Specific increase in p85alpha expression in response to dexamethasone is associated with inhibition of insulin-like growth factor-I stimulated phosphatidylinositol 3-kinase activity in cultured muscle cells.

The stimulation of phosphatidylinositol (PI) 3-kinase by insulin-like growth factor I (IGF-I) in L6 cultured skeletal muscle cells is inhibited by the glucocorticoid dexamethasone. The objective of this study was to investigate the mechanism of dexamethasone action by determining its effects on the expression of the p85α and p85β regulatory subunit isoforms of PI 3-kinase, their coupling with the p110 catalytic subunit, and their association with insulin receptor substrate 1 (IRS-1) in response to IGF-I stimulation. Dexamethasone induced a 300% increase in p85α protein content in the L6 cultured myoblast cell line, whereas it increased p110 content by only 38% and had no effect on p85β. The increase in p85α protein was associated with a coordinate increase in p85α mRNA. Stimulation with IGF-I induced the association of p85α and p85β with IRS-1, and this was accompanied by increased amounts of the p110 catalytic subunit and markedly increased PI 3-kinase activity in IRS-1 immunoprecipitates. In cells treated with dexamethasone, greater amounts of p85α and lower amounts of p85β, respectively, were found in IRS-1 immunoprecipitates, such that the α/β ratio was markedly higher than in control cells. In spite of the increase in both total and IRS-1-associated p85α following dexamethasone treatment, IRS-1-associated p110 catalytic subunit and PI 3-kinase activity were decreased by approximately 50%. Thus, dexamethasone induces a specific increase in expression of the p85α regulatory subunit that is not associated with a coordinate increase in the p110 catalytic subunit of PI 3-kinase. As a consequence, in dexamethasone-treated cells, p85α that is not coupled with p110 competes with both p85α·p110 and p85β·p110 complexes for association with IRS-1, leading to increased p85α but decreased p85β, p110, and PI 3-kinase activity in IRS-1 immunoprecipitates.

Growth factor activation of transmembrane tyrosine kinase receptors results in rapid recruitment of phosphatidylinositol (PI) 1 3-kinase activity to tyrosine-phosphorylated proteins. In intact cells, PI 3-kinase catalyzes the phosphorylation of PI 4,5-bisphosphate (PI-4,5-P 2 ) at the 3Ј-position of the inositol ring, thus leading to elevation in intracellular PI 3,4,5trisphosphate (PI-3,4,5-P 3 ) (reviewed in Ref. 1). Unlike the products of PI kinases in the classical PI cycle (PI-4-P and PI-4,5-P 2 ), 3-phosphorylated phosphoinositides are not cleaved by phospholipase C-␥ (2,3), and it has been suggested that they may serve as intracellular second messengers for yet unidentified in vivo targets (1,4). Extensive experimental evidence has established a key role for PI 3-kinase in the signal transduction mechanisms of a number of peptide growth factors, including epidermal growth factor, platelet-derived growth factor (PDGF), insulin, and insulin-like growth factor-I (IGF-I). PI 3-kinase has thus been implicated in the regulation of multiple general and specialized cellular processes, including membrane ruffling (5), receptor endocytosis (6), mitogenesis (7,8), cell differentiation (9), and insulin stimulation of glucose transport (10 -12) and glycogen synthesis (13,14).
Mammalian PI 3-kinase is a heterodimer composed of an 85-kDa (p85) regulatory subunit and a 110-kDa (p110) catalytic subunit (15,16). Two distinct and closely related 85-kDa protein isoforms, p85␣ and p85␤, have been cloned and shown to be the products of separate genes (17)(18)(19)(20). Both of these p85 proteins have the capacity to form stable high affinity complexes with the p110 component of PI 3-kinase (21,22). The two p85 isoforms have a multidomain structure, containing two SH2 (src homology 2) domains, one SH3 domain, and a region with significant sequence similarity to a GTPase-activating protein domain of the product of the breakpoint cluster region gene (23). The presence of several functional domains suggests that the p85 proteins may have multiple interactive and regulatory roles. At present, functional differences between the p85␣ and p85␤ protein isoforms have not been established.
Two forms of p110 also have been cloned, one from bovine brain designated p110 (20), and a second human variant designated p110␤ (24). Expression studies have demonstrated that both p110 proteins have intrinsic PI 3-kinase activity and can associate with the p85 component in intact cells (20,24). The domains in p85 and p110 required for subunit interaction have been identified and mapped to an amino acid sequence between the two SH2 domains of p85 and an NH 2 -terminal amino acid sequence of p110, respectively (20,(25)(26)(27). These studies have established a structural model of the PI 3-kinase complex, in which the p110 subunit contains catalytic activity and is tightly associated with the p85 subunit, which acts as an adaptor and/or regulatory subunit. Integrity of the p85⅐p110 complex appears to be necessary for p110 catalytic activity (26). Thus, overexpression of the p85 subunit or a portion of the p85 protein, such as an intact p85 SH2 domain, through transfection or microinjection of cells results in inhibition of PI 3-kinase activation and cell signaling (28,29). The physiological occurrence of selective up-regulation of p85 expression as a mechanism of inhibition of PI 3-kinase activity has not been investigated.
The best characterized mode of PI 3-kinase activation in response to peptide growth factors involves changes induced in the p85 protein upon binding to certain phosphorylated tyrosine residues, which are then transmitted to the associated p110 catalytic subunit and cause its activation. In the case of the epidermal growth factor and PDGF receptors, the p85 protein binds directly to phosphorylated tyrosines in the receptor molecule through its SH2 domains (reviewed in Ref. 1). In the case of the receptors for insulin and IGF-I, only a limited fraction of the total cell PI 3-kinase associates directly with the receptor, while most of PI 3-kinase interacts with specific tyrosine-phosphorylation sites of the receptor substrates IRS-1 and IRS-2 (30 -32). Binding of p85␣ to tyrosine-phosphorylated IRS-1 or phosphorylated IRS-1-related YMXM peptide sequences results in increased catalytic activity of the PI 3-kinase complex (33,34). Tyr-608 and Tyr-939 of IRS-1 appear to be the predominant sites for interaction with the amino-terminal SH2 domain of p85␣ (35). Although these studies have provided important insight into the mechanism of PI 3-kinase activation in response to insulin and IGF-I, limited information is available on the relative abundance of p85␣ and p85␤ within the IRS-1⅐PI 3-kinase complex and the coupling of IRS-1-associated p85 isoforms with catalytically active p110 subunits.
In a previous report (36), we described the inhibition of IGF-I activated PI 3-kinase activity by the glucocorticoid dexamethasone in the L6 skeletal muscle cell line. The objective of this study was to investigate the effects of dexamethasone on the expression of p85␣ and p85␤ isoforms of PI 3-kinase in L6 cells, their coupling with the p110 catalytic subunit, and their association with IRS-1 in response to IGF-I stimulation. We show that L6 myoblasts express both p85␣ and p85␤ and that the expression of p85␣, but not that of p85␤, is specifically increased by dexamethasone. The increase in the cellular pool of p85␣ correlates with an increased amount of this protein associated with IRS-1 after IGF-I stimulation. However, despite the increase in IRS-1-associated p85␣, both p110 and PI 3-kinase activity associated with IRS-1 are diminished. These data support the concept that a substantial fraction of p85␣ is "free" (i.e. not coupled with p110) in dexamethasone-treated cells. We propose that the selective increase in p85␣ expression may represent a novel physiological mechanism leading to inhibition of PI 3-kinase activity by glucocorticoids. Polyclonal phosphotyrosine (anti-PY) antibody was prepared in rabbits by injection of phosphotyrosine polymerized by 1-ethyl-3(3-dimethyl-aminopropyl)carbodiimide with alanine, threonine, and keyhole limpet hemocyanin, and purified by affinity chromatography on a phosphotyramine-Sepharose column as described previously (37). Polyclonal anti-peptide antibodies against a Tyr-Ala-Ser-Ile-Asn-Phe-Gln-Lys-Gln-Pro-Glu-Asp-Arg-Gln peptide, corresponding to the last 14 amino acids in the COOH-terminal region of rat IRS-1 (38), were generated and purified on specific peptide affinity columns as described previously (36,37). Polyclonal anti-p85 (anti-p85) antibodies that recognize both ␣ and ␤ isoforms of p85 were purchased from Upstate Biotechnology Inc. Monoclonal antibodies specific to p85␣ (anti-p85␣) were from Transduction Laboratories. Monoclonal antibodies to the p110 subunit of PI 3-kinase (anti-p110) were a generous gift of Dr. L. T. Williams (University of California, San Francisco, CA).

Reagents and
Cell Culture-The line of L6 rat skeletal muscle cells has been described previously (36). Cells were cultured in MEM supplemented with 10% donor calf bovine serum, 2 mM glutamine, and nonessential amino acids in a 5% CO 2 atmosphere at 37°C. Cells were plated in MEM containing 10% donor calf bovine serum in 150-mm culture dishes. On day 4 after plating, when the cells were 50 -60% confluent, the medium was replaced with serum-free MEM containing 0.5% bovine serum albumin, and cells were incubated in the presence or absence of 1 M dexamethasone (concentration confirmed by absorbance at 242 nm using a molar absorbance coefficient of 1.5 ϫ 10 4 M Ϫ1 cm Ϫ1 ) for the indicated times. All experiments were carried out with undifferentiated myoblasts.
Analysis of p85␣ and p85␤ mRNA Content-L6 cell monolayers were rinsed twice with ice-cold phosphate-buffered saline and solubilized directly on the tissue culture plates with 4 M guanidinium isothiocyanate, 0.1 M Tris-HCl, pH 7.5, 0.66% N-laurylsarcosine, and 5% ␤-mercaptoethanol. Total cellular RNA was isolated by low temperature 4 M guanidinium isothiocyanate-phenol-chloroform extraction, followed by cold ethanol precipitation (39), and quantitated by spectrophotometry at 260 nm. In all samples, intact ribosomal RNA bands were visualized after electrophoresis. To quantitate p85␣ and p85␤ mRNA content in L6 cells, a polymerase chain reaction (PCR) amplification method was used with oligonucleotide primers complementary to sequences that are identical or highly homologous in p85␣ and p85␤ mRNA but flank regions of different sizes, such that amplified cDNA fragments from the two p85 isoforms could be separated by polyacrylamide gel electrophoresis (40). Specific first strand cDNA copies of p85␣ and p85␤ mRNAs were synthesized using 100 units of Moloney murine leukemia virus-H Ϫ Reverse Transcriptase in a 20-l reaction volume containing 3-5 g of total cell RNA and 0.75 M reverse primer 5Ј-GTACAGGTT-GTAGGGCTC-3Ј at 42°C. This primer is complementary to a nucleotide sequence that is identical in bovine p85␣ and p85␤ (nucleotides 2064 -2047 and 2046 -2029 in p85␣ and p85␤ cDNA sequences, respectively) and in bovine and mouse p85␣ (17,19). The reverse transcription products were combined with 2.5 units of Ampli-Taq, 1 ϫ PCR buffer, 100 M dNTPs, 4 mM MgCl 2 , and 0.15 M oligonucleotide primers in a 100-l final reaction volume for PCR amplification. The sense primer for PCR amplification was a (1:1) mixture of the oligonucleotides 5Ј-GACAAACGCATGAACAG-3Ј and 5Ј-GACAAGCGCATGAACAG-3Ј, corresponding to nucleotides 1678 -1694 and 1657-1693 of bovine p85␣ and bovine p85␤ cDNAs, respectively. The oligonucleotide sequence 5Ј-GACAAACGCATGAACAG-3Ј is identical in bovine and mouse p85␣ (17,19). The antisense primer was the same one used for reverse transcription. Based on p85␣ and p85␤ cDNA sequences, PCR amplification with these primers should generate cDNA products of 386 and 389 bases for p85␣ and p85␤, respectively. 30 cycles of PCR were performed with denaturation at 95°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. PCR products were 35 S-labeled by using a sense primer phosphorylated with T4 polynucleotide kinase (Life Technologies, Inc.) in the presence of [ 35 S]ATP. Amplified cDNA fragments were precipitated with ethanol, resolved on a 6% denaturing polyacrylamide gel, and visualized by autoradiography. Scanning optical densitometry (Molecular Dynamics, Sunnyvale, CA) was performed to quantify the relative amounts of amplified cDNA species.
Cell Lysis and Immunoprecipitation of PI 3-Kinase-After incubation in serum-free medium without or with dexamethasone for the indicated times, cells were left untreated or stimulated with IGF-I (100 nM) for 10 min, washed once with ice-cold phosphate-buffered saline containing 100 M sodium orthovanadate and twice with 20 mM Tris-HCl (pH 7.6) containing 137 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , and 100 M sodium orthovanadate (Buffer A). The cells were lysed in Buffer A (1 ml/ 150-mm dish) containing 1% Nonidet P-40, 10% glycerol, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 4 g/ml leupeptin, and 2 mM sodium orthovanadate (lysis buffer). Insoluble material was removed by centrifugation at 12,000 ϫ g for 10 min at 4°C, the protein concentration in the resulting supernatant was determined with the Bradford dye binding assay (41), and the final protein concentration was adjusted to 2 mg/ml with lysis buffer.
Immunoprecipitation was carried out by incubation of the cell lysate overnight at 4°C with anti-IRS-1 antibodies and adsorption of resulting immune complexes to protein A-Sepharose beads for 2 h at 4°C. The pelleted beads were washed successively in phosphate-buffered saline containing 1% Nonidet P-40 and 100 M sodium orthovanadate (three times), 100 mM Tris-HCl, pH 7.6, containing 500 mM LiCl and 100 M sodium orthovanadate (three times), and 10 mM Tris-HCl, pH 7.6, containing 100 mM NaCl, 1 mM EDTA, and 100 M sodium orthovanadate (twice).
PI 3-Kinase Assay-The washed immunoprecipitates were resuspended in 50 l of 10 mM Tris-HCl, pH 7.6, containing 100 mM NaCl, 1 mM EDTA, and 100 M sodium orthovanadate, and then combined with 10 l of 100 mM MgCl 2 and 10 l of 2 g/l phosphatidylinositol (Avanti) that had been sonicated in 10 mM Tris-HCl, pH 7.6, containing 1 mM EGTA. The PI 3-kinase reaction was started by the addition of 10 l of 10 mM ATP containing 30 Ci of [␥-32 P]ATP. After 10 min at 22°C with constant vortexing, the reaction was stopped by the addition of 20 l of 8 N HCl and 160 l of chloroform/methanol (1:1, v/v). The mixture was vigorously mixed and centrifuged to separate the phases, and the lower organic phase was removed and applied to a silica gel TLC plate precoated with 1% potassium oxalate (Merck, Darmstadt, Germany). The plates were developed in chloroform/methanol/water/ammonia (60: 47:11.3:2, v/v), as described previously (36). The PI-3 product was identified by its comigration with a PI-4 standard and quantitated by scanning densitometry.
Immunoblotting-For identification and quantitation of specific proteins, cell lysates were prepared with 1% Nonidet P-40 as described above and analyzed by immunoblotting either directly or following immunoprecipitation with anti-IRS-1, anti-p85, or anti-p110 antibodies, as indicated. Immunoprecipitates or total cell lysates for immunoblotting were boiled in Laemmli buffer with 100 mM dithiothreitol for 4 min, electrophoresed on 8% SDS-polyacrylamide gels, and transferred by electroblotting onto nitrocellulose sheets (Schleicher & Schuell) as described previously (37). The transfer buffer used was 10 mM Tris, 192 mM glycine, 20% methanol (v/v), and 0.02% SDS, and blotting was carried out at 80 volts for 2.5 h. The membranes were incubated with anti-IRS-1, anti-p85, anti-p85␣, or anti-p110 antibodies as appropriate under conditions reported previously (37). Specific protein bands on autoradiographic images were quantified by scanning optical densitometry, and the data were expressed using arbitrary units normalized to control values within each gel.
Metabolic ([ 35 S]Methionine) Labeling of L6 Cells-Subconfluent cell monolayers in 100-mm dishes were washed twice in phosphate-buffered saline and incubated in serum-free, methionine-free MEM containing 0.1 mCi/ml Tran 35 S-label at 37°C for 20 h. Cell lysates then were prepared as described above and precleared by mixing for 16 h at 4°C with 1 l of normal goat serum (pre-adsorbed to 30 l settled volume of protein A-Sepharose) for each ml of lysate. Anti-IRS-1 antibody was added (5 g adsorbed for 2 h at 4°C to 30 l of protein A-Sepharose for each ml of precleared supernatant) and mixed for 4 h at 4°C. The Sepharose beads were washed as described previously, boiled in Laemmli buffer with 100 mM dithiothreitol for 4 min, and the solubilized proteins electrophoresed on 8% SDS-polyacrylamide gels. Gels were prepared for fluorography using Amplify TM (Amersham Int., Little Chalfont, United Kingdom), dried at 80°C under vacuum, and exposed to Kodak XAR-5 film at Ϫ80°C for 6 -36 h.
Statistical Analysis-Data are presented as mean Ϯ S.E. Statistical analysis was performed by paired and unpaired Student's t tests as appropriate.

Identification of p85␣ and p85␤ Isoforms of PI 3-Kinase in L6
Myoblasts-Two isoforms of the p85 regulatory subunit of PI 3-kinase have been described, p85␣ and p85␤, that are expressed in several, although not all, cell types (16,19). Previous work has demonstrated that p85 is expressed in rat skeletal muscle, but the isoform pattern in this tissue is not known (42). To investigate the expression of p85 isoforms in rat L6 skeletal muscle cells, we employed polyclonal antibodies to the fulllength 85-kDa subunit of p85␣ that detect both p85␣ and p85␤ (anti-p85) and a monoclonal antibody raised against a 19-kDa fragment corresponding to the COOH terminus of p85␣ that specifically recognizes p85␣ (anti-p85␣). As shown in Fig. 1A, two distinct protein species of 85 and 87 kDa were detected by direct immunoblotting with the isoform-nonspecific anti-p85 antibodies. The apparent molecular weights of these two protein species are similar to the reported size of p85␣ and p85␤ expressed in a reticulocyte lysate expression system (19). Immunoprecipitation of Nonidet P-40 extracts of L6 cells with anti-p85 prior to immunoblotting produced similar results, although the ratio of immunoreactive p85␣ to immunoreactive p85␤ was considerably greater as compared to direct immunoblotting ( Fig. 1A), likely reflecting the relatively higher affinity of anti-p85 antibodies for native as compared to denatured p85␣. Immunoblotting of these immunoprecipitates with the ␣ isoform-specific antibody anti-p85␣ confirmed the identity of the lower 85-kDa protein as p85␣ (Fig. 1B). These data indicate that both p85␣ and p85␤ are expressed in rat L6 myoblasts, migrating as 85-and 87-kDa proteins, respectively, on SDSpolyacrylamide gels.
Regulation of p85␣, p85␤, and p110 Expression-To investigate the effects of the glucocorticoid dexamethasone on the cellular content of p85␣ and p85␤, serum-starved L6 myoblasts were incubated in the presence or absence of 1 M dexamethasone for different times, and the total cell content of p85␣ and p85␤ was determined by immunoblotting with the polyclonal anti-p85 antibody. The content of p85␣ and p85␤ did not change in control L6 myoblasts over the 72-h study period ( Fig.  2A). In cells treated with 1 M dexamethasone, the amount of p85␣ progressively increased in a time-dependent manner ( Fig.  2A). After 72 h of treatment, p85␣ content was approximately 4-fold higher in dexamethasone-treated L6 myoblasts compared to control ( Fig. 2B; p Ͻ 0.05). By contrast, dexamethasone did not alter the amount of p85␤ (Fig. 2, A and B), indicating that the effect on the ␣ isoform of p85 was specific.
To determine whether the increase in the p85␣ regulatory subunit of PI 3-kinase was associated with a coordinate increase in the catalytic p110 subunit, similar L6 myoblast preparations were analyzed by immunoblotting with anti-p110, a specific monoclonal antibody raised against mouse p110 (26). As shown in Fig. 3A, one protein band of 110 kDa was recognized by anti-p110 in myoblast extracts. Dexamethasone treatment for 72 h increased p110 content by 38% ( Fig. 3B; p Ͻ 0.05). However, the magnitude of this response was much less than the 300% increase in p85␣ ( Fig. 2A), indicating that the effects of dexamethasone on the regulatory subunit p85␣ and the catalytic subunit p110, respectively, were not coordinate.  1. Expression of p85␣ and p85␤ isoforms of PI 3-kinase in L6 myoblasts. Total cell lysates from L6 myoblasts were obtained as described under "Experimental Procedures" and either left untreated or immunoprecipitated with antibodies to the p85 subunit of PI 3-kinase (anti-p85). Total cell lysates or Sepharose-bound immune complexes were resolved by SDS-PAGE on 8% polyacrylamide gels and analyzed by protein immunoblotting using (A) anti-p85 antibodies (Ab) or (B) a monoclonal antibody specific to p85␣ only (anti-p85␣). The p85 proteins were detected with 125 I-protein A.
To assess whether augmentation of p85␣ versus p85␤ by dexamethasone could involve a selective increase in p85␣ mRNA, p85␣ and p85␤ mRNA levels were measured by quantitative PCR. For this purpose, total RNA was extracted from cells incubated in the presence or absence of 1 M dexamethasone for 48 h and subjected to reverse transcription and PCR amplification. The p85␣ and p85␤ mRNAs were co-amplified in the same PCR reaction using primers complementary to identical or highly homologous nucleotide sequences in the p85␣ and p85␤ genes that flank intervening fragments of different length. 35 S-dATP was included in the PCR mixture during the reaction and resulting 35 S-labeled PCR products were resolved on a denaturing acrylamide gel and visualized by autoradiography. The correct size of the PCR fragments was confirmed by comparison with a sequencing reaction run on the same gel (not shown). A representative autoradiogram of the PCR products is illustrated in Fig. 4A. The products of 386 and 389 bases correspond to the PCR products obtained by amplification of p85␣ and p85␤ mRNA, respectively. With this method, the p85␤ PCR product was predominant over the p85␣ PCR product, likely reflecting higher p85␤ than p85␣ mRNA levels in control L6 myoblasts (ratio of 10:1, with a similar number of 35 S-labeled adenylic acid molecules in either PCR fragment). Dexamethasone treatment did not alter p85␤ mRNA levels, but induced a 3-fold increase in the amount of p85␣ mRNA compared to control (Fig. 4B). These results are consistent with the observed effects of dexamethasone on p85␣ and p85␤ protein levels, indicating that the specific augmentation of p85␣ protein content in L6 myoblasts can be explained by increased levels of p85␣ mRNA.
PI 3-Kinase Activity Associated with IRS-1-The mechanism of PI 3-kinase activation in L6 cells stimulated with IGF-I involves association of the p85 subunit to tyrosine-phosphorylated residues of the substrate IRS-1 and subsequent activation of the lipid kinase intrinsic to the p110 subunit. Although IGF-I also increases the amount of PI 3-kinase activity recoverable in anti-IGF-I receptor immunoprecipitates, receptor-associated activity represents a minor fraction compared to IRS-1-associated PI 3-kinase activity in L6 myoblasts (36). Since dexamethasone treatment induced a marked increase in the cellular content of p85␣ not paralleled by similar changes in either p85␤ or p110, the cellular pool of p85␣ should be in excess relative to other PI 3-kinase components. The consequences of the cellular excess of p85␣ on activation of IRS-1-associated PI 3-kinase were studied by measuring PI 3-kinase activity in IRS-1 immunoprecipitates. Antibody to the carboxyl terminus of rat IRS-1 precipitated 70 -80% of immunoreactive IRS-1 from total cell lysates of L6 myoblasts (not shown). In control cells, IGF-I stimulation induced a rapid and marked (42-fold) increase in the amount of PI 3-kinase activity assayed in vitro with [ 32 P]ATP and phosphatidylinositol in reconstituted IRS-1 immunoprecipitates (Fig. 5A). In cells treated with 1 M dexa- FIG. 4. Dexamethasone effects on p85␣ and p85␤ mRNA content in L6 myoblasts. Cells were grown in serum-free MEM ϩ 0.5% bovine serum albumin in the presence or absence of 1 M dexamethasone (Dex) for 48 h. Total RNA was extracted and p85␣ and p85␤ mRNAs were co-amplified in the same PCR reaction by using primers designed to be complementary to identical or highly homologous nucleotide sequences in the p85␣ and p85␤ genes that flank fragments of different lengths. 35 S-dATP was included in the PCR mixture, and resulting 35 S-labeled PCR products were resolved on a denaturing acrylamide gel and visualized by autoradiography, as shown in A. The correct size of the PCR fragments was confirmed by comparison with a sequencing reaction which was run on the same gel (not shown). The products of 386 and 389 bases correspond to the PCR products obtained by amplification of p85␣ and p85␤ mRNA, respectively. B, quantitation of p85␣ and p85␤ mRNAs in multiple experiments (n ϭ 3). Open bars, control cells; shaded bars, cells treated with 1 M dexamethasone for 48 h. *, p Ͻ 0.05 versus control cells, paired t test. methasone for 72 h, there was no difference in the basal level of PI 3-kinase activity associated with IRS-1, but IGF-1 stimulation induced only a 23-fold increase in IRS-1-associated PI 3-kinase activity (Fig. 5A). Thus, in comparison with control cells, dexamethasone reduced by 45% the level of IGF-I-stimulated PI 3-kinase activity associated with IRS-1 (Fig. 5B).
Tyrosine Phosphorylation and Cellular Content of IRS-1-The reduced levels of IRS-1-associated PI 3-kinase activity despite the marked increase in p85␣ protein content in dexamethasone-treated L6 myoblasts could be explained by several mechanisms, including decreased tyrosine phosphorylation and/or cellular content of IRS-1 protein, impaired association between p85 proteins and tyrosine-phosphorylated IRS-1, altered "coupling" between p85 and the catalytic subunit p110, or a combination of these factors. To investigate the tyrosine phosphorylation and cellular content of IRS-1, Nonidet P-40 extracts from control and dexamethasone-treated L6 myoblasts were resolved on 7% polyacrylamide gels, transferred to nitrocellulose, and subjected to immunoblotting with anti-PY or anti-IRS-1 antibodies. The effects of dexamethasone on IGF-I stimulation of IRS-1 tyrosine phosphorylation are illustrated in the autoradiograph in Fig. 6A and quantitated in the bar graph in Fig. 6B. Maximum IRS-1 tyrosine phosphorylation was modestly but significantly decreased in dexamethasone-treated compared with control cells (25% decrease, p Ͻ 0.05). In addition, the amount of IRS-1 protein was markedly reduced to 35% of control levels (p Ͻ 0.05) by dexamethasone (Fig. 6C), as reported previously (36). The decrease in IRS-1 protein levels occurred more rapidly than the increase in p85␣ protein levels, such that IRS-1 protein was reduced to 40% of control after only 12 h of exposure to dexamethasone (not shown). The greater decrease in protein content than tyrosine phosphorylation of IRS-1 resulted in an approximately 2-fold increase in the ratio of tyrosine-phosphorylated IRS-1 to total IRS-1 in L6 myoblasts treated with dexamethasone (Fig. 6C).
Association of PI 3-Kinase Subunits with IRS-1-The reduced level of tyrosine-phosphorylated IRS-1 in L6 myoblasts treated with dexamethasone could result in decreased associ-ation of this protein with p85, leading to impaired activation of PI 3-kinase. To investigate this possibility, the association of IRS-1 with PI 3-kinase regulatory and catalytic subunits was studied both with metabolic labeling experiments in which anti-IRS-1 immunoprecipitates were isolated from [ 35 S]methionine-labeled cells (Fig. 7), and with immunoblotting analysis of IRS-1 immunoprecipitates using antibodies specific to p85␣, p85␤, or p110 (Figs. 8 and 9).
For metabolic labeling studies, L6 myoblasts were labeled with [ 35  shown). IRS-1 immunoprecipitates were then obtained as described under "Experimental Procedures" and subjected to SDS-PAGE followed by fluorography. As compared with the basal state, IGF-I stimulation induced the association of three protein species of 85, 87, and 110 kDa, respectively, with IRS-1 in control cells (Fig. 7, first and second lanes). These protein species were found to be the only detectable 35 S-labeled proteins in the molecular mass range from 40 to 230 kDa undergoing increased association with IRS-1 upon IGF-I stimulation.
Based on their electrophoretic mobilities, the 85-, 87-, and 110-kDa proteins are likely to represent the p85␣, p85␤, and p110 subunits of PI 3-kinase. In consideration of the similar number of methionine and cysteine residues contained in p85 isoforms (9 methionines and 6 cysteines in p85␣, 8 methionines and 6 cysteines in p85␤) (19), the stoichiometry of p85␣ and p85␤ in the IRS-1 complex upon IGF-I stimulation appears to be 1:1.7 (Fig. 7, second lane), indicating that more p85␤ than p85␣ associates with IRS-1 after stimulation with IGF-I in L6 myoblasts. The 110-kDa band induced by IGF-I appears as a single protein species and shows higher labeling compared to the p85 proteins (Fig. 7, second lane), likely reflecting the greater number of methionine and cysteine residues in p110 compared to p85 (a total of 72 in bovine p110) (20) and the potential association of multiple p110 molecules with IRS-1 through distinct p85 proteins (19). IGF-I stimulation also induced the association of 35 S-labeled p85␣, p85␤, and p110 with IRS-1 in dexamethasone-treated L6 myoblasts (Fig. 7, third  and fourth lanes). However, the amounts of IRS-1 associated 35 S-labeled p85␣ and 35 S-labeled p85␤ in IRS-1 immunoprecipitates were approximately 200 and 70%, respectively, in L6 myoblasts treated with dexamethasone compared to control cells, such that the p85␣/p85␤ ratio was increased. In spite of the increase in IRS-1 associated p85␣, the amount of 35 Slabeled p110 was found to be decreased by 30% in cells treated with dexamethasone (Fig. 7, fourth lane). Thus, the increase in IRS-1 associated p85␣ was not associated with a coordinate increase in IRS-1 associated p110.
The levels of IRS-1 associated p85␣ and p85␤ in control and dexamethasone-treated L6 myoblasts also were determined by immunoblotting with anti-p85 antibody (Fig. 8A, left panel). To specifically detect p85␣, similar immunoprecipitates were also analyzed with a monoclonal antibody to p85␣ (Fig. 8A, right  panel). Quantitation of the two p85 isoforms in IRS-1 immunoprecipitates is presented in Table I. Small amounts of both p85␣ and p85␤ were associated with IRS-1 in the basal state, and the amount of immunoreactive p85␤ was approximately  9. Effects of dexamethasone on IRS-1/p110 complex formation. Total lysates were prepared from control or dexamethasone (Dex)-treated L6 myoblasts and subjected to immunoprecipitation with anti-IRS-1 antibody. Immunoprecipitates were then resolved on 8% polyacrylamide gels and subjected to immunoblotting with anti-p110 antibody, as described under "Experimental Procedures." Panel A shows a representative experiment, and the bar graph in panel B shows the quantitation of p110 in IRS-1 immunoprecipitates from multiple experiments (n ϭ 3). Open bars represent control cells and shaded bars represent cells treated with 1 M dexamethasone for 72 h. *, p Ͻ 0.05 versus control cells, paired t test. In panel C, lysates from control and dexamethasone-treated L6 myoblasts were subjected to immunoprecipitation with anti-p110 antibody followed by immunoblotting with anti-IRS-1 antibody.
2-fold greater than the amount of p85␣ (␣/␤ ratio ϭ 0.5). IGF-I stimulation increased the amount of p85␣ and p85␤ associated with IRS-1 by 9.4-and 5.5-fold, respectively, resulting in a slight increase in the ␣/␤ ratio to 0.85. Although this experiment does not provide direct quantitation of the absolute amounts of p85␣ and p85␤, it should be noted that the ␣/␤ ratio detected by direct immunoblotting with anti-p85 antibody was considerably greater than the ␣/␤ ratio in IRS-1 immunoprecipitates (compare Figs. 1A and 8A). This indicates that a greater proportion of cellular p85␤ was engaged in complexes with IRS-1 than the comparable fraction of cellular p85␣. As shown in Fig. 8A and Table I, in dexamethasone-treated L6 myoblasts, a considerably greater amount of p85␣ and a moderately lower amount of p85␤ were associated with IRS-1 in the absence of IGF-I stimulation (430 and 78% of control levels, respectively, p Ͻ 0.05), such that the ␣/␤ ratio was increased to 2.69. IGF-I stimulation increased the amounts of both p85␣ and p85␤ in IRS-1 immunoprecipitates of dexamethasonetreated cells (4.4-and 4.1-fold, respectively). However, in comparison with IGF-I stimulated cells not treated with dexamethasone, IRS-1-associated p85␣ was increased and p85␤ was decreased (205 and 60% of control levels, respectively, p Ͻ 0.05). Thus, the ␣/␤ ratio in IGF-I stimulated cells was further increased to 2.88 (Table I). These results indicate that IRS-1 is complexed to greater amounts of p85␣ and reduced amounts of p85␤ both in the basal state and after stimulation with IGF-I in dexamethasone-treated compared with control L6 myoblasts. It should be noted that complex formation between IRS-1 and p85␣ was increased in dexamethasone-treated L6 myoblasts, even though the levels of total and tyrosine-phosphorylated IRS-1 in the immunoprecipitates analyzed in these experiments were decreased by 68 Ϯ 15% and 27 Ϯ 12%, respectively (Fig. 8B, p Ͻ 0.5), in agreement with the results presented in Fig. 6. These findings were confirmed by immunoprecipitation with antibodies to p85␣ and subsequent immunoblotting with anti-IRS-1 or anti-PY antibodies, as illustrated in Fig. 8C. Taken together, these results indicate that considerably greater amounts of p85␣ and moderately lower amounts of p85␤, respectively, were associated with IRS-1 in L6 myoblasts treated with dexamethasone, in agreement with the results from metabolic labeling studies (Fig. 7). Thus, the increase in total p85␣ observed in L6 myoblasts treated with dexamethasone correlates with an increase in IRS-1-associated p85␣. This indicates that the inhibition of PI 3-kinase activation cannot be attributed to reduced complex formation between IRS-1 and p85 proteins.
Decreased IRS-1-associated PI 3-kinase activity (Fig. 5) in spite of the marked increase in IRS-1-associated p85␣ in L6 myoblasts treated with dexamethasone raises the possibility that a significant amount of the p85␣ in IRS-1 immunoprecipitates may not exist as a complex with the p110 catalytic subunit of PI 3-kinase. To directly measure the amount of the p110 subunit associated with IRS-1, proteins from control or dexa-methasone-treated L6 myoblasts were subjected to immunoprecipitation with IRS-1 antibody followed by immunoblotting with monoclonal antibody specific for p110 (26) (Fig. 9A). In the absence of IGF-I stimulation little p110 was detectable in IRS-1 immunoprecipitates from control or dexamethasone-treated cells. Stimulation of control cells with 100 nM IGF-I for 10 min increased the amount of p110 associated with IRS-1 by 3.9-fold (Fig. 9, A and B). In dexamethasone-treated L6 myoblasts, although the amount of p110 in IRS-1 immunoprecipitates also increased upon IGF-I stimulation, the amount of p110 associated with IRS-1 was decreased by 41% in comparison with control cells (Fig. 9, A and B, p Ͻ 0.05). These results are similar to the results presented in Fig. 7, showing reduced levels of 35 S-labeled p110 associated with IRS-1 upon IGF-I stimulation in dexamethasone-treated L6 myoblasts. If related to the level of tyrosine-phosphorylated IRS-1 in IRS-1 immunoprecipitates (Fig. 8B), IRS-1 associated p110 was still decreased by 22% in dexamethasone-treated cells compared to control. This correlates with the finding of a 25% decrease in the level of IRS-1 associated PI 3-kinase activity corrected for the level of tyrosine-phosphorylated IRS-1 in response to dexamethasone treatment. These results support the concept that PI 3-kinase inhibition by dexamethasone cannot be entirely explained by decreased tyrosine phosphorylation of IRS-1. The reduced complex formation between IRS-1 and p110 in dexamethasone-treated cells was confirmed by carrying out the reverse experiment with p110 immunoprecipitation followed by anti-IRS-1 immunoblotting (Fig. 9C). These observations indicate that IRS-1 is complexed with p85␣ molecules that are not coupled stoichiometrically with p110 in L6 myoblasts treated with dexamethasone. The decreased amounts of the p110 catalytic subunit in IRS-1 immunoprecipitates from L6 myoblasts treated with dexamethasone correlate with and can account for the observed decrease in the levels of PI 3-kinase activity. DISCUSSION This study demonstrates that both the cellular amounts of the various subunits constituting the PI 3-kinase enzyme complex and PI 3-kinase subunit association with tyrosine-phosphorylated IRS-1 are differentially regulated by the glucocorticoid dexamethasone in undifferentiated L6 skeletal muscle cells. Dexamethasone markedly increased p85␣ in L6 myoblasts, but did not alter the levels of p85␤ and induced only a modest increase in cellular p110 content. Under these conditions, a greater amount of p85␣ and reduced amounts of both p85␤ and p110 were recruited to the IGF-I receptor substrate IRS-1 upon hormone stimulation. In addition, the activity of PI 3-kinase measured in IRS-1 immune complexes was significantly decreased by dexamethasone, likely reflecting the reduced amounts of IRS-1-associated p110 catalytic subunit.
Glucocorticoids have been reported to increase the amount of p85 protein in rat skeletal muscle (42) and in F442A adipocytes  (43). In these previous studies, the p85 isoform pattern was not determined and, therefore, it is not known whether the effects of dexamethasone were isoform-specific. In L6 myoblasts, the increase in p85␣ protein content was associated with an increase in p85␣ mRNA and no change in p85␤ mRNA, suggesting that dexamethasone may act by specifically increasing expression of the p85␣ gene. The p85␣ and p85␤ isoforms possess 62% overall identity at the amino acid level and 58% nucleotide identity and, thus, are thought to be encoded by two distinct but related genes (19). Information on p85 gene structure is very limited at present and, in future studies, it will be important to identify the gene regulatory elements that dictate the tissue distribution of the two p85 isoforms as well as the glucocorticoid responsiveness limited to p85␣. Our data would indicate that glucocorticoid response elements may be identified exclusively in the p85␣ gene (and not in the p85␤ gene). The selective regulation of the ␣ isoform of p85 by dexamethasone supports the concept that p85␣ and p85␤ may have distinct biological roles. Although the related p85␣ and p85␤ regulatory subunits both have been shown to form stable complexes with the catalytic p110 component of PI 3-kinase (21,22), functional differences of p85␣ as compared to p85␤ previously have been reported. Studies conducted in T-lymphocytes have demonstrated that the two p85 isoforms have a different phosphorylation pattern upon T-cell activation (44). Following activation of the CD3 antigen complex in T-cells, rapid serine phosphorylation of p85␣ was observed, whereas phosphorylation of p85␤ was unchanged. In addition, the catalytic subunit p110 was shown to undergo rapid threonine phosphorylation when associated with p85␤ but not with p85␣. It has recently been reported that much larger stimulation of PI 3-kinase is found in p85␣ compared to p85␤ immunoprecipitates upon insulin stimulation of CHO-T cells, even though both p85␣ and p85␤ associate with IRS-1 (45). This has led to the suggestion that insulin causes recruitment of both p85␣ and p85␤ regulatory subunits to IRS-1 signaling complexes, but the activity of IRS-1-associated PI 3-kinase is stimulated only in the p85␣⅐p110 complex, with little or no stimulation of the p85␤⅐p110⅐PI 3-kinase complex. The results in the current study demonstrating specific augmentation of p85␣ protein content by dexamethasone indicate for the first time that the control of p85␣ and p85␤ expression represents an additional level of differential regulation of the two p85 isoforms.
IGF-I stimulation of L6 myoblasts induced a severalfold increase in the association of both p85␣ and p85␤ isoforms with IRS-1 immune complexes. Both p85 isoforms have reportedly been shown to associate with IRS-1 signaling complexes upon insulin stimulation in COS-1 cells transiently transfected with the insulin receptor and in CHO-T cells stably overexpressing the insulin receptor (45). In the latter study, the ␣/␤ ratio in IRS-1 immune complexes generally reflected the ␣/␤ ratio in the total cell lysate, indicating no preferential recruitment of a given p85 isoform to IRS-1 (45). By contrast, our results indicate that p85␤ may preferentially associate with IRS-1 in signaling complexes, since the association of p85␤ with IRS-1 immune complexes was greater quantitatively than that of p85␣ in response to IGF-I stimulation (Figs. 7 and 8). It is possible that association of p85␣ and p85␤ with IRS-1 signaling complexes may be modulated in a cell context specific manner and differ in cell lines expressing high levels of insulin receptors in which hyperphosphorylation of IRS-1 may occur. Interestingly, the amount of p85␣ in anti-phosphotyrosine immunoprecipitates from PDGF-stimulated L6 myoblasts was significantly higher than the amount of p85␤ and thus the ␣/␤ ratio was much greater than in IRS-1 immune complexes. 2 This suggests that association of p85␣ and p85␤ with tyrosine-phosphorylated proteins in L6 skeletal muscle cells may differ depending on the specific protein target (i.e. IRS-1 versus the PDGF receptor).
Ample experimental evidence has established the concept of IRS-1 acting as a "docking protein" capable of simultaneously binding multiple protein components through specific amino acid motifs containing phosphorylated tyrosine residues. GST fusions of the NH 2 -terminal SH2 domain of p85␣ were found to bind strongly to Tyr 608 -Met-Pro-Met and Tyr 939 -Met-Asn-Met and, to a lesser extent, to Tyr 461 -Ile-Cys-Met and Tyr 987 -Met-Tyr-Met in IRS-1 (35). At present, it is not known whether the same sites serve also for the two SH2 domains present in p85␤. Although both p85␣ and p85␤ were shown to bind simultaneously to a single IRS-1 molecule in COS-1 cells transiently transfected with the insulin receptor, this was not the case in CHO-T cells stably expressing the insulin receptor (45). In the PDGF receptor, Tyr 751 is a common binding site for both Nck and p85␣, indicating that SH2 domains of different signaling molecules can compete for binding to the same phosphorylated tyrosine motif (46). In L6 myoblasts treated with dexamethasone, the increase in IRS-1-bound p85␣ was associated with a coordinate decrease in IRS-1-associated p85␤, which could potentially be explained by p85␣ and p85␤ competing for the same binding site(s) on tyrosine-phosphorylated IRS-1.
Dexamethasone induced a cellular excess of p85␣ and a greater number of IRS-1⅐p85␣ complexes in L6 myoblasts. That the pool of IRS-1-associated p85␣ was largely composed of regulatory subunit lacking the p110 catalytic subunit is indicated by the detection of reduced amounts of IRS-1-associated p110 (demonstrated by both p110 immunoblotting and metabolic labeling experiments) and a corresponding decrease in PI 3-kinase activity. Although there was an increase in p110 protein in total cell lysates following treatment of L6 myoblasts with dexamethasone, this was much less pronounced than the increase in p85␣ protein (38 versus 300%, respectively). In interpreting these results, it is important to note that two p110 proteins (p110␣ and p110␤) have been identified (20,24). We have used a monoclonal antibody raised against bovine p110 that recognizes only the p110␣ isoform. Therefore, it is not known whether or not dexamethasone affects cellular levels of p110␤ and what fraction of IRS-1-associated p85␣ in dexamethasone-treated cells was coupled with p110␤. However, the decrease in IRS-1-associated p110␣ and IRS-1-associated PI 3-kinase activity in response to dexamethasone were coordinate. In addition, a single 35 S-labeled protein of 110 kDa, possibly representing both p110␣ and p110␤, was detectable in IRS-1 immunoprecipitates upon IGF-I stimulation. This band was less intense in L6 myoblasts treated with dexamethasone, confirming that the total amount of IRS-1-associated catalytic p110 subunit was decreased.
A p85 subunit lacking a p110 subunit may have the capacity to bind to the specific tyrosine-phosphorylated motif in target proteins without localizing catalytic activity in the proteinprotein signaling complex. Generation of non-coupled (i.e. monomeric or free) p85␣ can be achieved experimentally by transfection and overexpression of the p85␣ cDNA in mammalian cells. When this experiment was performed in 293 cells overexpressing the PDGF receptor, overexpression of p85␣ was shown to completely abrogate activation of PDGF receptorassociated PI 3-kinase activity (28). In addition, microinjection of the p85␣ NH 2 -terminal SH2 domain into rat 1 fibroblasts overexpressing the insulin receptor was shown to inhibit insu-lin-and IGF-I-induced DNA synthesis by competing with endogenous PI 3-kinase for binding to IRS-1 (29). These studies support the concept that cellular overexpression of the p85␣ regulatory subunit of the PI 3-kinase complex can lead to inhibition of PI 3-kinase activity and impairment of cell signaling through activation of this enzyme. Accordingly, the excess of free p85␣ induced by dexamethasone in L6 myoblasts may compete with both p85␣⅐p110 and p85␤⅐p110 complexes for binding to IRS-1. If there are functional specificities for IRS-1bound p85␣ and p85␤, respectively, this may have distinct effects by disrupting specific signaling responses not only through IRS-1⅐p85␣⅐p110 complexes, but also through IRS-1⅐p85␤⅐p110 complexes.
There is evidence that free p85␣ binds to tyrosine-phosphorylated proteins with greater avidity than the intact p85⅐p110 complex (19,29). Thus, preferential complex formation between IRS-1 and p85␣ may have occurred in L6 myoblasts treated with dexamethasone. It recently has been suggested that in situ concentrations of PI 3,4,5-P 3 synthesized locally by the catalytic subunit of PI 3-kinase may bind to the SH2 domain of p85 and dissociate PI 3-kinase from the tyrosine phosphoprotein (47). The lack of PI 3,4,5-P 3 production by the catalytically inactive p85␣ monomer induced by dexamethasone could provide a mechanism conferring greater affinity to this protein for binding to tyrosine-phosphorylated IRS-1 compared to p85␣⅐p110 and p85␤⅐p110 complexes. Such a mechanism could also lead to displacement of IRS-1-bound p85␤⅐p110 by free p85␣.
Activation of PI 3-kinase by receptor and nonreceptor protein tyrosine kinases has been implicated in a broad spectrum of cellular responses, including mitogenesis (1,7,8), chemotaxis (48,49), membrane ruffling (5), activation of p70 S6 kinase (50), insulin-dependent GLUT-4 translocation (51), glycogen synthesis (13,14), activation of integrins in platelets (52), histamine release (53), receptor down-regulation (6), and inhibition of apoptosis (54). All of these biological responses require a functionally active PI 3-kinase enzyme complex that reflects the coordinate expression of both p85 and p110 PI 3-kinase subunits in the cell. The generation of excess p85␣ regulatory subunit relative to the other components of the PI 3-kinase complex along with inhibition of IRS-1-associated PI 3-kinase activity in response to dexamethasone demonstrated in this study suggests a novel mechanism of PI 3-kinase regulation. This mechanism may not be limited to muscle cells and may occur in other cell types under physiological circumstances and/or in disease states characterized by high levels of endogenous cortisol or exogenously administered glucocorticoids.