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J. Biol. Chem., Vol. 277, Issue 21, 18640-18648, May 24, 2002

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-Thrombin Induces Rapid and Sustained Akt Phosphorylation by -Arrestin1-dependent and -independent Mechanisms, and Only the Sustained Akt Phosphorylation Is Essential for G₁ Phase Progression^*

Reema Goel, Polly J. Phillips-Mason^§, Daniel M. Raben^¶, and Joseph J. Baldassare

From the Department of  Pharmacological and Physiological Sciences, St. Louis University School of Medicine, St. Louis, Missouri 63104 and the ^¶ Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, September 18, 2001, and in revised form, February 13, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In Chinese hamster embryonic fibroblasts (IIC9 cells) -thrombin activates the MAPK(ERK) and phosphatidylinositol 3-OH-kinase (PI 3-kinase)/Akt pathways, and both are essential for progression through the G₁ phase of the cell cycle. We investigated in IIC9 cells, the role of -arrestin1 in -thrombin signaling to these pathways. -Thrombin stimulates rapid and sustained PI 3-kinase and Akt activities. Expression of a dominant negative -arrestin1 (-arrestin1(V53D)) inhibits rapid but not sustained PI 3-kinase and Akt activities. Surprisingly, expression of -arrestin1(V53D) does not block activation of the MAPK(ERK) pathway. PI 3-kinase and Akt activities are also inhibited by expression of a -arrestin1 mutant, which impairs binding to c-Src (-arrestin1(P91G-P121E)), indicating the involvement of c-Src in the rapid stimulation of the PI 3-kinase/Akt pathway. Consistent with these results, PP1, a selective inhibitor of c-Src family kinases, prevents -thrombin-stimulated Akt phosphorylation. Expression of - arrestin1(V53D) does not prevent G₁ progression, as its expression has no effect on [³H]thymidine incorporation into DNA. In agreement with the ineffectiveness of -arrestin1(V53D) to block G₁ progression, cyclin D1 protein amounts and CDK4-cyclin D1 activity is unaffected by expression of -arrestin1(V53D). Thus in IIC9 cells, -thrombin activates rapid -arrestin1-dependent and sustained -arrestin1-independent Akt activity, suggesting that two mechanisms are involved. Furthermore, although blocking the -arrestin1-independent PI 3-kinase/Akt pathway prevents G₁ progression, inhibition of the -arrestin1-dependent pathway does not, indicating different roles for the rapid and sustained activities.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In a variety of cell types (1-4), including Chinese hamster embryonic fibroblasts (IIC9 cells), -thrombin is a potent mitogen. The -thrombin receptor, a member of the 7-membrane-spanning superfamily of G-protein-coupled receptors (GPCRs),¹ mediates its responses by a novel mechanism that involves cleavage of the amino-terminal exodomain to generate a new amino terminus, which binds to and activates the receptor (5-7). The -thrombin receptor activates to several heterotrimeric G proteins (8-12) and transduces signals to a complex network of signaling proteins that stimulates cell cycle re-entry and proliferation. We have found previously that in IIC9 cells -thrombin induces both the ERK and PI 3-kinase pathways, and both signaling pathways are important regulators of progression through the G₁ into S phase of the cell cycle (13, 14).

PI 3-kinase phosphorylates the hydroxyl group of phosphoinositides, leading to the formation of phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. Lipid products of PI 3-kinase are involved in a wide variety of cellular events including mitogenic signaling, survival, vesicular trafficking, and control of the cytoskeleton (15-19)). Akt is a serine-threonine kinase that functions as part of a wortmannin-sensitive signaling pathway and is located downstream of PI 3-kinase (13, 20-23). Recent data in IIC9 cells (13) and NIH 3T3 cells (24) show that Akt kinase mediates the accumulation of cyclin D1 by inhibiting glycogen synthase kinase-3, the kinase that phosphorylates cyclin D1 and targets cyclin D1 for degradation via a ubiquitin-mediated mechanism (25). Inhibition of glycogen synthase kinase-3 therefore results in the accumulation of cyclin D1 and the activation of CDK4-cyclin D1, which is critical for G₁ progression of IIC9 cells (13) and NIH 3T3 cells (24).

Originally -arrestin was thought to be involved in receptor desensitization of GPCR due its role in uncoupling the receptor from the G protein and targeting the receptor for endocytosis (26). Recent data, however, demonstrate that -arrestins are also important in the activation of the MAPK pathways, by acting as scaffolding proteins (27, 28). Scaffolding proteins mediate protein-protein interactions that could prevent or minimize cross-talk between related pathways and also increase the responsiveness of the signaling cascade to the incoming signals. -Arrestin2 specifically interacts with both JNK3 and ASK1 to stimulate JNK3 phosphorylation following activation of angiotensin type 1A receptor by angiotensin II (29). It also scaffolds c-Raf1, MEK1, and ERK2 onto the angiotensin type 1A receptor (30). -Arrestin1 binds c-Src through interactions involving both the Src homology 3 and catalytic domain of c-Src (31, 32). This association results in the recruitment of c-Src to ₂-adrenergic receptor and is essential for activation of ERK2 by angiotensin type 1A receptor (31). In addition to angiotensin II, substance P and interleukin-8 mediate ERK1/2 activation by recruitment of c-Src or c-Src family members to the activated neurokinin through -arrestin (33) and interleukin-8 (34) receptors, respectively. Furthermore, stimulation of the PAR2 receptor, a member of the protease-activated receptor family, results in complex formation with -arrestin1; the internalized receptor--arrestin1 complex associates with Raf1 and ERK1/2 and results in the activation of ERK1/2 and its cytosolic retention (35).

We investigated the role of -arrestin in -thrombin-induced ERK1/ERK2 and Akt phosphorylation in IIC9 cells. We find that -thrombin stimulates -arrestin1-dependent Akt phosphorylation. Both expression of dominant negative -arrestin1 and -arrestin1 mutants that impair binding to c-Src block the rapid phosphorylation of Akt but not ERK1/2. Surprisingly, the inhibition of the rapid activation of Akt does not prevent -thrombin-induced G₁ progression of IIC9 cells as determined by [³H]thymidine incorporation and CDK4-cyclin D1 activity. Consistent with its inability to block CDK4-cyclin D1 activation, expression of dominant negative -arrestin is not involved in sustained activation of Akt phosphorylation. These data indicate that in IIC9 cells, -thrombin stimulates Akt phosphorylation by two mechanisms, and only one is -arrestin1-dependent. Furthermore, the -arrestin1-dependent pathway is not important for -thrombin-induced G₁ cell cycle progression.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Reagents-- IIC9 cells, a subclone of Chinese hamster embryo fibroblasts, were maintained in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and 2 mM L-glutamine (BioWhittaker, Walkersville, MD) supplemented with 5% (v/v) fetal calf serum. Subconfluent IIC9 cells (80%) were growth-arrested by washing once with -minimum Eagle's medium (Invitrogen), containing 2 mM L-glutamine (BioWhittaker) followed by a 48-h incubation in the same media. Human -thrombin isolated from plasma (Sigma) was used at 1 unit/ml in all experiments. Unless otherwise stated, PP1 (Biomol, Plymouth Meeting, PA) and LY294002 (Calbiochem) were used at 1 and 10 µM, respectively.

Transient Transfection-- The cDNA encoding pcDNA3.1 (Invitrogen), -arrestin1, -arrestin1(V53D) (a kind gift from Marc Caron) or MAS-GRK3-ct (a kind gift from Stephen R. Ikeda), or myristoylated Akt (Myr-Akt) (a kind gift from Tung O. Chan), or -arrestin1(P19G-P121E) (a kind gift from Robert J. Lefkowitz) were transfected into subconfluent (60-80%) IIC9 cells using LipofectAMINE (Invitrogen) following the manufacturer's protocol. In each experiment 0-3 µg of each plasmid was mixed with 10 µl of LipofectAMINE per 1 ml of media. Six hours post-transfection an equal volume Dulbecco's modified Eagle's medium supplemented with 0.2% (v/v) fetal calf serum was added to the transfection mixture, and the cells were incubated overnight. The following day, cells were growth-arrested by washing once with -minimum Eagle's medium followed by a 48-h incubation in the same media prior to agonist stimulation. Transient transfection using LipofectAMINE resulted in 80-90% expression efficiency as visualized by -galactosidase staining (data not shown).

Western Blot Analysis-- Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml -thrombin for the times indicated in the figure legends. At the indicated times, cells were washed twice in cold PBS and harvested by scraping into 150 µl of cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The lysates were sonicated briefly, and the insoluble material was pelleted by centrifugation at 14,000 × g at 4 °C for 5 min. Protein concentrations of the supernatants were determined using Coomassie Plus (Pierce) as recommended by the manufacturer. Protein lysates (10-25 µg) were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Boston, MA). Membranes were probed with polyclonal antibodies to Akt (Santa Cruz Biotechnology), phospho-AktSer-473 (New England Biolabs), phospho-ERK1/2 (New England Biolabs), phospho--arrestin1 (Cell Signaling), -arrestin1 (Transduction Laboratories), and cyclin D1 monoclonal (Santa Cruz Biotechnology). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) (Amersham Biosciences) as recommended by the manufacturer.

Immune Complex Kinase Assay for ERK1-- Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml -thrombin. At the indicated times, cells were washed twice in cold PBS, harvested by scraping into 100 µl of cold ERK lysis buffer, and assayed as described previously (14).

Preparation of IIC9 Membranes-- IIC9 lysates were prepared by scraping into 300 µl of cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% (v/v) glycerol, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). Cells were disrupted by 50 strokes with a hand-driven Dounce homogenizer. The lysates were sonicated briefly, and the insoluble material was pelleted by centrifugation at 14,000 × g at 4 °C for 5 min. Non-nuclear membranes were obtained by centrifuging the resulting supernatant at 100,000 × g for 3 h. Pellets were resuspended in 40 µl of cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The protein amounts of -arrestin1 were determined by Western blot analysis.

[³H]Thymidine Incorporation-- Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml -thrombin for 17 h. Following the 17-h incubation, 1 µCi/ml [³H]thymidine (PerkinElmer Life Sciences) was added, and [³H]thymidine incorporation into DNA was determined as described previously (13).

Ras Activation Assay-- Growth-arrested IIC9 cells or transfected cells were labeled for 4 h with ³²P_i at 0.2 mCi/100-mm dish in phosphate-free Dulbecco's modified Eagle's medium (BioWhittaker). Cells were then washed twice with phosphate-free media and once with a saline buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). Cells were incubated in the presence or absence of 1 NIH unit/ml -thrombin for 5 min. After stimulation, cells were washed two times with PBS and harvested by scraping into 500 µl of IP buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 150 mM NaCl, 1% Triton X-100, 2 mM p-nitrophenyl phosphate, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), and GDP and GTP ³²P-labeled fractions were quantified using a Molecular Dynamics PhosphorImager as described previously (13).

CDK4-Cyclin D1 Assay-- Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml -thrombin after preincubation in the absence or presence of 10 µM LY294002 for 30 min. After 17 h, cells were washed twice with cold PBS and lysed in cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM PMSF, 2 µM sodium vanadate, 20 mM sodium fluoride, 50 µM -glycerophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The lysates were sonicated briefly, and insoluble material was pelleted by centrifugation at 14,000 × g at 4 °C for 4 min. Protein concentrations were determined using Coomassie Plus (Pierce) as recommended by the manufacturer. Cell lysates (50 µg of protein) were incubated with 2 µg of monoclonal cyclin D1 antibody (Santa Cruz Biotechnology) at 4 °C with gentle rocking for 3 h. The CDK4 immune complexes were then immunoprecipitated by overnight incubation with protein G-agarose (Sigma) at 4 °C with gentle rocking. The CDK4-cyclin D1 immune complexes were pelleted by microcentrifugation at 14,000 × g and washed twice with cold wash buffer (50 mM HEPES, pH 7.5, 1 mM DTT, and 10 mM MgCl₂). The CDK4-cyclin D1 immune complexes were resuspended in 30 ml of reaction buffer (50 mM HEPES, pH 7.5, 1 mM DTT, 10 mM MgCl₂, 2.5 mM EGTA, 50 µM -glycerol phosphate, 1 mM sodium fluoride, and 20 µM ATP) and incubated with 2 µg/ml soluble GST-retinoblastoma fusion protein (retinoblastoma sequence encoding amino acids 379-928 inserted into pGEX-2T plasmid is a kind gift of Dr. Mark Ewen, Harvard University), 5 µCi of [-³²P]ATP at 37 °C for 30 min. The reaction was stopped by the addition of 10 µl of 4× Laemmli sample buffer. Samples were subjected to SDS-PAGE. The gels were dried and CDK4-cyclin D1 activity was quantified using a PhosphorImager (Molecular Dynamics).

Phosphatidylinositol 3-Kinase Assay-- Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml -thrombin for 5 min. After stimulation, cells were washed twice with PBS and harvested by scraping into 400 ml of cold lysis buffer assayed as described previously. Briefly, the lysates were sonicated and the insoluble material pelleted by centrifugation. p85 immune complexes were immunoprecipitated from lysates containing 300-400 mg of protein by incubation with polyclonal p85 antibody (Upstate Biotechnology Inc.) at 4 °C for 3 h, followed by an incubation with protein A-agarose (Sigma) at 4 °C overnight. The reaction was started by the addition of 5 ml of reaction buffer (0.88 mM ATP, 10 mCi of [-³²P]ATP, and 20 mM MgCl₂). Samples were incubated for 10 min at 37 °C, and the reactions were stopped by the addition of 20 ml of 6 N HCl. Lipids were extracted by adding 160 ml of CHCl₃/MeOH (1:1) to the samples and vortexing briefly. 50 ml of labeled lipids were resolved by spotting on a silica TLC plate (J. T. Baker Inc.) and developed with CHCl, MeOH, 4 N NH₄OH (9:7:2 v/v). Phosphatidlyinositol-3-phosphate production was quantified using a PhosphorImager (Molecular Dynamics). Phosphatidylinositol 4-phosphate isolated from bovine brain (Avanti Polar Lipids, Inc.) was included as a standard for TLC resolution of the lipids and visualized by iodine vapor.

Akt Kinase Activity Assay-- Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml -thrombin for 5 min. Cells were then washed twice in cold PBS and harvested by scraping into 100 µl of cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM PMSF, 2 µM sodium vanadate, 20 mM sodium fluoride, 50 µM -glycerophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The lysates were sonicated briefly, and the insoluble material was pelleted by centrifugation at 14,000 × g at 4 °C for 5 min. Protein concentrations of the supernatants were determined using Coomassie Plus (Pierce) as recommended by the manufacturer. Cell lysates (200 µg of protein) were incubated with 2 µg of anti-Akt (C-20) (goat antibody from Santa Cruz Biotechnology) at 4 °C with gentle rocking for 3 h. The Akt immune complexes were then immunoprecipitated by overnight incubation with protein G-agarose (Sigma) at 4 °C with gentle rocking. The Akt immune complexes were pelleted by microcentrifugation at 14,000 × g and washed twice with cold wash buffer (50 mM HEPES, pH 7.5, 1 mM DTT, and 10 mM MgCl₂). The immune complexes were resuspended in 30 ml of reaction buffer (50 mM HEPES, pH 7.5, 1 mM DTT, 10 mM MgCl₂, 2.5 mM EGTA, 50 µM -glycerol phosphate, 1 mM sodium fluoride, and 20 µM ATP) and incubated with 1 mg/ml histone 2B (Sigma) and 5 µCi of [-³²P]ATP at 37 °C for 30 min. The reaction was stopped by the addition of 10 µl of 4× Laemmli sample buffer. Samples were subjected to 15% SDS-PAGE. The gels were dried, and Akt activity was quantified using a PhosphorImager (Molecular Dynamics).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Dominant Negative -Arrestin1 Selectively Inhibits PI 3-Kinase and Akt Activities and Not ERK1/2-- In IIC9 cells -thrombin-induced MAPK (ERK) and PI 3-kinase pathways are dependent on Ras and inhibited by transient expression of G sequestrants (14, 36), suggesting that both signaling pathways are regulated in a similar manner. Because several G protein-coupled receptors (GPCR), including a member of the protease-activated receptor family, stimulate ERK activity by a -arrestin-dependent mechanism (35), we examined whether transient expression of a dominant negative -arrestin1 (-arrestin1(V53D) (31, 37, 38) blocks -thrombin-stimulated MAPK(ERK) and PI 3-kinase pathways. As reported previously (13), -thrombin stimulates a rapid increase of ERK and PI 3-kinase activities (Fig. 1). Surprisingly, although transient expression of -arrestin1(V53D) does not inhibit the rapid increases in ERK1/2 phosphorylation (Fig. 1A) or ERK1/2 activities (data not shown), it does block Akt phosphorylation (Fig. 1B). The inhibition is specific for -arrestin1(V53D), because expression of dominant negative -arrestin2 is ineffectual (data not shown). In addition the inhibition is dependent on the concentration of -arrestin1(V53D) (Fig. 1B). We also quantified the effect of transient expression of -arrestin1(V53D) on Akt activity (Fig. 1C). Consistent with our results on Akt phosphorylation, -thrombin stimulates a rapid 5-fold increase in Akt activity, and expression of -arrestin1(V53D) blocks the increase (Fig. 1C). We (13) found previously that changes in Akt activation are dependent on activation of the PI 3-kinase pathway and, therefore, quantified PI 3-kinase activity (Fig. 1D). In agreement with the ability of ectopic expression of -arrestin1(V53D) to inhibit -thrombin-induced Akt activity, transient expression of -arrestin1(V53D) also blocks PI 3-kinase activity (Fig. 1D).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Expression of dominant negative -arrestin1 selectively inhibits rapid Akt phosphorylation and not ERK1/2. IIC9 cells were untransfected or transfected with either -arrestin1(V53D) (2 µg) or dominant negative -arrestin2 (2 µg) as described under "Experimental Procedures" and serum-arrested for 48 h. In those cells treated with LY294002 (10 µM), LY294002 was added 30 min prior to stimulation. The cells were incubated for 5 min in the absence or presence of -thrombin (1 NIH unit/ml), and cells lysates were prepared as described under "Experimental Procedures." Lysate proteins (20 µg) were separated by SDS-PAGE (15%) and immunoblotted with either anti-phospho-ERK polyclonal antibody (A) or with anti-phospho-Akt or anti-Akt polyclonal antibodies (B). Phospho-ERK1/2 and phospho-Akt were quantified using a Molecular Dynamics densitometer. C, Akt immune complexes were immunoprecipitated from lysates containing equal protein with an anti-Akt antibody and analyzed for their ability to phosphorylate histone 2B as described under "Experimental Procedures." Lysate proteins were separated by SDS-PAGE (10%) and immunoblotted with anti-Akt polyclonal antibodies. D, PI 3-kinase immune complexes were immunoprecipitated from lysates containing equal proteins using an anti-p85 polyclonal antibody and assayed for their ability to phosphorylate phosphatidylinositol in vitro as described under "Experimental Procedures." Phosphatidylinositol 3-phosphate production was quantified using a PhosphorImager (Molecular Dynamics). Data in A-D are presented as fold stimulation above unstimulated cells (value = 1). Data are representative of four independent experiments.

-Thrombin Induces -Arrestin1 Translocation to Membranes-- Because the rapid -thrombin-induced Akt activation is inhibited by expression of dominant negative -arrestin1, we reasoned that -thrombin stimulates the translocation of -arrestin1 to the plasma membrane. Furthermore, GRK3 and -arrestin1 regulate PAR1 desensitization, suggesting that -thrombin stimulates the phosphorylation of PAR1 by GRK3, followed by the binding of -arrestin1 to phosphorylated PAR1 at the plasma membrane. We next examined -thrombin-induced membrane translocation of endogenous -arrestin1 and the effect of an inhibitor of GRK3 activation on this translocation (Fig. 2). An approximate 2-fold increase in membrane-associated -arrestin1 occurred within 5 min after -thrombin addition (Fig. 2). Furthermore, expression of a membrane-anchored GRK3 carboxyl-terminal polypeptide (MAS-GRK3ct), which encodes the G  binding domain (39), blocks the increase in the amount of -arrestin1 in the membrane fraction of GRK3 (Fig. 2) and the rapid increase in Akt activity (data not shown). These data are consistent with a role for -arrestin1 in PAR1 signaling.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   -Thrombin induces -arrestin1 translocation to membranes. IIC9 cells were untransfected or transfected with either Mas-GRK3ct (2 µg) as described under "Experimental Procedures" and serum-arrested for 48 h. Serum-arrested IIC9 cells were incubated for 5 min in the absence or presence of -thrombin (1 NIH unit/ml) and non-nuclear membranes prepared as described under "Experimental Procedures." A, non-nuclear membrane proteins (50 µg) were separated by SDS-PAGE (15%) and immunoblotted with an anti--arrestin1 antibody. B, serum-arrested IIC9 cells were incubated for the indicated times in the absence or presence of -thrombin (1 NIH unit/ml). Lysate proteins (100 µg) were separated by SDS-PAGE (15%) and immunoblotted with either an anti-phospho--arrestin1 antibody or anti--arrestin1 antibody.

Ras Activation Is Independent of -Arrestin1-- We found previously that both ERK and PI 3-kinase are dependent on Ras (13, 14). Because in IIC9 cells, -thrombin-induced ERK activation is independent of -arrestin1 and is downstream of Ras (14), we reasoned that Ras activation also should be independent of -arrestin1. As shown previously (13), -thrombin stimulates an approximate 3-fold increase in activated Ras as determined by the ratio of GTP/(GTP + GDP) bound to Ras (Fig. 3). We next examined the effect of expression of dominant negative -arrestin1. Consistent with its inability to block ERK1/2 phosphorylation, expression of -arrestin1(V53D) does not affect Ras activation (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Ras activation is independent of -arrestin1. IIC9 cells were transiently transfected with 3 µg of -arrestin1(V53D) as described under "Experimental Procedures." Transfected IIC9 cells were 32P-labeled and growth-arrested for 48 h prior to the addition of -thrombin (1 NIH unit/ml). -Thrombin-treated IIC9 cells were harvested after 5 min of -thrombin addition by scraping into cold Ras lysis buffer (see "Experimental Procedures"). Ras immune complexes were immunoprecipitated with a monoclonal antibody and analyzed for 32P-labeled guanine nucleotides by TLC as described under "Experimental Procedures." Resolved nucleotides were quantified using a Molecular Dynamic PhosphorImagerTM and reported as percent maximal stimulation. Data represent two separate experiments.

Rapid PI 3-Kinase and Akt Activities Are Inhibited by Expression of -Arrestin1(P91G/P121E)-- Recent studies (30, 31) show that -arrestin1 initiates the activation of mitogenic signaling pathways by its association and recruitment of c-Src. Expression of -arrestin1 containing mutations in its amino-terminal prolines 91 and 121 (P91G/P121E), whose association with c-Src is impaired, blocks ₂-adrenergic receptor activation of ERK1/2 (31). Because -thrombin-induced activation of PI 3-kinase and Akt are dependent on -arrestin1, we reasoned that c-Src should be critical for PI 3-kinase and Akt activities. To examine whether the association of c-Src with -arrestin1 is important for rapid PI 3-kinase and Akt activities, we transiently expressed -arrestin1(P91G/P121E) and quantified ERK1/2 phosphorylation, PI 3-kinase, and Akt activities (Fig. 4). Expression of -arrestin1(P91G/P121E) inhibits -thrombin-induced PI 3-kinase (Fig. 4A), Akt activities (Fig. 4B), and Akt phosphorylation (data not shown). Furthermore, in agreement with the inability of expression of dominant negative -arrestin1(V53D) to block ERK1/2 phosphorylation, ERK1/2 phosphorylation is insensitive to transient expression of -arrestin1(P91G/P121E) (Fig. 4C). These data indicate that the association of c-Src with -arrestin1 is necessary for -thrombin-induced PI 3-kinase activity and Akt phosphorylation and suggests a role for c-Src tyrosine kinase in the stimulation. We next examined the effect of Src family tyrosine kinase inhibitor, PP1, on -thrombin-induced PI 3-kinase activity and Akt phosphorylation (40). Treatment of IIC9 cells with PP1 blocks these activations (Fig. 4D) in a dose-dependent manner. Treatment with PP1 has no effect on ERK1/2 phosphorylation (Fig. 4D). Taken together, these data indicate that in IIC9 cells c-Src is required for -thrombin-induced Akt but not ERK1/2 activation.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Rapid Akt phosphorylation is inhibited by expression of -arrestin1(P91G/P121E). IIC9 cells were transiently transfected with pcDNA 3.1 (3 µg) or -arrestin1(P91G-P121E) (3 µg) as described under "Experimental Procedures." The transfected IIC9 cells were serum-arrested cells for 48 h, and the serum-arrested cells were incubated for 5 min in the absence or presence of -thrombin (1 NIH unit/ml) and then harvested by scraping into cold lysis buffer. A, PI 3-kinase immune complexes were immunoprecipitated from lysates containing equal protein using an anti-p85 polyclonal antibody and assayed for their ability to phosphorylate PI in vitro described under "Experimental Procedures." B, Akt immune complexes were immunoprecipitated from lysates containing equal protein with an anti-Akt antibody and analyzed for their ability to phosphorylate histone 2B as described under "Experimental Procedures." C, lysate proteins (20 µg) were separated by SDS-PAGE (15%) and immunoblotted with anti-phospho-ERK polyclonal antibody. The data were quantified using a Molecular Dynamics densitometer and presented as fold stimulation above unstimulated cells (value = 1). D, serum-arrested IIC9 cells were treated with varying concentrations of PP1, 30 min prior to the addition of -thrombin (1 NIH unit/ml). 30 min after -thrombin addition, the cells were harvested by scraping into cold lysis buffer, lysate proteins (20 µg) separated by SDS-PAGE (15%), and immunoblotted with anti-phospho-Akt, anti-Akt or anti-phospho ERK antibodies. Data are representative of 3 independent experiments.

Expression of Dominant Negative -Arrestin1 Does Not Affect G₁ Progression-- Previous data from our laboratory show that Akt activity is required for -thrombin-stimulated DNA synthesis in IIC9 cells (13). Inhibition of Akt activation blocks the accumulation of cyclin D1 and CDK4-cyclin D1 activities that is required for progression into the S phase of cell cycle. Because -arrestin1 is required for the rapid increase in Akt activity, we postulated that expression of dominant negative -arrestin1(V53D) would inhibit -thrombin-mediated DNA synthesis as determined by [³H]thymidine incorporation (Fig. 5). -Thrombin stimulates a 12-fold increase in [³H]thymidine incorporation (Fig. 5). Previous studies (24) report that inhibition of Akt activity blocks progression through G₁ into S phase. In agreement with these data, inhibition of PI 3-kinase activity with a selective inhibitor, LY294002, prevents G₁ progression in IIC9 cells (13), indicating that in IIC9 cells progression through G₁ into S phase is sensitive to the inhibition of PI 3-kinase and Akt activities. Surprisingly, transient expression of -arrestin1(V53D) does not inhibit DNA synthesis, suggesting that inhibition of rapid Akt activity does not prevent G₁ progression.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Expression of dominant negative -arrestin1 does not affect -thrombin-stimulated DNA synthesis. IIC9 cells were untransfected or transfected with -arrestin1(V53D) (2 µg) or myristoylated Akt (Myr-Akt) (2 µg) and serum-arrested for 48 h as described under "Experimental Procedures." The serum-arrested cells were incubated in the absence or presence of 10 µM LY294002 for 30 min prior to addition of -thrombin (1 NIH unit/ml). The IIC9 cells were incubated for an additional 3 h with 1 µCi of [3H]thymidine 17 h after -thrombin addition. Cells were washed, and the DNA was precipitated as described under "Experimental Procedures." [3H]DNA was quantified by scintillation counting. Data are representative of four independent experiments done in triplicate.

Expression of Dominant Negative -Arrestin1 Does Not Affect CDK4-Cyclin D1 Activity-- Several laboratories (13, 24) have found that Akt phosphorylates and inhibits glycogen synthetase kinase 3 activity. Because glycogen synthetase kinase 3 phosphorylates and prevents the accumulation of cyclin D1 and CDK4-cyclin D1 activity (25), we next examined CDK4-cyclin D1 activity, which in IIC9 cells also is sensitive to Akt activity (13). -Thrombin induces a 10-fold increase in CDK4-cyclin D1 activity determined 8 h after -thrombin addition (Fig. 6A). CDK4-cyclin D1 activity is insensitive to the transient expression of -arrestin1(V53D) (Fig. 6A). These data are consistent with our results on [³H]thymidine incorporation (Fig. 4) and suggest that -arrestin1-dependent activation of Akt is not important for CDK4-cyclin D1 activity. In agreement with the data for CDK4-cyclin D1, expression of -arrestin1(V53D) does not affect cyclin D1 accumulation as determined 8 h after the addition of -thrombin (Fig. 6B). Although these results are in agreement with our data on [³H]thymidine incorporation (Fig. 5) and indicate that inhibition of Akt phosphorylation by -arrestin1 is not critical for -thrombin-stimulated progression through G₁ into S phase, they suggest that sustained activation of Akt is independent of -arrestin1.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Expression of dominant negative -arrestin1 does not affect CDK4-cyclin D1 activity. IIC9 cells were untransfected or transfected with -arrestin1(V53D) (3 µg) and serum-arrested for 48 h. Serum-arrested cells were incubated in the absence or presence of -thrombin (1 NIH unit/ml) for 8 h. Cells were harvested by scraping into cold retinoblastoma lysis buffer and cyclin D1-CDK4 immune complexes immunoprecipitated from lysates containing equal amounts of lysate protein with a monoclonal cyclin D1 antibody (A). The immune complexes were assayed for their ability to phosphorylate soluble GST-Rb fusion protein in vitro as described under "Experimental Procedures." B, lysate proteins (20 µg) were separated by SDS-PAGE (10%) and immunoblotted with a monoclonal cyclin D1 antibody. The data were quantified using a Molecular Dynamics densitometer and are presented as fold stimulation above unstimulated cells (value = 1). Data are representative of two independent experiments.

-Thrombin-stimulated Sustained PI 3-Kinase Activity and Akt Phosphorylation Is Not Dependent on -Arrestin1-- To determine whether Akt and PI 3-kinase activations are sustained, PI 3-kinase activity and Akt phosphorylation were determined at several times throughout the early G₁ phase of the cell cycle. -Thrombin-stimulated PI 3-kinase activity and Akt phosphorylation are sustained for up to 6 h after -thrombin addition (Fig. 7A). We next examined the effect of expression of -arrestin1 on sustained PI 3-kinase activity and phosphorylation of Akt. In IIC9 cells expression of -arrestin1(V53D) reduces PI 3-kinase activity and Akt phosphorylation to near unstimulated levels at 5 min but does not inhibit Akt phosphorylation at 4 or 8 h (Fig. 7B). These data indicate that whereas rapid PI 3-kinase activity and Akt phosphorylation are sensitive to -arrestin1, sustained PI 3-kinases are insensitive. These results suggest that the rapid and sustained PI 3-kinase activities and Akt phosphorylations are regulated by different mechanisms.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   -Thrombin stimulates sustained PI 3-kinase activity and Akt phosphorylation independent of -arrestin1. IIC9 cells were untransfected or transfected with -arrestin1(V53D) (3 µg) and serum-arrested for 48 h. A, serum-arrested IIC9 cells were incubated for 2, 4, 6, and 8 h in the presence of -thrombin (1 NIH unit/ml). Lysates were prepared as described under "Experimental Procedures," and lysate proteins (20 µg) were separated by SDS-PAGE (9.75%) and immunoblotted with either anti-phospho-Akt or anti-Akt antibodies. B and C, serum-arrested cells were incubated for 5 min and 4 or 8 h in the absence or presence of -thrombin (1 NIH unit/ml). Lysate proteins (20 µg) were separated by SDS-PAGE (15%) and immunoblotted with anti-phospho-Akt, anti-phospho-ERK1/2, or anti-Akt antibodies (B) and anti--arrestin1 antibody (C). Data for A and B were quantified using a Molecular Dynamics densitometer and are presented as fold stimulation above unstimulated cells (value = 1). Data for A and B are representative of 2 and 3 independent experiments, respectively.

Sustained PI 3-Kinase/Akt Pathway Is Required for G₁ Progression-- Taken together, our results indicate that inhibiting the rapid -arrestin1-dependent activation of PI 3-kinase/Akt pathway does not prevent -thrombin-induced progression through the G₁ phase of the cell cycle in IIC9 cells. To confirm that sustained activation of PI 3-kinase is required for G₁ progression, IIC9 cells were treated with LY294002 at 0, 2, and 4 h after -thrombin addition. Addition of LY294002 at 2 and 4 h post-stimulation blocks sustained Akt phosphorylation (Fig. 8A). Consistent with a role for sustained Akt activity in regulating G₁ progression, treatment with LY294002 ~2 and 4 h after -thrombin addition inhibits [³H]thymidine incorporation (Fig. 8B). Because Akt is downstream of PI 3-kinase activity and LY294002 inhibits PI 3-kinase, we reasoned that transient expression of constitutive Akt would rescue -thrombin-induced G₁ to S phase progression. To determine the effects of sustained Akt activity on G₁ progression, we examined whether expression of constitutive Akt rescues the effect of treatment with LY294002 (Fig. 8C). Consistent with our interpretation of LY294002 treatment, transient expression of constitutive Akt does rescue [³H]thymidine incorporation (Fig. 8C). Taken together these data indicate that sustained Akt activity is downstream of PI 3-kinase activity, and both sustained PI 3-kinase and Akt activities are essential for -thrombin-induced G₁ progression in IIC9 cells.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Sustained PI 3-kinase/Akt pathways are required for G1 progression. Serum-arrested IIC9 cells were incubated for 17 h in the absence or presence of -thrombin (1 NIH unit/ml). The -thrombin-stimulated cells were treated with 10 µM LY294002 at the times indicated after the addition of -thrombin. IIC9 cells were untransfected or transiently transfected with Myr-Akt (2 µg) and serum-arrested for 48 h as described under "Experimental Procedures." A, Akt immune complexes were immunoprecipitated from lysates containing equal protein with an anti-Akt antibody and analyzed for their ability to phosphorylate histone 2B as described under "Experimental Procedures." B and C, after 17 h the cells were incubated for an additional 3 h with 1 µCi of [3H]thymidine and [3H]DNA quantified as described under "Experimental Procedures." Data are representative of 3 experiments done in triplicate.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This is the first study to demonstrate a critical role for -arrestins in the activation of the PI 3-kinase/Akt pathway. In IIC9 cells, -thrombin-induced Akt phosphorylation increases within 5 min and is sustained for up to 6 h. The rapid increase in Akt phosphorylation is sensitive to expression of dominant negative -arrestin1, whereas sustained phosphorylation is insensitive. Because PtdIns(3,4,5)P₃s bind -arrestins with high affinity and are important regulators of receptor internalization (41), it has been thought that -arrestins are downstream targets of PI 3-kinase activity. Our data clearly show that -thrombin-induced rapid PI 3-kinase and Akt activities are downstream of -arrestin1. Our data also demonstrate a role for c-Src or a Src family member in the phosphorylation of Akt. Transient expression of -arrestin1(P91G-P121E), which prevents the binding of c-Src to -arrestin1 but does not inhibit internalization (31), also blocks -thrombin-induced phosphorylation of Akt. In agreement with a role for c-Src or an Src family member in the phosphorylation of Akt, treatment of IIC9 cells with PP1, a selective inhibitor of the Src family kinases (40), prevents -thrombin-induced rapid Akt phosphorylation. Taken together, these results strongly suggest that in IIC9 cells the association of -arrestin1 with c-Src is essential for -thrombin-induced Akt phosphorylation.

Although expression of dominant negative -arrestin1 blocks the PI 3-kinase/Akt pathway, it does not inhibit stimulation of the MAPK(ERK) pathway. The inability of -arrestin1 to affect the MAPK(ERK) pathway is surprising, because others (30, 33-35, 42) have shown that -arrestin1 can mediate MAPK(ERK) pathway activation. We found previously that -thrombin-induced stimulation of the MAPK(ERK) pathway is downstream of Ras (13, 14). Consistent with the ineffectiveness of dominant negative -arrestin1 to prevent ERK1/2 phosphorylation, Ras activation is unaffected by expression of dominant negative -arrestin1. Importantly, ERK1/2 phosphorylation is insensitive to treatment with PP1. These results suggest that the ability of -arrestin1 to assemble selective signaling components into functional complexes varies depending on the receptor and cell types.

In IIC9 cells -thrombin activation of the PI 3-kinase/Akt appears to be complex in IIC9 cells. We found that rapid and sustained Akt phosphorylation is differentially regulated. Rapid phosphorylation of Akt is dependent on -arrestin1; however, the sustained is not. Previous data from our laboratory (13, 36) found that the rapid increase in PI 3-kinase activity can be determined by generation of PtdIns(3,4,5)P₃ in p85 immune complexes, suggesting a role for class 1A PI 3-kinase in the rapid increase. At present the PI 3-kinase important for sustained Akt phosphorylation is unknown and requires further study.

PI 3-kinase is required for DNA synthesis in response to several mitogens (43) including -thrombin (13). In IIC9 cells, inhibition of PI 3-kinase activity and Akt phosphorylation by pretreatment with the selective PI 3-kinase inhibitor, LY42009, prevents the accumulation of cyclin D1 protein and the increase in CDK4-cyclin D1 activity in response to -thrombin (13). Because expression of dominant negative -arrestin1 inhibits PI 3-kinase/Akt pathway activation, we expected it to prevent passage through G₁ into S phase. Surprisingly, expression of the mutant is ineffective in blocking G₁ progression as measured by [³H]thymidine incorporation into DNA. Also -thrombin-induced increases of cyclin D1 protein and CDK4-cyclin D1 activity are not affected by expression of dominant negative -arrestin1. Consistent with the dependence of G₁ progression on the PI 3-kinase/Akt pathway, expression of dominant negative -arrestin1 inhibits the rapid phosphorylation of Akt but not sustained phosphorylation. These data suggest that rapid and sustained phosphorylation of Akt have distinct mechanisms of activation and different sets of targets. Interestingly, the -arrestin-dependent increases in phosphorylated JNK3 (29) and ERK (35) are localized to the cytosol, suggesting that activating these kinases through a -arrestin scaffold limits the subcellular targets of these kinases. Determining the different mechanisms for activating the PI 3-kinase/Akt pathway will be important for understanding how these mechanisms affect function.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant R01 DK46814 (to J. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Present address: Dept. of Molecular Biology and Microbiology, Case Western University, Cleveland, OH 44106.

To whom correspondence should be addressed. Tel.: 314-577-8543; E-mail: baldasjj@slu.edu.

Published, JBC Papers in Press, March 18, 2002, DOI 10.1074/jbc.M108995200

¹ The abbreviations used are: GPCRs, G protein-coupled receptors; PI 3-kinase, phosphatidylinositol 3OH-kinase, MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; JNK, c-Jun amino-terminal kinase; CDK, cyclin-dependent kinase; GRK, GPCR kinase; PMSF, phenylmethylsulfonyl fluoride; PtdIns(3,4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; PBS, phosphate-buffered saline; DTT, dithiothreitol.

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