©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Control of PHAS-I by Insulin in 3T3-L1 Adipocytes
SYNTHESIS, DEGRADATION, AND PHOSPHORYLATION BY A RAPAMYCIN-SENSITIVE AND MITOGEN- ACTIVATED PROTEIN KINASE-INDEPENDENT PATHWAY (*)

(Received for publication, May 5, 1995; and in revised form, June 6, 1995)

Tai-An Lin (1) Xianming Kong (1) Alan R. Saltiel (2) Perry J. Blackshear (3) John C. Lawrence , Jr. (1)(§)

From the  (1)Department of Molecular Biology and Pharmacology Washington University School of Medicine, St Louis, Missouri 63110, the (2)Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105, and the (3)Howard Hughes Medical Institute Laboratories, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-Ibulleteukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H^6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H^6]PHAS-I when the [H^6]PHAS-IbulleteIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-IbulleteIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.


INTRODUCTION

The stimulation of protein synthesis by insulin is the net result of changes in both transcriptional and translational processes (for review, see (1) and (2) ). In liver, heart, skeletal muscle, and adipose tissue, insulin increases total protein by more than it increases total mRNA(2) , indicative of the importance of increased translation in the stimulation of overall protein synthesis by the hormone. The synthesis of certain proteins, such as those encoding ribosomal proteins(3, 4) , is increased disproportionately by insulin. A highly structured (G/C-rich) 5`-nontranslated region (5) or a polypyrimidine motif near the 5` cap (6) are two structural motifs that may contribute to the selective increase in translation in response to insulin. The presence of the cap, m^7GPPPN (where N is any nucleotide)(7) , appears to be required for insulin-stimulated translation to occur(8) .

Generally, the rate-limiting step for translation is the initiation phase, which involves recognition of capped mRNA, melting of secondary structure in the 5` region of the mRNA, and binding to the 40 S ribosomal subunit(9, 10, 11, 12) . These processes require eIF-4F, a complex containing a large subunit (p220) whose function is not fully defined, an ATP-dependent helicase (eIF-4A), (^1)and the mRNA cap binding protein, eIF-4E. The activity of eIF-4E is controlled by PHAS-I(13) , a widely expressed heat- and acid-stable protein that is phosphorylated in response to insulin and growth factors(14, 15, 16) . PHAS-I binds to eIF-4E and inhibits translation of capped mRNA, both in vitro and in intact cells(17, 18) . When phosphorylated in the appropriate sites in response to insulin, PHAS-I does not bind to eIF-4E(16, 17, 18) . Thus, release of eIF-4E from inhibition by PHAS-I may increase eIF-4F activity.

Recombinant PHAS-I is an excellent substrate for MAP kinase(19) , and phosphorylation of PHAS-I by MAP kinase in vitro dramatically decreases binding of PHAS-I to eIF-4E(16) . Moreover, the major site (Ser) phosphorylated by MAP kinase in vitro is phosphorylated in response to insulin in adipocytes(19) . However, other kinases are involved in phosphorylating PHAS-I, as sites in addition to Ser are phosphorylated in the protein (19) . Insulin activates multiple protein kinases in cells(20) . p70 is one such kinase that is part of a signal transduction pathway that is distinct from the MAP kinase signaling pathway(21, 22) . Because MAP kinase and p70 are regulated by many of the same hormones and growth factors, assigning specific functions to the kinases has been difficult. Recently, reagents have become available that allow discrimination between the pathways. The immunosuppressant, rapamycin (for review, see (23, 24, 25) ), blocks activation of p70 by insulin without affecting the activation of MAP kinase or the downstream kinase, p90(26, 27, 28) . An inhibitor (PD 098059) of MEK, the protein kinase that phosphorylates and activates MAP kinase(29) , selectively inhibits the MAP kinase signaling pathway (30) .

The experiments described in this report were performed to investigate mechanisms by which insulin controls the levels of PHAS-I and regulates the phosphorylation of protein.


EXPERIMENTAL PROCEDURES

3T3-L1 Adipocytes

3T3-L1 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum on plastic culture dishes (10-cm diameter) and converted to adipocytes by using differentiation medium as described previously (33) . Unless otherwise stated, experiments were performed on adipocytes 12-16 days after withdrawal from differentiation medium. The growth medium was replaced with Low P(i) buffer containing 135 mM NaCl, 5.4 mM KCl, 1.4 mM CaCl(2), 1.4 mM MgSO(4), 5 mM glucose, 5 mg/ml bovine serum albumin, 0.2 mM NaP(i), and 10 mM HEPES (pH 7.4). In P-labeling experiments, the Low P(i) buffer was supplemented with NaP(i) (2 mCi/5 ml). The cells were homogenized after 3 h of incubation in Low P(i) buffer. Additions of insulin and other agents were made at the appropriate time prior to the end of this incubation period to give the desired treatment. To terminate the incubation, the medium was aspirated, and the cells were rinsed twice with phosphate-buffered saline and homogenized (1 ml of buffer/dish) in a glass homogenization tube with a Teflon pestle driven at 1,000 rpm. Phosphate-buffered saline contained 145 mM NaCl, 5.4 mM KCl, and 10 mM NaP(i) (pH 7.4). Homogenization buffer contained 1 mM EDTA, 5 mM EGTA, 10 mM MgCl(2), 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM benzamidine, 10 mM KP(i), and 50 mM beta-glycerophosphate (pH 7.3). Homogenates were centrifuged at 10,000 g for 20 min, and the supernatants were retained for analyses. The protein content was determined by using bicinchoninic acid(34) .

S Labeling

Adipocytes were rinsed twice and incubated for 1 h at 37 °C in methionine-free DMEM. To measure the initial rate of incorporation of [S]methionine into PHAS-I, the medium was replaced with methionine-free DMEM supplemented with 10 µM [S]methionine (10 µCi/ml) and incubated for increasing times (±20 nM insulin). In pulse-chase experiments, cells were incubated with 10 µM [S]methionine (10 µCi/ml) for 3 h at 37 °C and then rinsed twice with DMEM and incubated (±20 nM insulin) for increasing times in DMEM supplemented with 2 mM methionine. The cells were homogenized as described above.

Antibodies and Immunoprecipitations

PHAS-I antibodies were generated by immunizing rabbits with a peptide (CSSPEDKRAGGEESQFE) having a sequence derived from the COOH-terminal region of PHAS-I. The antibodies were affinity-purified as described previously(13) . Antiserum to eIF-4E was generated in rabbits and was provided by Nahum Sonenberg (McGill University). GLUT-4 antibody was prepared by immunizing rabbits with a peptide having the sequence of the last 12 residues in the COOH terminus of GLUT-4 and was affinity-purified before use(35) . For immunoprecipitation, PHAS-I antibodies were coupled to protein A-agarose beads (0.5 mg of antibody/ml of beads) by incubation at 23 °C for 60 min in Homogenization buffer. The beads were then washed 3 times with Homogenization buffer. Samples (100 µl) of extract were incubated with beads (10 µl) for 30 min at 23 °C with constant mixing. After washing the beads 5 times, proteins were eluted with SDS sample buffer.

Electrophoretic Analyses

Samples were subjected to SDS-PAGE using the method of Laemmli(36) . To determine the relative S contents of proteins, gels were dried and radioactivity was measured using a PhosphorImager (Molecular Dynamics). For immunoblotting, proteins were electrophoretically transferred to nylon membranes (Immobilon, Millipore). The membranes were immersed in phosphate-buffered saline containing 5% powdered milk (Carnation) and incubated for at least 1 h. Sheets were then incubated with PHAS-I antibody (2 µg/ml) or GLUT-4 antibody (2 µg/ml) in phosphate-buffered saline plus milk for 2 h and then washed as described previously(37) . Antibody binding was detected by enhanced chemiluminescence (Tropix system) using alkaline phosphatase conjugated to either goat anti-rabbit IgG or protein A. The intensities of the bands corresponding to PHAS-I were determined by two-dimensional scanning using a laser densitometer (Molecular Dynamics).

Two-dimensional electrophoresis, with isoelectric focusing in the first dimension and SDS-PAGE in the second, was performed as described by Blackshear et al.(38) . PHAS-I was detected by immunoblotting, essentially as described above, except that antibody binding was detected using horseradish peroxidase conjugated to goat anti-rabbit IgG and a colorimetric stain as directed by the supplier (Bio-Rad, horseradish peroxidase color development reagent).

Cloning of 3T3L1 PHAS-I cDNA

A 3T3-L1 adipocyte cDNA library (in Zap II) provided by Dr. Fred Fiedorek (University of North Carolina) was used to obtain mouse PHAS-I cDNA by expression cloning(39) . Briefly, phage (10^6) were grown at 42 °C until small plaques became visible. Nitrocellulose membranes impregnated with isopropyl-1-thio-beta-D-galactopyranoside to induce expression of proteins were applied, and the phage were allowed to grow for 3 h at 37 °C. Membranes were incubated in blocking solution and PHAS-I antibodies and then washed essentially as described for detecting PHAS-I by immunoblotting. Clones that bound antibodies were detected by enhanced chemiluminescence. After plaque-purification, inserts were subcloned into pBluescript SK (Stratagene) for nucleotide sequencing, which was performed using an automatic sequencing apparatus and sequencing kit (part 901497) as directed by the supplier (Applied Biosystems). Out of the 25 positive clones isolated, 15 represented partial-length cDNAs that had sequences identical to regions of the PHAS-I sequence (see Fig. 1), which was derived separately from two independent clones.


Figure 1: Nucleotide sequence of mouse PHAS-I cDNA (A) and the deduced amino acid sequence of PHAS-I protein from 3T3 L1 cells (B).



Preparation of Recombinant Proteins

``Histidine-tagged'' PHAS-I ([H^6]PHAS-I) was expressed in bacteria and purified as described previously(19) . Human eIF-4E cDNA cloned into pET-11d (Novagen) was provided by Dr. Rosemary Jagus (Maryland Biotechnology Institute, Baltimore, MD). eIF-4E was expressed in Escherichia coli and purified as described by Stern et al.(40) . [H^6]PHAS-IbulleteIF-4E complexes were generated by allowing eIF-4E to renature in the presence of excess [H^6]PHAS-I. This was accomplished by slight modification of the eIF-4E purification method(40) . E. coli (BL21(DE3)pLysS strain, 800 ml) expressing [H^6]PHAS-I was pelleted by centrifugation (7,000 g for 15 min) and homogenized in 30 ml of 8 M urea, 50 mM dithiothreitol, and 50 mM HEPES (pH 7.6). After centrifugation at 39,000 g for 20 min, the supernatant containing [H^6]PHAS-I was added to 20 ml of a solution containing eIF-4E that had been extracted from inclusion bodies (from 800 ml of bacteria) by using this buffer. Subsequent steps, including renaturation by sequential dialysis to remove urea and affinity chromatography with m^7GTP-Sepharose, were performed as described by Stern et al.(40) . Because [H^6]PHAS-I does not bind directly to m^7GTP-Sepharose(16) , only [H^6]PHAS-I bound to eIF-4E is recovered with the cap affinity resin. To estimate the relative amounts of [H^6]PHAS-I and eIF-4E in the complex, samples were subjected to SDS-PAGE, and proteins were stained with Coomassie Blue (for example, see Fig. 12). Based on staining intensities determined by optical density scanning, the preparation contained 0.5 mg of [H^6]PHAS-I (predicted M(r) = 15,094) per mg of eIF-4E (predicted M(r) = 25,117), which is similar to the proportion of 0.6 mg of [H^6]PHAS-I per mg of eIF-4E that would be expected of a 1:1 complex of [H^6]PHAS-I and eIF-4E.


Figure 12: Resolution of PHAS-I kinases by Mono Q chromatography. Adipocytes were incubated for 10 min without additions (), with 20 nM EGF (▴), or with 20 nM insulin (▪). Extracts were applied to a Mono Q column, and proteins were eluted with an increasing gradient of NaCl. PHAS-I kinase activities were measured using [-P]ATP and either 50 µg/ml [H^6]PHAS-IbulleteIF-4E (A) or 25 µg/ml [H^6]PHAS-I (B) as substrates. After incubating for 20 min at 30 °C, samples were subjected to SDS-PAGE, and amounts of P incoroporated into PHAS-I were determined by scintillation counting of gel slices. A Coomassie Blue-stained gel of the preparations of [H^6]PHAS-I (lane1) and [H^6]PHAS-IbulleteIF-4E (lane2) used is shown as an inset in B. Protein kinase activities in fractions 56-60 were also assayed in the absence and presence of heparin (from porcine intestine, 0.1 mg/ml) with phosvitin (1.5 mg/ml) as substrate (C). Protein kinase activities (A-C) are expressed as pmol of phosphate incorporated into either PHAS-I or phosvitin/min/ml column fraction.



Anion-exchange Chromatography

Extracts (5 ml) were diluted with H(2)O (5 ml) and applied at 21 °C to a Mono Q HR 5/5 column (Pharmacia Biotech Inc.) equilibrated with Buffer A (1 mM EGTA, 0.1 mM Na(3)VO(4), and 50 mM beta-glycerol phosphate, pH 7.3) as described previously(41) . The flow rate was maintained at 1 ml/min, and fractions were collected each min. Proteins were eluted by increasing Buffer B (Buffer A plus 800 mM NaCl) to produce the following gradient of NaCl: 0 mM for 15 min, 0-350 mM in 50 min, and 350-800 mM in 5 min.

Measurements of Protein Kinase Activities

The activities of the ERK-1 and ERK-2 isoforms of MAP kinase activity were measured using [-P]ATP and myelin basic protein as substrates in the gel renaturation method described by Wang et al.(42) . Ribosomal protein S6 kinase activity was measured using intact 40 S ribosomes as substrate as described previously(41) .

Other Materials

Porcine insulin (27 units/mg) was supplied by Eli Lilly Co. PD 098059 was provided by Parke-Davis. EGF was purchased from Boeringer Mannheim. [-P]ATP and [S]methionine were from DuPont NEN. Rapamycin and FK506 were obtained from Calbiochem and Fujisawa Pharmaceuticals, respectively. Most commonly used chemicals were from Sigma. Oligonucleotides and peptides were synthesized by the Molecular Biology Core Laboratory of the Washington University Diabetes Center.


RESULTS

Mouse PHAS-I

PHAS-I cDNA from 3T3-L1 adipocytes (Fig. 1) was highly homologous to rat PHAS-I cDNA(13) . Overlapping regions of the G/C-rich 5`-nontranslated regions were identical in the rat and mouse cDNAs, and the deduced amino acid sequences of the proteins from the two species were 97% identical. As in the rat protein, 27 out of 117 amino acids in mouse PHAS-I are Ser/Thr residues (Fig. 1), which represent potential sites of phosphorylation. The major site (Ser) phosphorylated by MAP kinase is conserved in mouse PHAS-I, as is the consensus PEST motif between Asp and Ser.

Control of PHAS-I Levels in 3T3-L1 Cells

Fat contained the highest levels of PHAS-I mRNA (13) and protein (16, 43) of several rat tissues analyzed, suggesting that PHAS-I was induced during the formation of fat cells. To investigate this possibility, levels of PHAS-I were measured by immunoblotting at different times as 3T3-L1 fibroblasts differentiated into adipocytes (Fig. 2). PHAS-I increased markedly as the cells differentiated. The increase in PHAS-I was first noted on the fourth day after adding differentiation medium. This rise preceded by 2 days the first detectable increase in levels of the insulin-responsive glucose transporter, GLUT4, which was monitored as a marker of the terminally differentiated state. By 8-days, PHAS-I reached a maximum, representing an increase of approximately 8-fold over the level of the protein found in fibroblasts.


Figure 2: Expression of PHAS-I and GLUT4 during differentiation of 3T3-L1 fibroblasts into adipocytes. To induce differentiation, the medium of confluent cultures of 3T3-L1 fibroblasts was replaced (day 0) with growth medium (10% fetal bovine serum in DMEM) supplemented with 0.5 mM isobutylmethylxanthine, 0.25 µM dexamethasone, and 350 nM insulin. This medium was replaced after 2 days with growth medium, which was then changed every other day. Cells were homogenized at increasing times after addition of the differentiation medium. Homogenates were centrifuged at 100,000 g for 45 min. Samples (25 µg of protein) of the supernatants and pellets were subjected to SDS-PAGE, and immunoblots were prepared for PHAS-I and GLUT4, respectively. Levels of the proteins are expressed as percentages of the maximum level detected, and are mean values ± half of the range of two experiments.



3T3-L1 adipocytes were used to investigate the regulation of PHAS-I synthesis and degradation in vitro (Fig. 3). Treating adipocytes with insulin for 2 days increased the amount of PHAS-I protein (measured by immunoblotting) relative to total protein by 3.3 ± 0.5-fold (n = 3 experiments). To investigate the mechanism of the increase in PHAS-I, [S]methionine-labeling experiments were performed. Relative rates of synthesis of PHAS-1 were determined by incubating 3T3-L1 adipocytes in the absence or presence of insulin for increasing times with [S]methionine before PHAS-I was immunoprecipitated (Fig. 3A). Incorporation of [S]methionine into PHAS-I increased in a linear manner for 3 h. Insulin increased the initial rate of incorporation of [S]methionine by approximately 3-fold (Fig. 3A). This effect of insulin was most likely due to increased translation of PHAS-I mRNA, as it was observed after incubating with the hormone for only 1 h, a time in which we have been unable to detect any change in PHAS-I mRNA. (^4)


Figure 3: PHAS-I synthesis (A) and degradation (B). A, 3T3-L1 adipocytes were incubated in medium containing [S]methionine in the absence and presence of 20 nM insulin for increasing times. PHAS-I was immunoprecipitated and subjected to SDS-PAGE. The gels were dried, and S was detected by using a PhosphorImager. The amount of S in PHAS-I is expressed in arbitrary units, where 1.0 represents the S content of PHAS-I found in control cells after 6 h of incubation with [S]methionine. The results are mean values ± S.E. from three experiments. B, 3T3-L1 adipocytes were incubated in medium containing [S]methionine in the absence and presence of 20 nM insulin for 3 h. The cells were then rinsed to remove [S]methionine, incubated (with or without insulin) in DMEM containing 2 mM methionine, and homogenized after increasing times. [S]PHAS-I was immunoprecipitated and subjected to SDS-PAGE before relative amounts of radioactivity were determined using a PhosphorImager. The results are expressed in arbitrary units, with 1.0 representing the S content of PHAS-I recovered in PHAS-I immediately after the 3-h incubation with [S]methionine. Mean values ± half of the range of two experiments are presented.



Pulse-chase experiments were performed to investigate the rate of degradation of PHAS-I (Fig. 3B). Cells were incubated with [S]methionine for 3 h to label the endogenous pool of PHAS-I. The cells were then rinsed and incubated in the absence and presence of insulin in medium supplemented with excess unlabeled methionine to limit reutilization of [S]methionine released as result of the degradation of S-labeled proteins. Assuming the disappearance of [S]PHAS-I to be a first order process, values for t of [S]PHAS-I in control and insulin-treated cells were approximately 1.7 and 2.6 days, respectively. In the presence of insulin, the t of PHAS-I was approximately 20 times higher than that of insulin receptor substrate-1 (2-3 h) (44) , another insulin-stimulated phosphoprotein (20) that contains multiple PEST domains(44) . The relatively slow rate of degradation of PHAS-I indicates that the PEST motif in PHAS-I does not target it for rapid degradation in these cells.

Control of PHAS-I Phosphorylation

PHAS-I (M(r) approx 12,400) migrates anomalously when subjected to SDS-PAGE (apparent M(r) = 20,000-24,000)(19) , probably because of its high content of proline and glycine(13) . Moreover, PHAS-I from most tissues appears as three bands, denoted alpha, beta, and (Fig. 4), representing protein phosphorylated to differing extents(16) . Because phosphorylation decreases the electrophoretic mobility of the protein, the shift to species of higher apparent M(r) is indicative of increased phosphorylation(16) . In nonstimulated cells, approximately 20% of the PHAS-I was present as the nonphosphorylated (alpha) form, which binds tightly to eIF-4E. As previously observed (16, 17, 18) , PHAS-I alpha was the predominate form found bound to the initiation factor when complexes were isolated from extracts of control cells by affinity purification with m^7GTP-Sepharose (Fig. 4). However, some PHAS-I beta was also recovered with the cap-affinity resin (Fig. 4), indicating that at least one site in PHAS-I may be phosphorylated without loss of eIF-4E binding.


Figure 4: Regulation of the phosphorylation of PHAS-I and the association of PHAS-I and eIF-4E by insulin and rapamycin. 3T3-L1 adipocytes were incubated without or with 20 nM rapamycin for 10 min. The cells were then incubated for increasing times without insulin, with 20 nM insulin, or with the combination of insulin plus rapamycin. Samples of extracts (20 µl) were dissolved in SDS sample buffer. Other samples (100 µl) were incubated with m^7GTP-Sepharose to determine the relative amounts of PHAS-I that copurified with eIF-4E. After washing the resins, proteins were eluted with SDS sample buffer. Representative immunoblots of extracts (Extract) and of proteins that bound m^7GTP-Sepharose (mGTP) in the regions containing PHAS-I are presented.



The effects of insulin on increasing PHAS-I phosphorylation occurred rapidly, with maximum effects observed after 10 min of incubation (Fig. 5). PHAS-I alpha and beta were decreased by 60% after this time (Fig. 5, A and B), and the more highly phosphorylated PHAS-I was increased 8-fold (Fig. 5C). The effects of insulin persisted for 60 min, although some dephosphorylation of PHAS-I occurred with the longer periods of incubation, as indicated by an increase in the beta form (Fig. 5B). Insulin also rapidly decreased the amount of PHAS-I bound to eIF-4E (Fig. 5D). The maximum effect was observed after 5 min and represented a decrease of approximately 60% in the amount of PHAS-I bound to eIF-4E. Partial reversal of the insulin effect was noted after 60 min (Fig. 5D) and was associated with increased binding of PHAS-I beta (Fig. 4).


Figure 5: Time course of insulin action on PHAS-I in the absence and presence or rapamycin. Experiments were conducted as described in the legend to Fig. 4. Effects of treatments on PHAS-I alpha (A), PHAS-I beta (B), and PHAS-I (C) were determined from differences in the optical densities of the appropriated bands, which were measured by scanning laser densitometry. Changes in PHAS-I binding to eIF-4E (D) represent differences in the amounts of PHAS-I (alpha plus beta forms) recovered when eIF-4E was partially purified using m^7GTP-Sepharose. The abscissa (A-D) represents the time of incubation after the addition of insulin. Note that because rapamycin was added 10 min before insulin, the actual time of incubation with rapamycin (in both the Rapamycin or Rapamycin + Insulin treatments) was 10 min longer than indicated by the scale on the x axes. The results are expressed as percentages of the respective control values and are means ± S.E. from three experiments.



The effects of insulin on the MAP kinase isoforms, ERK-1 (Fig. 6A) and ERK-2 (Fig. 6B), and on ribosomal protein S6 kinase activity (Fig. 6C) also reached peaks after 10 min of incubation. As noted previously(33) , the effects of insulin on the MAP kinases declined with longer times of incubation with the hormone, although activities were still approximately 2 times higher than the control after an hour of incubation (Fig. 6, A and B). The effect of insulin on increasing ribosome protein S6 kinase activity was more prolonged than the effect on MAP kinase, and S6 kinase was more than 6-fold higher than the control after 1 h with insulin (Fig. 6C). Thus, the time course of PHAS-I phosphorylation in response to insulin correlated better with that of S6 kinase activation than that of MAP kinase activation. Although p70 itself does not phosphorylate PHAS-I in vitro(19) , these findings suggest that other elements in the p70 pathway might be involved in regulating PHAS-I.


Figure 6: Time courses of the activation of MAP kinase and ribosomal protein S6 kinases by insulin in the absence and presence of rapamycin. Cells were incubated with rapamycin and insulin as described in the legend to Fig. 4. Activation of the ERK-1 (A) and ERK-2 (B) isoforms of MAP kinase was monitored using an in-gel assay with myelin basic protein as substrate (42) . The activity of S6 protein kinases (C) was measured using intact 40 S ribosomes as substrate. The x axes represent the times of incubation with insulin (see legend to Fig. 5). The results are expressed as percentages of the respective control values and are means ± S.E. from three experiments.



Attenuation of Insulin-Stimulated Phosphorylation by Rapamycin

Results obtained with rapamycin also suggest a role of the p70 pathway in regulating the phosphorylation of PHAS-I. Incubating cells with rapamycin alone decreased the phosphorylation of PHAS-I, as indicated by decreased PHAS-I (Fig. 5C), and increased PHAS-I alpha (Fig. 5A). Rapamycin also attenuated the effects of insulin on increasing the phosphorylation of PHAS-I. In the presence of rapamycin, insulin did not decrease the level of PHAS-I alpha below the control level, even after an hour of incubation (Fig. 5A). As might be expected, rapamycin markedly inhibited the effect of insulin on decreasing the amount of PHAS-I bound to eIF-4E, although an effect of the hormone was observed with longer times of incubation (Fig. 5D). After 60 min, PHAS-I binding to eIF-4E was decreased by approximately 25% in cells incubated with the combination of insulin plus rapamycin. Moreover, in the presence of rapamycin, insulin still increased PHAS-I by 3-4-fold (Fig. 5C), indicating that the hormonal effect on PHAS-I phosphorylation was not completely inhibited. In contrast, rapamycin abolished the increase in ribosomal protein S6 kinase activity (Fig. 6C). This finding indicates that under the conditions of these experiments, essentially all of the ribosomal protein S6 protein kinase activity measured in extracts was due to p70, as rapamycin inhibits activation of this kinase without affecting p90(26, 27, 28) . MAP kinase activity was also affected by rapamycin, but the direction of the effect depended on the time of incubation with insulin. In cells incubated for 5 min with insulin, prior treatment with rapamycin increased the amount of ERK-1 and ERK-2 activity; however, with longer times of incubation with insulin, the activities of the MAP kinase isoforms were slightly less in extracts of rapamycin-treated cells (Fig. 6, A and B).

Rapamycin appears to act in cells by binding to one or more members of a family of FK506 binding proteins (FKBPs)(23, 24, 25) . The inhibitory action of rapamycin on p70 can be partially reversed by the structurally related drug, FK506(26, 27, 28) , which competes with rapamycin for binding to FKBPs(23, 24, 25) . Consistent with previous findings with p70(26, 27, 28) , FK506 attenuated the inhibitory effect of rapamycin on the activation of ribosomal protein S6 kinase by insulin (Fig. 7B). FK506 was without effect on the amount of PHAS-I alpha in either the absence or presence of insulin; however, FK506 abolished the effects of rapamycin on PHAS-I alpha in both the presence and absence of the hormone (Fig. 7A). These results indicate that the effects of rapamycin on PHAS-I phosphorylation are mediated by FKBPs.


Figure 7: Reversal of the effects of rapamycin on PHAS-I and S6 protein kinases by FK506. 3T3L1 adipocytes were incubated in duplicate without additions, with 20 nM rapamycin, with 12 µM FK506, or with the combination of rapamycin plus FK506 for 10 min. After adding insulin to half of the cells, the incubations were continued for 10 min. A, samples were subjected to SDS-PAGE, and the relative amounts of PHAS-I alpha were determined by immunoblotting. The results are expressed as a percentage of the control and are mean values + S.E. from three experiments. B, ribosomal protein S6 kinase was measured in cell extracts. The results are expressed as pmol of phosphate incorporated into protein S6 per min/culture, and are mean values ± S.E. of three experiments.



Failure of the MEK Inhibitor, PD 098059, to Prevent Insulin-stimulated Phosphorylation of PHAS-I

PD 098059 acts noncompetitively with respect to ATP to inhibit MEK1 and MEK2 in vitro.^2 The inhibitor blocks the activation of MEK by growth factors in a variety of cells and inhibits the hormonal stimulation of MAP kinase activity without inhibiting other protein or lipid kinases(30) . Incubating 3T3L1 adipocytes with 50 µM PD 098059 decreased the effects of insulin on ERK-1 and ERK-2 by approximately 80% (Fig. 8, A and B). Experiments were conducted with P-labeled adipocytes to investigate the effects of the MEK inhibitor on PHAS-I phosphorylation (Fig. 9). P-Labeled PHAS-I immunoprecipitated from extracts appeared as two bands corresponding to PHAS-I beta and (Fig. 9A). Incubating cells with the inhibitor was without effect on the P-content of PHAS-I. Rapamycin attenuated the insulin-stimulated phosphorylation of PHAS-I (Fig. 9A), confirming the results from the immunoblotting analyses (Fig. 5C). Rapamycin abolished phosphorylation of the M(r) = 31,000 extract protein (Fig. 9A), previously shown to be ribosomal protein S6(45) . Phosphorylation of S6 was also slightly decreased by the MEK inhibitor, as was insulin-stimulated ribosomal protein S6 kinase activity (Fig. 9B).


Figure 8: Effects of rapamycin and MEK inhibitor on the activation of MAP kinase and ribosomal protein S6 kinases. Adipocytes were incubated in medium without additions, with 20 nM rapamycin for 10 min, with 50 µM PD 098059 for 30 min or with PD 098059 for 20 min, followed by rapamycin plus PD 098059 for 10 min. Insulin was then added to the medium, and incubations were continued for 10 min. Activities of ERK-1 (A), ERK-2 (B), and ribosomal protein S6 kinases (C) are expressed as a percentage of the respective control values. Means ± S.E. of six experiments are presented.




Figure 9: Effects of rapamycin and MEK inhibitor on insulin-stimulated phosphorylation of ribosomal protein S6 and PHAS-I. A, P-Labeled adipocytes were subjected to the treatments indicated in the legend to Fig. 8. Samples of extract and of immunoprecipitated PHAS-I were subjected to SDS-PAGE. An autoradiogram of the dried gel of extract samples is presented. S6 denotes P-labeled ribosomal protein S6, which has an apparent M(r) = 31,000. The picture (PHAS-I IP) of immunoprecipitated PHAS-I was obtained using a PhosphorImager. B, samples of extracts from cells incubated without P were subjected to SDS-PAGE. Other samples were incubated with m^7GTP-Sepharose. After washing, the resin proteins were eluted with SDS-sample buffer and subjected to electrophoresis. PHAS-I immunoblots are presented.



Consistent with its failure to affect the P content of PHAS-I, PD 098059 did not change the electrophoretic pattern of PHAS-I determined by immunoblotting (Fig. 9B). Quantitation of results from six such experiments indicated that the MEK inhibitor was without effect on the proportions of PHAS-I alpha (Fig. 10A), beta (Fig. 10B), or (Fig. 10C). Moreover, the inhibitor did not attenuate the effect of insulin on dissociation of the PHAS-IbulleteIF-4E complex assessed by m^7GTP binding (Fig. 9B and 10D), although in some experiments PD 098059 itself decreased the amount of PHAS-I recovered with the m^7GTP resin.


Figure 10: Effects of rapamycin and MEK inhibitor on PHAS-I. Cells were incubated as described in the legend to Fig. 8. Relative amounts of PHAS-I alpha (A), PHAS-I beta (B), and PHAS-I (C) forms were determined by optical density scanning, and are expressed as a percentage of the total PHAS-I present. The relative amount of PHAS-I bound to eIF-4E (D) was assessed by determining how much PHAS-I (alpha and beta forms) was recovered when eIF-4E was partially purified using m^7GTP. Results in D are expressed as a percentage of the control. The results presented (A-D) are mean values ± S.E. from six experiments.



Multiple Forms of PHAS-I alpha, beta, and Identified by Two-dimensional Electrophoresis

To investigate the complexity of PHAS-I phosphorylation, extracts from control and insulin-treated cells were analyzed by two-dimensional electrophoresis (Fig. 11). Consistent with increasing PHAS-I phosphorylation, insulin promoted a general shift to species of lower pI and electrophoretic mobility. PHAS-I alpha from control cells appeared as a doublet, containing the spots labeled alpha1 and alpha2. As the alpha band did not appear to be labeled with P in extracts of control cells (Fig. 9),^4 alpha1 and alpha2 probably represent PHAS-I species that differ in covalent modification other than phosphorylation. Consequently, the doublets containing beta1 and beta2 and beta7 and beta8, and the minor doublet containing beta4 and beta5 may represent corresponding pairs differing by such a modification, although it is not feasible to determine if this is the case. The relatively large shifts in mobility produced by insulin are almost certainly due to phosphorylation. A total of eight major spots and six minor spots were detected by immunoblotting. Even allowing for other covalent modifications, it seems clear that several sites in PHAS-I are phosphorylated in response to insulin.


Figure 11: Resolution of multiple forms of PHAS-I alpha, beta, and by two-dimensional electrophoresis. Adipocytes were incubated without (A, control) or with (B) 20 nM insulin for 10 min. Extract proteins were resolved by isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. The composite drawing (C) identifies spots detected in gels of the control and insulin-treated samples.



Insulin-stimulated PHAS-I Kinases

To attempt to identify PHAS-I kinases, extracts from 3T3L1 adipocytes that had been incubated with insulin or EGF were fractionated by Mono Q chromatography before PHAS-I kinase activity was measured using recombinant [H^6]PHAS-I or a complex of [H^6]PHAS-I and eIF-4E as substrates. In agreement with previous results obtained with native recombinant PHAS-I(41) , both insulin and EGF increased [H^6]PHAS-I kinase activities that eluted in the positions of ERK-1 and ERK-2 (Fig. 12B). In contrast, neither insulin nor EGF increased kinase activity toward [H^6]PHAS-I bound to eIF-4E (Fig. 12A). We were also unable to detect insulin- or EGF-stimulated PHAS-I kinase activity in unfractionated extracts when the [H^6]PHAS-IbulleteIF-4E complex was used as substrate.^4

A kinase that acted on both free [H^6]PHAS-I and [H^6]PHAS-I bound to eIF-4E eluted at relatively high concentrations of NaCl (fraction 58). This enzyme phosphorylated phosvitin and was inhibited by heparin (Fig. 12C). These properties(46) , and the position of elution from Mono Q(47) , are suggestive of casein kinase II, which has been shown previously to phosphorylate PHAS-I(19, 48) .


DISCUSSION

The present findings demonstrate that the stimulation of PHAS-I phosphorylation by insulin involves at least one pathway that is independent of the MAP kinase signaling pathway. The complex two-dimensional electrophoretic pattern indicates that several sites in PHAS-I are phosphorylated in response to insulin (Fig. 11). As MAP kinase phosphorylates only one site, Ser, at a reasonable rate in vitro(19) , other kinases must be involved in the insulin response to account for multisite phosphorylation of PHAS-I. Moreover, by using rapamycin and PD 098059, the activation of MAP kinase could be dissociated from PHAS-I phosphorylation. Rapamycin markedly reduced the effect of insulin on phosphorylating PHAS-I without inhibiting MAP kinase (Fig. 4-6), and the MEK inhibitor markedly decreased MAP kinase activity under conditions in which the phosphorylation of PHAS-I was not detectably changed (Fig. 8-10). Thus, it seems clear that MAP kinase is not the only mediator of PHAS-I phosphorylation.

Role of MAP Kinase

Our results do not eliminate MAP kinase as a mediator of PHAS-I phosphorylation in cells. Supporting a role of MAP kinase is an increasing list of agents that both activate MAP kinase and increase the phosphorylation of PHAS-I in adipocytes. In addition to insulin, this list includes EGF(14, 16, 38) , platelet-derived growth factor(14, 38) , phorbol 12-myristate 13-acetate(38) , and vanadate.^4 However, these agents also activate other kinases. Moreover, in considering the role of the MAP kinase in regulating the association of PHAS-I and eIF-4E, it is necessary to address the inability of ERK-1 and ERK-2 to phosphorylate PHAS-I when the protein was bound to eIF-4E (Fig. 12). Although the rate of dissociation of nonphosphorylated PHAS-I and eIF-4E has not been measured directly in cells, it appears to be relatively slow, as complexes of PHAS-I and eIF-4E can be isolated from cell extracts. The stability of the complex implies that the bound form of PHAS-I would have to be phosphorylated to account for the rapid dissociation of PHAS-I and eIF-4E observed in response to insulin ( Fig. 4and Fig. 5). Phosphorylation of the free PHAS-I might still serve an important function, as phosphorylation of Ser should prevent reassociation of the protein with eIF-4E(16) . Thus, MAP kinase might function in this capacity. It is also possible that MAP kinase acts on the phosphorylated form of PHAS-I found in the complex. To test this hypothesis, it will be necessary to determine how to generate the PHAS-I betabulleteIF-4E complex.

Potential Mechanisms Involved in Rapamycin-sensitive Regulation of PHAS-I

An important finding is that the control of PHAS-I phosphorylation involves a rapamycin-sensitive pathway. The dephosphorylation of PHAS-I in response to rapamycin was associated with increased PHAS-I binding to eIF-4E (Fig. 5D), indicative of the functional importance of this pathway. Rapamycin appears to exert its action in cells by binding to FKBP12, a protein with peptidyl-prolyl isomerase activity(23, 24, 25) . Rapamycin and FK506 compete for binding to the same site in FKBP12, and both drugs inhibit the isomerase activity. This activity is not involved in the effects of rapamycin on PHAS-I phosphorylation, since FK506 did not affect PHAS-I phosphorylation (Fig. 7A). However, FK506 abolished the effects of rapamycin on PHAS-I phosphorylation (Fig. 7A), consistent with the interpretation that the drug effects were mediated by FKBP12.

When bound to rapamycin, FKBP12 binds tightly to a much larger protein, designated TOR (target of rapamycin)(23, 24, 25) . TOR was originally identified in yeast, but mammalian counterparts, termed FRAP(49) , RAFT1(57) , and mTOR (50) have been recently cloned. TOR proteins have regions of homology with the lipid kinase, phosphatidylinositol-3`-OH kinase, although it has not been demonstrated that the TORs possess lipid kinase activity. Sensitivity to rapamycin is generally viewed as indicative of the p70 pathway, since rapamycin blocks activation of the kinase by insulin and growth factors. Activation of p70 is also blocked by wortmannin, a potent inhibitor of phosphatidylinositol-3`-OH kinase(51) . We have found that wortmannin potently inhibits the phosphorylation of PHAS-I in response to insulin.^4 Thus, effects of both wortmannin and rapamycin are suggestive a role of the p70 pathway in mediating the phosphorylation of PHAS-I.

Purified p70 from cycloheximide-treated rat liver did not phosphorylate recombinant PHAS-I in vitro(19) . Moreover, no insulin-stimulated PHAS-I kinase activity was detected in the elution position (see (41) ) of p70 when extracts of 3T3-L1 adipocytes were fractionated by Mono Q (Fig. 12). Therefore, it is unlikely that direct phosphorylation of PHAS-I by p70 explains PHAS-I phosphorylation in cells. However, other elements in the p70 pathway are candidates. Activation of p70 is associated with phosphorylation of four clustered sites having a Ser/Thr-Pro motif(52) . Interestingly, PHAS-I contains seven such motifs (Fig. 1), and one of these (Ser) has been shown to be phosphorylated in response to insulin in rat adipocytes(19) . Thus, PHAS-I and p70 might be phosphorylated by the same kinase.

Another possibility is that rapamycin promotes dephosphorylation of PHAS-I, not by inhibiting a PHAS-I kinase, but by stimulating a phosphatase. This has been suggested as a mechanism for regulation of p70 as rapamycin caused dephosphorylation of sites in p70 distinct from those phosphorylated in response to mitogens(53) . A rapamycin-stimulated phosphatase would provide a means for increasing phosphorylation independently of p70. It is important to keep this in mind when using rapamycin sensitivity to implicate the p70 pathway in the phosphorylation of proteins such as PHAS-I and ribosomal protein S6. However, there is still no direct evidence that rapamycin increases phosphatase activity.

The possibility that insulin inhibits a PHAS-I phosphatase should also be considered. That insulin-stimulated PHAS-I kinase activity was not detected when the [H^6]PHAS-IbulleteIF-4E complex was used as substrate would be consistent with phosphatase regulation, although there are other explanations for the inability to detect insulin-stimulated PHAS-I kinase activity. For example, the stimulated activities of kinases regulated by noncovalent mechanisms might not persist after homogenization. Nevertheless, inhibition of a phosphatase could allow PHAS-I kinases not activated by insulin to contribute to the regulation of PHAS-I. Casein kinase II and protein kinase C are the only kinases other than MAP kinase that have been shown to phosphorylate recombinant PHAS-I(19) . Purified casein kinase appears to phosphorylate PHAS-I in the [H^6]PHAS-IbulleteIF-4E complex at least as well as it phosphorylates free [H^6]PHAS-I.^4

Implications of the Dephosphorylation of PHAS-I in Response to Rapamycin

In lymphoid cells, rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins(54) . Specificity for the inhibitory effect of rapamycin on translation initiation appears to be a polypyrimidine motif located just downstream of the 5` cap site(6) . The correlation between this effect and the inhibition of ribosomal protein S6 phosphorylation by rapamycin in Swiss mouse 3T3 cells led to the suggestion that S6 phosphorylation was involved in the increased translation of these messages(6) . Our findings indicate that the effect of rapamycin on inhibiting translation initiation involves dephosphorylation of PHAS-I and inhibition of eIF-4E. The initiation factor promotes selective translation of certain mRNAs, particularly those having highly structured 5`-nontranslated regions(55) . While experiments are needed to determine whether eIF-4E increases translation of mRNAs containing the polypyrimidine motif, it might be noted that the motif is located near the cap site where eIF-4E binds.

In addition to its role in translation, eIF-4E has potent mitogenic actions. Increasing eIF-4E in cells, either by cDNA transfection (31, 56) or by microinjecting the protein(32) , not only stimulated cell growth but also caused the cells to acquire a transformed phenotype. A final speculative idea is that by inhibiting eIF-4E action, PHAS-I dephosphorylation might contribute to the antiproliferative effects of rapamycin.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK28312 and AR41180 and by the Washington University Diabetes Research and Training Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Ave., St Louis, MO 63110. Tel.: 314-362-3936; Fax: 314-362-7058.

^1
The abbreviations used are: eIF, eukaryotic initiation factor; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; PHAS, phosphorylated heat- and acid-stable protein; PAGE, polyacrylamide gel electrophoresis; FKBP, FK506 binding protein; FKBP12, FKBP of M(r) = 12,000; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase kinase; TOR, target of rapamycin.

^2
D. R. Alessi, A. Cuenda, P. Cohen, D. T. Dudley, and A. R. Saltiel, submitted for publication.

^3
D. F. Lazar, M. J. Brady, R. J. Wiese, C. C. Mastick, S. B. Waters, K. Yamauchi, J. E. Pessin, P. Cuatrecasas, and A. R. Saltiel, submitted for publication.

^4
T.-A. Lin, X. Kong, S. Pang, and J. C. Lawrence, Jr., unpublished observations.


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