protein synthesis in skeletal muscle independently of augmenting substrate availability.
Of the amino acids, leucine appears unique as a regulator of protein synthesis in skeletal muscle. The cellular pathways by which leucine in particular modulates protein synthesis are beginning to be elucidated. Amino acids fail to stimulate phosphatidylinositol (PI) 3-kinase or PKB (Akt), indicating that signaling pathways that become activated by insulin and growth factors may not be necessary or sufficient for mediating the effects of amino acids on protein synthesis (19, 26, 32, 33). Alternatively, amino acids, and leucine in particular, consistently activate the 70-kDa ribosomal protein S6 kinase-1 (S6K1) and the translation repressor eukaryotic initiation factor (eIF)4E-binding protein (4E-BP1) through enhanced phosphorylation using both in vitro (24, 25, 35, 47) and in vivo models (13, 5, 32, 33, 49). Phosphorylating S6K1 and 4E-BP1 is associated with an acceleration of mRNA translation initiation, leading to a stimulation of protein synthesis. S6K1 and 4E-BP1 are phosphorylated by a common upstream kinase, the mammalian target of rapamycin (mTOR), suggesting a role for mTOR in mediating, in part, the effects of leucine to phosphorylate these two proteins (13, 19). Indeed, structure-activity relationships indicate that leucine was the most potent amino acid in augmenting phosphorylation of 4E-BP1 in adipocytes (31) or H4IIE hepatocytes (36) maintained in culture.
However, not all the effects of leucine on protein synthesis can be attributed to a stimulation of mTOR activity and increased phosphorylation of S6K1 and 4E-BP1. Orally administered leucine via gavage stimulates protein synthesis in skeletal muscle of rats with experimental diabetes without increasing the phosphorylation of 4E-BP1 or S6K1 (3). Likewise, oral administration of leucine by gavage stimulates phosphorylation of 4E-BP1 and accelerates protein synthesis but fails to increase the phosphorylation of S6K1 in skeletal muscle following acute alcohol intoxication (27). Therefore, other signaling pathways may be involved in the leucine-mediated stimulation of protein synthesis in muscle.
The protein kinase C (PKC) family is a group of at least 12 known members that contain phospholipid-dependent serine/threonine kinase activity. The PKC family is further divided into three subfamilies on the basis of lipid and cofactor requirements. The three subfamilies are the conventional (PKC, -
I, -
II, and -
), the atypical (PKC
and -
), and the novel (n)PKC
, -
, -
, and -
). PKC has been implicated as an intracellular mediator of several neurotransmitters, growth factors, and tumor promoters through multiple signal transduction pathways. PKC activity is regulated by two broad mechanisms, one relating to the subcellular compartmentation and the other to the phosphorylation of the kinases themselves. The response of the PKC isoforms to the same stimuli may not be uniform, but rather is dependent on the individual cell type.
The various PKC isoforms exhibit common domains required for catalytic activity and regulatory function. Three key serine and threonine residues in the catalytic domain undergoing reversible phosphorylation have been described in the 12 isoforms. Phosphorylation sites can reside in the so-called activation loop [e.g., PKC(Thr505)] and are the rate-limiting steps in activation. Activation loop phosphorylation introduces a negative charge that critically aligns the catalytic site and is required for catalytic competence of the enzyme. This phosphorylation enables two subsequent phosphorylations at conserved reside [e.g., PKC
(Ser643) or PKC
(Ser729)] in the autophosphorylation domain (COOH-terminal V5 domain turn motif and an additional phosphorylation in the hydrophobic FXXFS/TF/Y motif). Phosphorylation in this region is believed to fix the enzyme in a catalytically active and protease/phosphatase-resistant confirmation, which correlates with increased PKC activity (for review see Ref. 36a).
Whereas nPKC phosphorylation is increasingly recognized as a potential mechanism to regulate nPKC catalytic activity, the studies are performed mainly in cells in culture. Moreover, phosphorylation of PKC and PKC
may be regulated in part by mTOR using cells in culture (34). However, there is relatively limited information regarding conditions that promote changes in phosphorylation of nPKC in vivo. Because our previous reports indicated that meal feeding may enhance mTOR signaling (15, 16, 20, 3033), we tested the hypothesis that meal feeding and leucine administration could modulate the phosphorylation and cellular distribution of PKC
and PKC
in skeletal muscle.
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METHODS |
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Meal feeding. For the meal feeding regimen, the rats were adapted to a reverse light cycle (the dark cycle began at 7:00 AM and the light cycle at 7:00 PM). Animals were caged in pairs rather than singly to reduce anxiety-induced changes in food intake. The animals were trained over a period of 12 days to consume a meal when presented. Food was provided in two metal food cups for 3 h beginning 30 min after the beginning of the dark cycle. The diet consisted of Teklad Diet 8604. The concentration of leucine in this diet is 2.04%, and protein concentration is 24.48% according to the manufacturer.
On day 12, animals were weighed (304 ± 3.9 g) and euthanized at six different times relative to the start of the meal. For baseline, a group of animals was sacrificed 0.5 h before presentation of the meal (t 0.5 h). After the meal was provided, animals were sampled at 0.5, 1, 3, 6, and 9 h after the start of the meal. Truncal blood was collected, and the gastrocnemius was removed and immediately frozen between clamps precooled to the temperature of liquid nitrogen.
Leucine gavage.
Animals were food deprived for 18 h and then allocated to one of four treatment groups, as follows: saline (Sal, control), carbohydrate (CHO), norleucine (Nor), or leucine (Leu). At time 0, the animals were randomly divided into the four groups and administered one of the following solutions by oral gavage (2.5 ml/100 g body wt) according to their designated treatment group: Sal (0.155 mol NaCl/l), CHO (262.5 g/l D-glucose mixed with 262.5 g/l D-sucrose), Nor (54.0 g/l L-norleucine), Leu (54.0 g/l L-leucine), as described previously (33). The dose of leucine is equivalent to the amount of leucine consumed by rats of this age and strain during 24 h of free access to a commercial rodent diet (2, 3, 33). The amount of carbohydrate given represents 15% of daily energy intake (2, 3). Thirty minutes later, animals were anesthetized and the gastrocnemius excised and immediately frozen between clamps precooled to the temperature of liquid nitrogen. At the time of tissue sampling, the plasma leucine concentrations were the following: Sal 127 ± 15; CHO 148 ± 13; Leu 2,273 ± 99; and Nor 2,390 ± 74 nmol/ml (33).
Insulin administration. To ascertain the effect of insulin on PKC phosphorylation, rats were anesthetized (Nembutal, 100 mg/kg body wt) and placed on a warming pad to maintain body temperature. A midline incision was made, and insulin (Humulin R, 10 U/kg body wt) was injected in the vena cava using a 27-gauge needle. After withdrawal of the needle, any bleeding was arrested by compression followed by application of a drop of super glue on the blood vessel. The abdomen was closed, and a saline-soaked gauze pad was placed over the wound. Twenty minutes later, the gastrocnemius was excised and frozen between clamps precooled to the temperature of liquid nitrogen.
Chronic leucine feeding.
Animals were allocated to one of the following groups: control (n = 12), leucine supplemented (n = 16), and norleucine supplemented (n = 12), as described previously (32). Animals were caged in pairs to reduce anxiety-induced changes in food intake. The mean starting body weights of animals in each group were not statistically different (control, 96.2 ± 2.6 g, n = 12; leucine supplemented: 97.8 ± 1.8 g, n = 16; norleucine supplemented 96.2 ± 2.4 g, n = 12). Each cage contained two ceramic food containers in separate corners of the cage to facilitate ad libitum feeding. Food remaining in dishes and crumbs that fell through the cage mesh were weighed daily starting 12 days before day 0. Food consumption was calculated as food consumed per cage and divided by 2. Food was provided because we suspected that if leucine were given alone without additional food, protein synthesis might not be stimulated, owing to limitations in amino acid availability. Water or leucine analog-containing water (114 mM leucine or norleucine) was provided starting on day 0. The amount of fluid consumed was measured daily. A filled drinking bottle was always hung on an empty cage per cage rack. This allowed an estimate of the amount of water lost each day from dripping due to placement of the drinking bottle or handling of the wheeled cage rack (12 ml/day). This amount lost was subtracted from the water consumption measurements. Net water consumption was measured per rat cage and divided by 2. Food and water/supplement were provided until the time of anesthetization (Nembutal, 100 mg/kg body wt) on the 12th day of the feeding regimen (32). The gastrocnemius was then excised and frozen between clamps precooled to the temperature of liquid nitrogen. At the time of tissue sampling, the plasma leucine concentrations were the following: Control 160 ± 15; Leucine 235 ± 19; and Norleucine 171 ± 24 (33).
Hindlimb perfusions. In some experiments, the hindlimb was perfused with buffer supplemented with leucine, according to previously described methods (21, 22, 40, 41). Food was not withdrawn before the perfusion experiments, and perfusions were begun by 10:00 AM. Animals were anesthetized (Nembutal, 100 mg/kg) and catheters placed in the descending aorta and vena cava below the level of the renal artery and vein. Perfusion was initiated immediately and the first 50 ml of perfusate were discarded. The perfusate was then recirculated. After an initial period of 15 min, perfusion was continued for an additional 60 min (40). The perfusate (250 ml) consisted of a modified Krebs-Henseleit bicarbonate buffer containing 30% (vol/vol) washed bovine erythrocytes, 4.5% (wt/vol) bovine serum albumin (BSA fraction V), 11 mM glucose, and amino acids present at plasma concentrations with leucine added at either 1 or 10 times the plasma concentration (40). Leucine at 1x concentration serves as the control group for the experiments. Leucine at 10x was selected because previous studies showed it to be a maximally effective concentration for stimulation of skeletal muscle protein synthesis (28, 40). At the conclusion of the perfusion, the gastrocnemius was quickly removed and frozen between clamps precooled to the temperature of liquid nitrogen.
PKC phosphorylation.
An aliquot (0.2 g) of frozen, powdered gastrocnemius was homogenized in 8 volumes of homogenization buffer A (in mM: 20 HEPES, pH 7.4, 2 EGTA, 50 NaF, 100 KCl, 0.2 EDTA, 50 -glycerophosphate, 1 DTT, 0.1 PMSF, 1 benzamidine, and 0.5 sodium vanadate, and 1 µM microcystin LR), using a Polytron homogenizer on ice. An aliquot of the supernatant was reserved for protein assay, and the rest was added to an equal volume of 2x Laemmli sodium dodecyl sulfate (SDS) sample buffer. Proteins were separated on 12.5% acrylamide gels. Following transfer to polyvinylidene difluoride (PVDF) membranes, the blots were probed using phosphospecific antibodies PKC
(Ser729) (Upstate Cell Signaling, Lake Placid, NY), PKC
(Ser643) (Upstate Cell Signaling), or PKC
(Thr505) (Cell Signaling Technology, Vancouver, CA). The blots were then developed using an ECL Western blotting kit (Amersham Pharmacia Biotech, Piscataway, NJ). The blots were exposed to X-ray film in a cassette equipped with DuPont Lightning Plus intensifying screens. Following development, the film was scanned (Microtek ScanMaker IV) and quantitated using NIH Image 1.6 software. After quantification of the relative intensity of the signal for phosphorylation on PKC
(Ser729), PKC
(Ser643), or PKC
(Thr505), the antibodies were removed from PVDF membranes by washing for 1020 min at 50°C with buffer containing 100 mM
-mercaptoethanol, 63 mM Tris·HCl (pH 7.4), and 2% SDS. The blots were then probed with an antibody that recognizes total PKC
or PKC
(i.e., both phosphorylated and unphosphorylated forms; Upstate Cell Signaling). The blots were developed using an ECL Western blotting kit and quantitated as described for phosphorylated forms of the proteins.
S6K1 phosphorylation.
For detection of total S6K1 and phosphorylation of S6K1 on Thr389, proteins in homogenates described above for PKC were separated on 7.5% acrylamide gels with reduced bisacrylamide concentration (30:0.19%) to improve separation of the various phosphorylated isoforms as described previously (32, 33, 41, 44). Following transfer to PVDF membranes, the blots were probed using phosphospecific antibodies raised to a peptide corresponding to p-Thr389 on S6K1 (Cell Signaling Technology, Beverly, MA). The blots were developed using an ECL Western blotting kit and quantitated as described for PKC phosphorylation. After quantification of the relative intensity of the signal for phosphorylation at Thr389, the antibodies were removed from PVDF membranes by washing for 1020 min at 50°C with buffer containing 100 mM -mercaptoethanol, 63 mM Tris·HCl (pH7.4), and 2% SDS. The blots were then probed with an antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes total S6K1 (i.e., both phosphorylated and unphosphorylated forms). The blots were developed using an ECL Western blotting kit and quantitated as described for PKC phosphorylation. Results are presented as the ratio of the densitometric analysis of blot for phosphorylated Thr389 divided by total S6K1 performed on the same gel.
4E-BP1 phosphorylation.
The phosphorylated forms of 4E-BP1 were measured in skeletal muscle extracts following boiling for 5 min of an aliquot (200 µl) of the homogenates obtained for measurement of PKC phosphorylation described above. The boiled homogenate was centrifuged in a microcentrifuge at room temperature, and the supernatant was decanted. An equal volume of 2x Laemmli SDS buffer (65°C) was then added to the supernatant. The various phosphorylated forms of 4E-BP1 (designated ,
, and
) were separated by SDS-PAGE electrophoresis and quantitated by protein immunoblot analysis as described previously (27, 45, 46).
PKC translocation. To assess translocation of PKC isoforms, cytosolic- and membrane-enriched fractions were prepared from gastrocnemius from different experimental conditions. An aliquot (0.2 g) of frozen, powdered gastrocnemius was homogenized in 8 volumes of homogenization buffer A, using a Polytron homogenizer on ice. The homogenate was centrifuged at 750 g for 10 min at 4°C. The supernatant was saved and used as cytosolic fraction. The pellet was resuspended in buffer A with 0.5% (vol/vol) Triton X-100 added and allowed to incubate on ice for 1 h. The sample was then centrifuged at 100,000 g for 60 min at 4°C. The supernatant was representative of the membrane-enriched fraction. The protein content of each sample was measured using the Pierce BCA procedure, as outlined in the manufacturer's instructions, with crystalline serum bovine albumin as a standard. Cytosolic and membrane fractions were stored at 80°C until Western blot analysis. For Western blot analysis of the fractions, equal amounts of protein (50 µg) were electrophoresed.
Statistics. Values are presented as means ± SE of multiple densitometric analyses for each group. Results were compared using a two-tailed, two-sample Student's t-test to assess differences between two treatment groups using the INSTAT program. ANOVA statistical analysis was performed with a Sidak post hoc test when ANOVA indicated a significant difference where more than two comparisons were necessary. Differences were considered significant when P < 0.05.
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RESULTS |
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Next, we examined the potential role of mTOR signaling in mediating the effects of meal feeding on phosphorylation of PKC. Activation of the mTOR signaling pathway is implicated in mediating the short-term effects of refeeding. The phosphorylation of S6K1 and 4E-BP1 following meal feeding was examined as indicators of downstream effectors of mTOR signaling (Fig. 2). At the beginning of the experiment, phosphorylation on S6K1(Thr389) was below the level of detection. Following presentation of the meal, the phosphorylation of S6K1(Thr389) increased. The extent of phosphorylation of S6K1 was significantly maximally elevated by t +0.5 h and remained increased throughout the period when food was provided (t +0.5 to +3 h). Following withdrawal of the meal, the extent of phosphorylation of S6K1 fell significantly by t +6 h and fell below the level of detection by t +9 h. The extent of 4E-BP1 in the
-phosphorylated form mirrored that observed in S6K1 phosphorylation, consistent with the role of mTOR as an upstream regulator of the phosphorylation both S6K1 and 4E-BP1.
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Insulin administration.
Meal feeding or leucine gavage on previously fasted rats would be expected to alter a number of plasma hormones, including insulin. We examined the possibility that a rise in the plasma insulin concentration following meal feeding or leucine gavage would mediate the effects of meal feeding to enhance the phosphorylation of PKC. To accomplish this, we injected fed rats with insulin and analyzed the gastrocnemius for changes in phosphorylation of PKC
. Injection of rats with insulin did not alter the phosphorylation of PKC
(Table 1). Likewise, insulin treatment did not alter the phosphorylation of either PKC
(Ser643) or PKC
(Thr505) in gastrocnemius (Table 1). Furthermore, insulin was unable to cause a shift in the distribution of PKC
from the cytosol to membrane fractions (Table 2).
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Chronic leucine administration.
We next attempted to determine whether the effect of leucine to stimulate PKC phosphorylation persisted if the plasma leucine concentration remained elevated. To accomplish this, we provided leucine in the drinking water for 12 days. Providing leucine in the drinking water raises the plasma leucine concentration by 50% (32) whereas the remainder of the plasma amino acids are relatively unaffected. In the present study, chronic dietary leucine supplementation increased the phosphorylation of PKC
by 71%, consistent with effects of leucine on PKC
phosphorylation observed following gavage (Fig. 4). As was observed with acute leucine administration, chronic dietary leucine supplementation was without effect on the phosphorylation of either PKC
(Ser643) or PKC
(Thr505) in gastrocnemius. Chronic feeding with leucine did not significantly alter the amount of PKC
or PKC
(data not shown). Because these animals were in the postprandial state, the extent of phosphorylation of S6K1 was not significantly different between the two groups (data not shown).
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DISCUSSION |
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However, the other signaling pathways stimulated by leucine remain unresolved in skeletal muscle. In the present set of investigations, we examined the potential effects of elevating leucine concentration both in vivo and in vitro on the novel PKC isoforms PKC and PKC
. Our initial interest in examining the regulation of these two nPKC isoforms by leucine stems from a report using HEK 293 cells in culture, suggesting that phosphorylation of PKC
(Ser662) is controlled by a rapamycin-dependent pathway involving mTOR (34). Furthermore, phosphorylation of PKC
mimicked that of PKC
(Ser662).
Control of protein kinases is necessary as a prerequisite in their operation as signal transducers. Regulation of the PKC family falls mainly into two categories, namely, changes in subcellular distribution and changes in the phosphorylation of the PKCs. The localization of PKC from one compartment to another has profound effects on their activation and specificity of action. Likewise, changes in phosphorylation serve to regulate the catalytic potential of PKC. In the present set of experiments, we studied the ability of meal feeding and leucine to stimulate the phosphorylation and cellular distribution of two of the nPKC isoforms PKC and PKC
.
PKC is a phosphatidylserine/diacylglyceride-dependent, calcium-independent PKC isoform. PKC
is controlled via phosphorylation at three sites in the catalytic domain (Thr566 in the activation loop, Thr710 in the turn motif, and Ser729 in the COOH-terminal hydrophobic motif). Phosphorylation of these sites is required for binding to and activation by diacylglyerol (9, 14, 23). Ser729 phosphorylation is mediated by PKC itself via autophosphorylation (11), a protein complex that includes the atypical PKC
(50) or a rapamycin-sensitive heterologous kinase (34). Regulation of the hydrophobic-site phosphorylation Ser729 may be antagonized by rapamycin, implying that PKC phosphorylation lies downstream of mTOR in an amino acid-sensing pathway(34).
Meal feeding results in a sustained increase in the phosphorylation of PKC that remained elevated as long as the animals consumed a meal. With the cessation of the meal, phosphorylation of PKC
returned to values observed before feeding. In contrast, we did not observe an enhancement of phosphorylation of PKC
following consumption of a meal. Hence, phosphorylation PKC
appears dependent on signals generated by meal feeding, whereas PKC
did not. We examined leucine as a potential mediator of the enhanced phosphorylation of PKC
in response to meal feeding. A role for leucine as a unique modifier of PKC
phosphorylation is supported by the following observations. First, providing animals leucine via gavage acutely or chronically in the drinking water could mimic the extent of stimulation of PKC
phosphorylation. Second, an analog of leucine, norleucine (30), that we have previously shown to stimulate protein synthesis in gastrocnemius (33), stimulated PKC
phosphorylation in vivo following gavage. Third, perfusion of hindlimb with buffer containing elevated concentrations of leucine at least equivalent to that observed following gavage stimulated phosphorylation of PKC
. Thus leucine appears to be a component of the meal associated with enhanced phosphorylation of PKC
.
The signaling pathways leading to a leucine-induced phosphorylation of PKC remain unresolved. In vivo, leucine is an insulin secretagogue, and this has raised the question of whether or not the effects of leucine in vivo are direct effects or rather reflect a leucine-mediated rise in plasma insulin after feeding. Three lines of evidence argue against a role for changes in the plasma insulin concentrations in mediating the leucine-induced phosphorylation of PKC
. First, insulin injection did not increase the phosphorylation of PKC
signaling through the PI 3-kinase-PKB-mTOR pathway to increase the phosphorylation of S6K1 and 4E-BP1. Second, a significant rise in plasma insulin occurs following the carbohydrate meal gavage (2), but providing carbohydrate alone is not associated with significant increases in phosphorylation PKC
. Third, a leucine mimetic, norleucine, does not stimulate insulin secretion or lead to a rise in plasma insulin concentrations following gavage (33). However, norleucine stimulated phosphorylation of PKC
with equal efficacy compared with leucine. These findings argue against a role of insulin in leucine-stimulated phosphorylation of PKC
in gastrocnemius. Taken together, the findings indicate that leucine, as a nutrient signal, acts directly on gastrocnemius independently of the signaling pathways used by insulin. Similar conclusions were reached by Cutherbertson et al. (12) in humans in whom a stimulation of skeletal muscle protein synthesis by essential amino acids occurred when insulin concentration were clamped at basal levels.
The activation of mTOR above basal conditions does not appear to be required for enhanced phosphorylation of PKC. Our reasoning for this conclusion is twofold. First, although elevation of leucine concentrations enhances PKC
phosphorylation in perfused hindlimb, it does so without significant increases in phosphorylation of S6K1 or 4E-BP1, both downstream targets of mTOR (8). Second, activation of mTOR signaling by insulin administration did not augment PKC
phosphorylation. Therefore, it would appear that mTOR activation and PKC
phosphorylation are not necessarily coupled. Last, the immunosuppressant drug rapamycin, an inhibitor of mTOR kinase, did not modify the extent of phosphorylation of PKC
in hindlimb muscles perfused with elevated (10x) plasma concentrations of leucine. However, we cannot preclude the possibility that some low level of mTOR activity is necessary for leucine to stimulate PKC
phosphorylation.
PKC is a phosphatidylserine/diglyceride-dependent, calcium-independent PKC isoform. It is activated by phosphorylation in the activation loop Thr505, presumably following translocation to the membrane (34). Once in an activated conformation, PKC
can be phosphorylated on Ser662 in the catalytic domain. Phosphorylation at residue Thr505 appears important in preventing efficient dephosphorylation of Ser662. Activation of PKC
appears dependent upon stimulation of PI 3-kinase activity in various cells (4, 37, 48). We have focused on the phosphorylation of Ser643 and Thr505. PKC
(Ser643) is a major autophosphorylation site in vitro, and autophosphorylation of PKC
on this site is required for its full enzymatic activity (29). In the present set of experiments, meal feeding or leucine administration in vivo and perfusion with elevated leucine concentrations in vitro failed to stimulate the phosphorylation of PKC
. Such a finding is consistent with our previous report (8) indicating that inhibition of PI 3-kinase with LY-294002 was without effect on the effect of leucine to stimulate protein synthesis in perfused hindlimb. Therefore, the ability of leucine to stimulate PKC
activation is specific and not a generalized effect of leucine on nPKCs.
Aside from phosphorylation, PKC activity can also be modulated through translocation to different cellular fractions. We also examined the effect of leucine on the distribution of PKC between the cytosolic and membrane fractions. We observed an increased abundance of PKC
in the cytosolic fraction in perfused hindlimb in the presence of elevated leucine concentrations. Accompanying this change, there was a corresponding decrease in the abundance of PKC
in the membrane fraction compared with values obtained in animals perfused with 1x leucine. There was a different response with regard to PKC
. Unlike PKC
, there were no significant differences in abundance in either cytosolic fraction or membrane fraction in muscle perfused with medium containing 10x leucine compared with 1x leucine. Thus, in addition to changes in phosphorylation state, leucine appears to have a specific effect on the distribution of PKC
between the cytosolic and the membrane fractions.
The results described herein provide evidence that meal feeding, and leucine in particular, leads to an activation (autophosphorylation) and subcellular redistribution of PKC, but not PKC
, in gastrocnemius both in vivo and in vitro, indicating that the response is specific. Rises in plasma insulin concentrations secondary to meal feeding or leucine gavage are probably not responsible for increased phosphorylation of PKC
. Furthermore, activation of the mTOR-signaling pathway above basal conditions does not appear to be necessary to induce phosphorylation or translocation of PKC
, suggesting that multiple signaling pathways become activated with leucine. As is the case for other PKC isoforms, the potential downstream effector molecules affected by increased phosphorylation of PKC
remain unknown in skeletal muscle. The observations of these experiments will allow investigators to focus future efforts on the physiological role of PKC
in nutrient signaling in skeletal muscle.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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