Disruption of the Actin Cytoskeleton Leads to Inhibition of Mitogen-Induced Cyclin E Expression, Cdk2 Phosphorylation, and Nuclear Accumulation of the Retinoblastoma Protein-Related p107 Protein

Experimental Cell Research 259, 35–53 (2000) doi:10.1006/excr.2000.4966, available online at http://www.idealibrary.com on

Disruption of the Actin Cytoskeleton Leads to Inhibition of MitogenInduced Cyclin E Expression, Cdk2 Phosphorylation, and Nuclear Accumulation of the Retinoblastoma Protein-Related p107 Protein Galina Reshetnikova,* ,† Rita Barkan,† Boris Popov,* ,† Nikolay Nikolsky,† and Long-Sheng Chang* ,1 *Department of Pediatrics, Children’s Hospital and The Ohio State University College of Medicine and Public Health, Columbus, Ohio 43205; and †Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia

INTRODUCTION The actin cytoskeleton has been found to be required for mitogen-stimulated cells to passage through the cell cycle checkpoint. Here we show that selective disruption of the actin cytoskeleton by dihydrocytochalasin B (H 2CB) blocked the mitogenic effect in normal Swiss 3T3 cells, leading to cell cycle arrest at mid to late G 1 phase. Cells treated with H 2CB remain tightly attached to the substratum and respond to mitogen-induced MAP kinase activation. Upon cytoskeleton disruption, however, growth factors fail to induce hyperphosphorylation of the retinoblastoma protein (pRb) and the pRb-related p107. While cyclin D1 induction and cdk4-associated kinase activity are not affected, induction of cyclin E expression and activation of cyclin E– cdk2 complexes are greatly inhibited in growth-stimulated cells treated with H 2CB. The inhibition of cyclin E expression appears to be mediated at least in part at the RNA level and the inhibition of cdk2 kinase activity is also attributed to the decrease in cdk2 phosphorylation and proper subcellular localization. The expression patterns of cdk inhibitors p21 and p27 are similar in both untreated and H 2CBtreated cells upon serum stimulation. In addition, the changes in subcellular localization of pRb and p107 appear to be linked to their phosphorylation states and disruption of normal actin structure affects nuclear migration of p107 during G 1-to-S progression. Taken together, our results suggest that the actin cytoskeleton-dependent G 1 arrest is linked to the cyclin– cdk pathway. We hypothesize that normal actin structure may be important for proper localization of certain G 1 regulators, consequently modulating specific cyclin and kinase expression. © 2000 Academic Press Key Words: retinoblastoma protein; p107; actin cytoskeleton; dihydrocytochalasin B; cyclins; cdks; inhibitors; subcellular localization; cell cycle.

The actin cytoskeleton has been shown to be involved in the processes of growth-factor-mediated signal transduction (for reviews, see [3, 28]). The receptors of several growth factors bind to actin filaments [27, 70, 71]. Many other proteins involved in growth signaling are also associated with the cytoskeleton [6, 19, 24, 56, 60, 72]. In addition, selective disruption of normal actin structure leads to inhibition of the ability of growth factors to promote S phase entry [5, 8, 31, 32, 43, 52]. Currently, the molecular mechanisms by which the actin cytoskeleton participates in the processes that take place between the events following the binding of growth factors to their receptors and initiation of DNA synthesis are not understood. The progression of eukaryotic cell cycle is regulated by a series of serine/threonine protein kinases termed cyclin-dependent kinases (cdks) (reviewed in [38, 65]). The cdk-associated kinase activities are dependent on the synthesis of and association with specific regulatory subunits known as cyclins. Among them, D-type cyclins (D1, D2, and D3), which associate with and activate cdk4 and cdk6, and cyclin E, which is required for the activation of cdk2, play important roles during G 1-to-S progression. When quiescent cells are stimulated to enter the cell cycle, expression of both cyclin D and cyclin E are induced. The cyclin-D-associated kinase activity reaches a maximum at mid G 1 phase [44, 46], while the cyclin-E-associated kinase activity reaches a maximum at late G1 to early S phase [15]. These cyclin D– cdk4/6 and cyclin E– cdk2 complexes promote G 1 progression by phosphorylating the retinoblastoma (Rb) protein (pRb) and pRb-related p107, important negative regulators of cell cycle progression [38]. These pRb family proteins can bind to the E2F family of transcription factors, necessary for many genes important for cell cycle control or DNA synthesis [51]. The pRb–E2F interaction not only blocks transcription activation by E2F but also forms an active transcription repressor

1 To whom correspondence and reprint requests should be addressed at Department of Pediatrics, Children’s Hospital and The Ohio State University, W230, 700 Children’s Drive, Columbus, OH 43205-2696. Fax: (614) 722-2774. E-mail: [email protected].

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0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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complex at the promoter of several cell cycle genes [10, 41, 42, 75]. Recent evidence shows that the cyclin D– cdk4/6 complexes only partially phosphorylate pRb, resulting in blocking active transcription repression by pRb but not releasing it from interaction with E2F [25, 40]. Subsequent hyperphosphorylation of pRb by the cyclin E– cdk2 complexes disrupts the pocket structure of pRb and prevents it from binding and inactivating E2F [25]. Such a progressive loss of pRb activities resulting from sequential phosphorylation of pRb by cyclin– cdk complexes may allow differential regulation of genes involved in cell cycle progression. In addition to the interplay between the positive and negative cell cycle regulators, recent studies suggest that additional cell cycle control involves the modulation of their subcellular localization. Both pRb and p107 change their intracellular localization during G 1to-S progression [1, 39, 47, 48, 50, 73]. In G 0/G 1, hypophosphorylated pRb is tightly associated with the nuclear structure, while in late G 1 into early S phase, pRb becomes hyperphosphorylated. The hyperphosphorylated pRb has a weak association with the nuclear compartment and is released from the nucleus into the cytoplasm [47, 48]. On the other hand, p107 expression is induced as cells progress through G 1 into S phase and the protein is accumulated in the nucleus [1, 39, 50, 73]. In addition, subcellular distribution of certain cyclins and cdks has been shown to be regulated by cell-cycle-dependent events [11, 14, 55, 57, 77]. For example, while cyclin E is typically localized in the nucleus, cdk2 compartmentalizes into the cytoplasm in quiescent cells [11, 55]. Upon serum stimulation, cdk2 rapidly enters the nucleus and the abundance of the cyclin E– cdk2 complexes increases as cell progress from G 1 into S. It is generally believed that the changes of intracellular localization of these cell cycle regulators are important for their growth regulatory functions. Although selective disruption of the actin cytoskeleton in mitogen-stimulated cells prevents S phase entry, currently it is not known whether such a block is directly linked to the cell cycle regulatory pathway and whether normal actin structure is important for the proper intracellular localization of certain G 1 regulators. In this work we present evidence that the actin cytoskeleton-dependent G 1 arrest correlates with the inhibition of hyperphosphorylation of pRb and p107. Disruption of the actin cytoskeleton leads to inhibition of cyclin E induction, cdk2 phosphorylation and subcellular localization, as well as nuclear accumulation of p107 in growth-stimulated cells. MATERIALS AND METHODS Cell culture, synchronization, and cell cycle analysis. Swiss 3T3 cells were obtained from Flow Laboratories (Rockville, MD) and

grown in Dulbecco’s modified Eagle’s (DME) medium supplemented with 10% fetal bovine serum (FBS). To maintain normal cell characteristics, the cells were routinely passaged prior to confluence. For serum starvation and stimulation experiments, Swiss 3T3 cells were seeded at a density of 5 ⫻ 10 5 cells per 100-mm dish or 1.5 ⫻ 10 5 cells per 60-mm dish in DME medium containing 10% FBS. On the next day, the medium was replaced with starvation medium (DME medium containing 0.1% FBS) and cells were arrested for 72 h. Quiescent cells were then restimulated to enter the cell cycle by the addition of 10% FBS. To disrupt the actin cytoskeleton, separate sets of cells were also treated with 10 ␮g/ml of dihydrocytochalasin B (H 2CB; Sigma). For flow cytometry analysis, quiescent or serumstimulated cells with or without H 2CB treatment were harvested and then fixed and permeabilized with ice-cold 70% ethanol. Fixed cells were stained in a solution containing 10 ␮g/ml of propidium iodide (PI) and 0.5 mg/ml of RNase A and analyzed on a Coulter EPICS Elite flow cytometer. For thymidine incorporation experiments, cells were plated out in 24-well dishes (Nunc) and growth arrested as described above. Quiescent cells were stimulated by the addition of 10% FBS or epidermal growth factor (EGF; 10 ng/ml). At the time of the addition of growth factors or at various times after stimulation, some cells were also treated with H 2CB. In some experiments, cultured medium containing H 2CB was removed after different time intervals by washing the cells with phosphate-buffered saline (PBS) and then replenishing them with growth medium containing 10% FBS or EGF. In the cases when H 2CB was removed from cultured medium later than 10 h after stimulation, medium supplemented with 0.5% FBS was used to prevent restimulation of cells [58]. At 20 h after stimulation, cells were pulse-labeled with 4 ␮Ci/ml of [ 14C]thymidine for 1 h. Labeled cells were washed with PBS three times and incubated with ice-cold 5% trichloroacetic acid at room temperature for 20 min to remove acid-soluble labels. After being washed with PBS twice, samples were solubilized in 0.5 ml of 0.1 M NaOH and the acid-insoluble radioactivity in each sample was determined by scintillation counting. Immunofluorescence microscopy. Cells were seeded on chamber slides (Nagle Nunc) and arrested for 72 h as described above. Quiescent cells were restimulated with 10% FBS with or without the presence of 10 ␮g/ml H 2CB. Eight hours after stimulation cells were fixed in 3.7% formaldehyde in PBS for 10 min, rinsed with PBS three times, and permeabilized for 5 min in 0.5% Triton X-100 in PBS. For visualization of actin filaments, slides were incubated with 100 ng/ml of TRITC-conjugated phalloidin (Sigma) for 15 min, washed with PBS three times, and mounted with buffered glycerol. Photographic images of stained cells were taken under a Zeiss UV microscope. Immunoblotting and subcellular fractionation analysis. Cells were washed twice with ice-cold PBS and extracted in lysis buffer A (50 mM Tris–HCl, pH 7.4, 250 mM NaCl, 2 mM EDTA, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 ␮g/ml aprotinin, 5 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, 50 mM sodium fluoride, 0.1 mM sodium orthovanadate) at 4°C for 30 min. Soluble protein extracts were obtained by centrifugation and their concentrations were determined using the Bio-Rad protein assay. Equal amounts of protein (100 ␮g) were fractionated in 7.5–12% SDS–polyacrylamide gels and electroblotted onto a Hybond-C extra membrane (Amersham). The filter membrane was then incubated for 1 h at room temperature with blocking buffer containing 5% powdered nonfat milk or 2% bovine serum albumin in TBST (20 mM Tris–Cl, pH 7.6, 150 mM NaCl, and 0.1% Tween-20). Specific antibody against pRb (C15 and G3-245), p107 (C-18), cyclin D1 (HD11), cyclin E (M20), cyclin H (FL-323), cdk2 (M2), cdk4 (C22), CDK7 (C-4), MAT1 (FL-309), p27 (C-19), or p21 (C-19) (all from Santa Cruz with the exception of G3-245 from Pharmingen) was diluted in blocking buffer and added to the membrane for 1 h at room temperature or overnight at 4°C. After being extensively washed with TBST, the enhanced chemiluminesence system with a secondary antibody conjugated with horse-

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FIG. 1. Analysis of the cell cycle distribution in serum-stimulated Swiss 3T3 cells with or without H 2CB treatment. Cells were grown, arrested, and restimulated with FBS as described under Materials and Methods. Flow cytometry analysis was performed on normal cycling cells (A), arrested cells deprived of serum for 3 days (B), and serum-stimulated cells without (C) or with H 2CB treatment for various times (D). DNA content as determined by linear PI fluorescence and cell numbers were plotted on the abscissa and ordinate, respectively. Duplicated experiments were performed and a representative set of flow cytometric profile was shown.

radish peroxidase (Amersham) was used to visualize the immunoblot signal. For detection of phosphorylated MAP kinases, the phosphop44/p42 MAP kinase (Thr 202/Tyr 204) E10 monoclonal antibody (New England BioLabs) was used. The expected molecular weight of each specific protein detected was compared to molecular weight standards. For subcellular fractionation, nuclear and cytoplasmic fractions were prepared according to Muller et al. [50] with slight modification. Briefly, cells were swollen for 20 min in 300 ␮l of ice-cold hypotonic buffer (10 mM Tris–HCl, pH 7.5, 10 mM KCl, 3 mM MgCl 2, 1 mM EGTA, 1 mM Na 3VO 4, 10 mM NaF, 1 mM PMSF, and protease inhibitor cocktail for mammalian cell extracts [Sigma]), subjected to 30 slow strokes with a Dounce homogenizer, and spun for 5 min at 375g and 4°C. The supernatant containing the cytoplasmic fraction was removed and saved on ice. The nuclear pellet was washed three times in hypotonic buffer and solubilized in lysis buffer A. Equal amounts of protein (50 ␮g) from each fraction were electrophoresed on SDS–polyacrylamide gels and Western blotted with appropriate antibodies as described above. In vitro kinase assay. For cyclin-E-associated kinase assay [64], cyclin-E-associated complexes were immunoprecipitated with an antibody against either cyclin E or cdk2, and the kinase activity was determined using histone H1 (Boehringer Mannheim) as the substrate. For cyclin-D-associated kinase assay [44], cyclin-D-associated

complexes were immunoprecipitated with an antibody against cdk4, and the kinase activity was determined using a glutathione S-transferase (GST)–Rb fusion protein containing amino acids 379 –928 of the human Rb protein [33] as the substrate. Briefly, cells were lysed in buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 10% glycerol, 1% NP-40, 10 mM ␤-glycerophosphate, 1 mM PMSF, 1 mM aprotinin, 1 mM leupeptin, 1 mM pepstatin, and 1 mM sodium orthovanadate. Soluble extracts (0.5 mg) were immunoprecipitated with 5 ␮l of specific antibodies for 1 h at 4°C with gentle shaking. Immune complexes were collected by incubation for 1 h at 4°C with protein A–Sepharose (Sigma) and washed three times in ice-cold lysis buffer and then twice at room temperature with kinase buffer (20 mM Hepes, pH 7.5, 20 mM MgCl 2, 2 mM MnCl 2, 1 mM DTT, and 10 mM ␤-glycerolphosphate). The final pellet was resuspended in kinase buffer supplemented with 25 ␮M ATP, 5 ␮Ci of [␥- 32P]ATP (6000 Ci/mol), and 2 ␮g of GST–Rb or histone H1 protein in a final volume of 20 ␮l. After incubation for 30 min at room temperature, the kinase reaction was stopped by the addition of equal volume of 2⫻ SDS gel–sample buffer. The extent of substrate phosphorylation was determined by SDS–polyacrylamide gel electrophoresis and autoradiography. For preparation of GST–Rb fusion protein, fresh overnight cultures of Escherichia coli DH5␣ carrying the pGT–RB(379 –928) plas-

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TABLE 1 Cell Cycle Analysis of Swiss 3T3 Cells Following Serum Stimulation a % Cells

G 1/G 0

S

G2 ⫹ M

Cycling cells Arrested, 3 days Arrested, 3.5 days Arrested, 4 days Serum stimulation ⫹FBS, 9 h ⫹FBS, 12 h ⫹FBS, 14 h ⫹FBS, 16 h ⫹FBS, 18 h ⫹FBS, 21 h ⫹FBS, 24 h ⫹FBS, 34 h

24.7 94.6 96.1 95.9

74.2 4.9 3.1 4.0

1.1 0.5 0.8 0.1

95.1 95.0 89.3 31.8 16.9 14.1 46.1 78.5

3.2 4.9 10.5 68.0 82.2 55.3 28.3 19.1

1.7 0.1 0.2 0.2 0.9 30.6 25.6 2.4

⫹FBS⫹H 2CB, 9h ⫹FBS⫹H 2CB,12h ⫹FBS⫹H 2CB,14h ⫹FBS⫹H 2CB,16h ⫹FBS⫹H 2CB,18h ⫹FBS⫹H 2CB,21h ⫹FBS⫹H 2CB,24h ⫹FBS⫹H 2CB,34h

G 1/G 0

S

G2 ⫹ M

95.5 95.3 93.3 86.3 76.3 69.4 69.8 66.0

3.4 3.6 6.5 13.0 22.2 25.2 18.8 7.2

1.1 1.1 0.2 0.7 1.5 5.4 11.4 26.8

a The cell cycle distribution of cells under various culture conditions as indicated was analyzed by a flow cytometer as described in Fig. 1. Extended analysis of DNA content was performed on Multicycle software (Phoenix Flow System).

mid, kindly provided by Dr. William G. Kaelin, Jr., of Harvard Medical School, was diluted 1:10 in LB broth containing ampicillin (50 ␮g/ml) and incubated for 1 h at 37°C with vigorous shaking. To induce fusion protein expression, isopropyl-␤-D-thiogalactopyranoside (Sigma) was added to a final concentration of 0.1 mM and the culture was grown for another 4 h at 37°C. Induced bacterial cells were harvested by centrifugation, resuspended in ice-cold buffer containing 20 mM Tris–Cl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP40, and lysed by sonication. After centrifugation at 10,000g for 5 min at 4°C, soluble lysate was incubated with glutathione– Sepharose (Pharmacia) and the GST–Rb fusion protein was eluted accordingly [33]. The size, purity, and concentration of the fusion protein were evaluated by Coomassie blue staining of SDS–polyacrylamide gels. The purified GST–Rb was found to migrate as a single 85-kDa band. RNase protection assay (RPA). Cytoplasmic RNA was isolated from cells as described previously [79]. In vitro transcription was performed in the presence of [␣- 32P]UTP using mCyc-1 and mCyc-2 mouse cell cycle regulator multiprobe template sets (Pharmingen). The mCyc-1 multiprobe template set allowed the detection of cyclin D RNA in addition to cyclin A, B, and C RNAs, while the mCyc-2 multiprobe template set detected cyclin E RNA in addition to cyclin F, G, H, and I RNAs. The 32P-labeled riboprobes were used to hybridize with cytoplasmic RNA (20 ␮g), using the RiboQuant multiprobe RPA system (Pharmingen). The protected product was analyzed on a 5 or 6% denatured polyacrylamide gel containing 8 M urea. The relative intensity of each protected band was quantified using a Storm 860 phosphoimager (Molecular Dynamics).

RESULTS

Effect of disruption of the actin cytoskeleton on G 1to-S progression. Cytochalasin B has been shown to inhibit actin polymerization by binding of the cytochalasin molecule to the fast polymerizing end of actin filament and has been widely used as a tool to identify the actin-dependent processes [12]. To understand the role of the actin cytoskeleton during mitogenic signal transduction, we examined the effect of treatment of the cytochalasin B derivative H 2CB on quiescent Swiss 3T3 cells following stimulation by growth factors.

To determine the cell cycle distribution following growth stimulation, normal Swiss 3T3 cells deprived of serum for 72 h were restimulated with FBS to reenter the cell cycle. Selective disruption of the actin cytoskeleton was performed by the addition of H 2CB to culture medium at the time of stimulation. Cells were harvested at various times after stimulation and analyzed by flow cytometry for DNA contents. As illustrated in Fig. 1, quiescent cells when stimulated by FBS exhibited synchronous progression through the cell cycle. Compared to normal cycling cells (Fig. 1A), almost all of the cells deprived of serum for 3 days remained in G 1/G 0 (Fig. 1B and Table 1). Upon serum stimulation, cells gradually moved into the cell cycle and a significant increase of S phase cells was observed at 14 h after stimulation (Fig. 1C and Table 1). These serumstimulated cells completed one round of the cell cycle between 24 and 34 h as shown by the majority of cells progressing through G 2/M and back to G 1 phase. In contrast, the majority of the cells treated with H 2CB following stimulation remained at G 0/G 1 phase and did not enter the cell cycle (Fig. 1D). Intriguingly, we also observed a small percentage of stimulated cells, which were able to enter into S phase in the presence of H 2CB (Table 1). Although the exact reason for the presence of such cell population is not known, it is possible that they might represent cells that were not arrested by serum deprivation under our experimental condition or they were resistant to the inhibitory effect of H 2CB during growth stimulation. In addition, we noted the presence of such cell population only in cells stimulated by serum but not by EGF in the presence of H 2CB (data not shown). Since serum contains multiple growth factors, some of the factors might trigger growth-signaling pathways that were not affected by disruption of the actin cytoskeleton. Overall, these results are consis-

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tent with previous observations that the actin cytoskeleton is important for the cell to progress from G 1 to S phase. To further investigate the involvement of the actin cytoskeleton during G 1-to-S progression, quiescent Swiss 3T3 cells were stimulated with FBS, and H 2CB was added at various times following serum stimulation. Cell cycle progression into S phase was monitored by the incorporation of [ 14C]thymidine into DNA 20 h after stimulation (Fig. 2). Compared to quiescent cells, serum-stimulated cells gave rise to about a 10-fold stimulation of DNA synthesis (Fig. 2A). Interestingly, when H 2CB was added together with serum or 2– 8 h after serum addition, the cell failed to undergo DNA synthesis. When H 2CB was added 10 h after serum addition, a slight increase of DNA synthesis was observed compared to that in quiescent cells. In contrast, when H 2CB was added 12 h or later after serum stimulation, high levels of DNA synthesis similar to those in stimulated cells without H 2CB treatment were detected (Fig. 2A). Similar results were also observed when EGF was used in place of FBS to stimulate cell growth (Fig. 2B). These results together with those from the flow cytometry analysis (Fig. 1 and Table 1) indicate that the actin cytoskeleton is not required for growth-stimulated cells to progress into S phase when the cells have passed mid G 1. Subsequently, we determined whether disruption of normal actin structure at G 0/early G 1 phase would affect the S phase entry. H 2CB was added together with FBS to quiescent Swiss 3T3 cells. After different time intervals, H 2CB was removed from cells and fresh medium was added. At 20 h after stimulation, thymidine incorporation experiments were carried out. When H 2CB was present in the medium within the first 6 h after serum stimulation, high levels of DNA synthesis similar to or even higher than those observed in the control stimulated cells were detected (Fig. 2C). On the contrary, when cells were treated with H 2CB for 8 h or longer after serum stimulation, little or no DNA synthesis was observed. These results indicate that disruption of the actin cytoskeleton at G 0/early G 1 phase does not inhibit the ability of growth factors to

FIG. 2. Effect of H 2CB treatment on cell cycle progression into S phase in growth-factor-stimulated cells. Quiescent Swiss 3T3 cells were stimulated with FBS (A) or EGF (B) to proliferate. H 2CB was added to the cell at indicated time after stimulation. Alternatively, H 2CB was added to the cell together with serum and removed from the cell culture at indicated times after stimulation (C). Entry into S phase was measured by [ 14C]thymidine incorporation 20 h after stimulation. Quiescent cells or cells stimulated by serum or EGF were also used as controls. Triplicate samples were used for each treatment and at least three independent experiments were performed. Data shown are the average of triplicate samples from a representative experiment.

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FIG. 3. Cell morphology and actin structure in Swiss 3T3 cells with or without H 2CB treatment. Quiescent cells (A), serum-stimulated cells (B), or stimulated cells in the presence of H 2CB (C) were fixed, stained with TRITC-conjugated phalloidin to label F-actin, and then photographed under a Zeiss UV microscope.

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FIG. 3—Continued

stimulate DNA synthesis. It should be noted that induction of DNA synthesis appeared to be higher in H 2CB-treated cells (especially in those treated for 4 – 6 h) than in stimulated cells without H 2CB treatment (Fig. 2C). Currently, the explanation for such an effect is not clear. It is possible that a short treatment with H 2CB may lead to a more synchronous initiation of S phase entry after growth stimulation. Taken together, these results suggest that the actin cytoskeleton is important for the transmission of some growth signals appearing only in the mid to late part of G 1 phase. H 2CB-treated cells remain attached to the substratum and responded to MAP kinase activation upon serum stimulation. Cytochalasins at different concentrations have been shown to have differential effects on cell morphology, attachment, and signaling [2, 68]. We compared the morphology of Swiss 3T3 cells with or without H 2CB treatment for 8 h, followed by staining with TRITC-labeled phalloidin, which binds to F-actin. As shown in Fig. 3A, quiescent cells remained with a flat morphology with some stress fibers; some appeared to retain more stress fibers than others which showed only very few filamentous actin. Upon serum stimula-

tion cells possessed many long stress fibers, mostly aligned parallel to each other (Fig. 3B). In the presence of H 2CB, the actin cytoskeleton was severely disrupted in serum-stimulated cells, while punctate distribution of cortical structures was retained (Fig. 3C). Without the actin cytoskeleton, these cells showed some different morphology and were not able to stretch out like those with normal actin structure (compare Fig. 3B to Fig. 3C). Note that under our H 2CB treatment condition cells remained tightly attached to the substratum and, after the removal of H 2CB, cells restored their actin structure. Also, the level of actin protein remained unchanged in cells with or without H 2CB treatment (Fig. 4). These results indicate that H 2CB at the dose that we used selectively disrupts the actin cytoskeleton but does not affect the adherent ability of the cell. Previous reports demonstrate that focal adhesion complex formation is required for efficient growth factor activation of p42 and p44-MAP kinases, also referred to as ERK1 and ERK2 [63]. We also examined the effect of cytoskeleton disruption by H 2CB on MAP kinase activation. Since activation of MAP kinases occurs through their phosphorylation by an upstream

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FIG. 4. Effect of cytoskeleton disruption by H 2CB on MAP kinase activation. (A) Quiescent cells were stimulated with FBS for 5 min or were treated with H 2CB for 2 h prior to serum stimulation for 5 min. Cells were harvested for Western blot analysis using the anti-phospho-p44/p42 MAP kinase monoclonal antibody or an anti-actin antibody. (B) Quiescent cells were stimulated with FBS in the presence or absence of H 2CB for 14 h prior to harvesting for Western blot analysis. Note that a similar level of phospho-MAP kinases was detected in serum-stimulated cells with or without H 2CB treatment.

MAP kinase (MEK) [59], we measured the level of phospho-p44/p42 MAP kinases in extracts prepared from serum-stimulated cells with or without the presence of H 2CB using the anti-phospho-p44/p42 E10 monoclonal antibody. Quiescent cells were serum stimulated for 5 min or were pretreated with H 2CB for 2 h prior to serum stimulation. In another experiment quiescent cells were serum stimulated with or without the presence of H 2CB for 14 h. Analogous to those reported previously [59], quiescent cells had little or no phoshop44/p42 MAP kinases (Fig. 4A, lane 1). Upon serum stimulation for 5 min, a doublet of phospho-p44/p42 MAP kinases was detected readily (lane 2). Importantly, cells pretreated with H 2CB for 2 h followed by serum stimulation for 5 min also showed a doublet of phospho-p44/p42 MAP kinases (lane 3). Similar results for the presence of phospho-p44/p42 MAP kinases were also obtained in serum-stimulated cells with or without the presence of H 2CB for 14 h (Fig. 4B). These results indicate that under our H 2CB treatment protocol the signaling pathway leading to MAP kinase activation remains intact in cells without the actin cytoskeleton, and they further suggest that the G 1 block resulting

from cytoskeleton disruption by H 2CB treatment in mitogen-stimulated cells is not linked to the adhesionmediated MAP kinase pathway. The actin cytoskeleton-dependent G 1 arrest correlates with inhibition of hyperphosphorylation of pRb and p107. pRb is initially phosphorylated at mid to late G 1 phase and the phosphorylation state increases as a function of cell cycle progression [38, 65]. To examine whether H 2CB-induced growth arrest correlated with the inhibition of pRb phosphorylation at mid to late G 1, Western blot analysis using an anti-pRb antibody was carried out. Quiescent Swiss 3T3 cells were stimulated by FBS or EGF to reenter the cell cycle. H 2CB was added together with growth factors and at various hours after stimulation cells were harvested and analyzed. As evidenced by the difference in their relative mobility in the gel, extracts prepared from quiescent cells exhibited mainly the hypophosphorylated form of pRb, which migrated faster in the gel (Fig. 5A). Hyperphosphorylated forms of pRb, which migrated more slowly in the gel, were first detected 9 h after serum stimulation and became predominant after 12–14 h. However, this hyperphosphorylation of pRb was greatly inhibited in stimulated cells in the presence of H 2CB, as evidenced by the presence of mostly one slow-migrating band (Figs. 5A and 5B). Similar results for the inhibition of pRb hyperphosphorylation were also observed in cells stimulated by EGF in the presence of H 2CB (Fig. 5B). These results indicate that the actin cytoskeleton-dependent G 1 arrest is linked to the pathway leading to pRb hyperphosphorylation. The pRb-related p107 protein has also been implicated in the control of cell cycle progression [38]. The p107 protein is expressed at low levels in quiescent cells and begins to accumulate as cells pass through G 1 [7, 30, 67]. In addition, phosphorylation of p107 is cell cycle regulated and is functionally important because it is associated with the loss of growth suppression activity. We also examined whether disruption of the actin cytoskeleton affected p107 phosphorylation by Western blot analysis using an anti-p107 antibody. As shown in Fig. 5C, quiescent cells expressed a small amount of p107 protein, which migrated as two bands in the gel with the fast-migrating, hypophosphorylated species predominantly. Upon serum stimulation for 6 h, p107 remained at low levels and treatment with H 2CB did not affect p107 expression pattern. Levels of p107 increased substantially 14 h after serum stimulation and the majority of the p107 protein exhibited as a slow-migrating, hyperphosphorylated form. Interestingly, although treatment with H 2CB did not reduce p107 levels, it considerably inhibited p107 hyperphosphorylation as indicated by the appearance of the predominant fast-migrating, hypophosphorylated species.

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FIG. 5. Inhibition of hyperphosphorylation of pRb and p107 in mitogen-stimulated Swiss 3T3 cells treated with H 2CB. (A) Equal amounts of soluble protein extracts prepared from quiescent cells or FBS-stimulated cells with or without H 2CB treatment for various times, as indicated, were fractionated on an SDS–polyacrylamide gel and analyzed by immunoblotting with an anti-pRb antibody. A doublet of pRb bands was visualized with the upper, slow-migrating species representing the hyperphosphorylated form. (B) Inhibition of pRb hyperphosphorylation is also observed in EGF-stimulated cells in the presence of H 2CB. (C) Expression and phosphorylation patterns of p107 were examined by Western blot analysis using an anti-p107 antibody. Similar to pRb, two major bands of p107 were detected. The slow-migrating species represents hyperphosphorylated p107.

Similarly, cells stimulated with serum for 16 h expressed high levels of p107, which existed mainly as the hyperphosphorylated form, and treatment with H 2CB inhibited p107 hyperphosphorylation (Fig. 5B). These results indicate that the actin cytoskeleton-dependent G 1 arrest also correlates with the inhibition of p107 hyperphosphorylation.

Cyclin D1– cdk4 kinase activity is not affected by cytoskeleton disruption. In fibroblasts, hyperphosphorylation of pRb during G 1 phase is thought to be mediated by the G 1 cyclin– cdk complexes, cyclin D– cdk4 and cyclin E– cdk2 [38, 65]. To investigate whether cytoskeleton disruption by H 2CB affected the expression of these G 1 cyclin– cdk complexes, we first

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FIG. 6. Expression of the cdk-4 and cyclin D1 proteins and cdk4associated kinase activity in cells with or without H 2CB treatment. (A) Western blot analysis of cell extracts from quiescent cells or serum-stimulated cells with or without H 2CB treatment for various times, as indicated, was conducted using the antibody against cdk-4 (top) or cyclin D1 (bottom). (B) An in vitro kinase assay was used to detect the cdk4-associated kinase activity. Equal amounts of protein extracts from cycling cells (lane 1), quiescent cells (lane 2), and serum-stimulated cells without (lane 3) or with H 2CB treatment (lane 4) for 10 (top) or 14 h (bottom) were immunoprecipitated with an anti-cdk4 antibody. In vitro kinase activity in the precipitate was measured using a GST–Rb(379 –928) fusion protein as the substrate (lanes 1– 4). As controls, extracts from cycling cells were assayed using a GST protein as the substrate (lane 5) or without any substrate (lane 6).

compared the expression of the cyclin D1 and cdk4 proteins by Western blot analysis. Consistent with previous observations [4, 44, 76], quiescent Swiss 3T3 fibroblasts expressed little or no detectable amount of the cyclin D1 protein, while cells stimulated by FBS for 6 or 14 h expressed significant amounts of the cyclin D1 protein (Fig. 6A). This induction of cyclin D1 protein expression appeared to persist at similar levels from 6 to 14 h. Treatment with H 2CB did not affect the induction of cyclin D1 at either 6 or 14 h after serum stimulation. Unlike cyclin D1 expression, quiescent cells expressed substantial amounts of the cdk 4 protein (Fig. 6A). Serum-stimulated cells for 6 or 14 h showed

similar levels of cdk4 protein expression as the unstimulated cells. Treatment with H 2CB also did not affect expression of the cdk4 protein at either 6 or 14 h after serum stimulation. Subsequently, we analyzed the cdk4-associated kinase activity by in vitro kinase assay. Extracts prepared from cycling, quiescent, or serum-stimulated cells with or without H 2CB treatment for 10 (Fig. 6B, top) or 14 h (Fig. 6B, bottom) were immunoprecipitated with an anti-cdk4 antibody. In vitro kinase activity of the immunoprecipitate was detected using a GST– Rb(379 –928) fusion protein as the substrate. In cycling cells the cdk4-associated kinase activity was readily detected, while in quiescent cells low levels of the kinase activity was noted (Fig. 6B). Similar to the induction of cyclin D1 protein expression, the cyclin D1– cdk4-associated kinase activity was induced in cells stimulated by serum for 10 or 14 h. Treatment of stimulated cells with H 2CB for 10 h only slightly reduced the induction of the cdk4 kinase activity (Fig. 6B, top) while no difference in the cdk4 kinase activity was seen in stimulated cells with or without H 2CB treatment for 14 h (Fig. 6B, bottom). All of these results indicate that disruption of the actin cytoskeleton during growth stimulation does not significantly affect cyclin D1 induction, cdk4 protein expression, and activation of the cdk4-associated kinase activity. Disruption of the actin cytoskeleton leads to inhibition of cyclin E induction and cdk2 kinase activity. Cyclin E binding to cdk2 and activation of the cyclin E– cdk2 complex are also involved in G 1-to-S progression. We carried out a similar Western blot analysis to compare the expression of cyclin E and cdk2 proteins in cells with or without H 2CB treatment for 14 h. Similar to those reported previously [11, 55, 80], low levels of cyclin E protein were detected in quiescent cells (Fig. 7A). Upon serum stimulation, cyclin E expression was induced to high levels. Interestingly, in contrast to cyclin D1 expression, treatment with H 2CB abolished the induction of cyclin E expression in serum-stimulated cells. Despite the fact that cyclin E expression is induced upon growth stimulation, the levels of cdk2 protein were found to be more or less constant among quiescent, serum-stimulated, or cycling cells (Fig. 7A). Treatment with H 2CB in serum-stimulated cells also did not alter the levels of cdk2 protein expression. These results indicate that disruption of the actin cytoskeleton leads to inhibition of cyclin E induction but not cdk2 expression in growth-stimulated cells. To confirm whether inhibition of cyclin E induction upon H 2CB treatment would result in a decrease of the cyclin E– cdk2-associated kinase activity, cell extracts prepared from quiescent or serum-stimulated cells with or without H 2CB treatment for 14 h were immunoprecipitated with either an anti-cyclin E or an anti-

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ulated cells with H 2CB resulted in substantial reduction of the cdk2-associated kinase activity (Fig. 7B, lanes 4 and 8), which was in parallel to the inhibition of cyclin E protein induction (Fig. 7A). Taken together, these results indicate that induction of cyclin E protein expression and activation of the cyclin E– cdk2 complex in growth-stimulated cells depend on the presence of the actin cytoskeleton. Disruption of the actin cytoskeleton affects cyclin E RNA expression. Expression of the cyclin E gene is cell cycle regulated and its promoter containing E2Fbinding sites is regulated by E2F [9, 21, 37, 53]. To examine whether inhibition of cyclin E protein expression by H 2CB in serum-stimulated cells was due to reduction of its gene transcription, we measured cyclin E RNA levels by RPA. Cytoplasmic RNAs were isolated from quiescent cells or cells stimulated by FBS with or without the presence of H 2CB for 14 h, and analyzed. Total yeast RNA was also used as a nonspecific control. Consistent with previous reports [15, 35], quiescent cells expressed little or no cyclin E mRNA (Fig. 8A,

FIG. 7. Effect of cytoskeleton disruption on cyclin E protein expression and cyclin-E-associated cdk2 kinase activity in serumstimulated cells. (A) Expression of the cyclin E but not cdk2 protein is affected by H 2CB treatment. Western blot analysis using an anticyclin E (top) or anti-cdk2 antibody (bottom) was conducted as described in Fig. 6. (B) Reduction of cyclin E– cdk2 kinase activity in serum-stimulated cells treated with H 2CB. Equal amounts of soluble protein extracts were immunoprecipitated with either an anti-cdk2 (lanes 1– 4) or an anti-cyclin E antibody (lanes 5– 8). In vitro kinase activity in the precipitate was measured using a histone H1 as the substrate.

cdk2 antibody, and in vitro kinase activity of the immunoprecipitates was measured using histone H1 protein as the substrate. Consistent with the pattern of cyclin E and cdk2 protein expression, high levels of cdk2-associated kinase activity were detected in cycling cells when either the anti-cyclin E or the anticdk2 antibody was used to immunoprecipitate the kinase complexes (Fig. 7B, lanes 1 and 5). In contrast, little or no cdk2-associated kinase activity was detected in quiescent cells (lanes 2 and 6). Upon serum stimulation, high levels of the cdk2-associated kinase activity were detected (lanes 3 and 7), analogous to those found in cycling cells. Treatment of serum-stim-

FIG. 8. Inhibition of cyclin E RNA expression upon cytoskeleton disruption in serum-stimulated cells. Analysis of cyclin E, F, G, H, and I RNAs was performed as described in the text using the mCyc-2 multiprobe RPA system.

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lane 2). Upon serum stimulation cyclin E mRNA levels were elevated (lane 3) compared to the RNA levels of two housekeeping genes, ribosomal L32 and glyceraldehyde-3-phosphate dehydrogenase. Treatment of serum-stimulated cells with H 2CB resulted in inhibition of cyclin E RNA expression (Fig. 8A, lane 4). Intriguingly, we also observed that cyclin F RNA expression was induced in serum-stimulated cells and its induction was also inhibited upon H 2CB treatment (Fig. 8A). No obvious changes in the RNA levels of cyclin G, H, and I were observed in serum-stimulated cells with or without H 2CB treatment. In addition, consistent to that found for cyclin D1 protein expression (Fig. 6A), cyclin D1 RNA level also was not changed in stimulated cells with or without H 2CB treatment (data not shown). These results indicate that the actin cytoskeleton-dependent inhibition of cyclin E expression is mediated at least in part at the RNA level. Expression of the cdk inhibitors p27 Kip1 and p21 Cip1 is not affected by cytoskeleton disruption. The cdk inhibitors p27 Kip1 and p21 Cip1 have been implicated in the mitogen-dependent regulation of cdks, including cdk2 [65]. In addition, p27 Kip1, while being a potential inhibitor of cyclin-E- and cyclin-A-dependent kinases, could act as a positive regulator for cyclin-D-dependent kinase [13]. p27 Kip1 is accumulated to high levels in quiescent cells and is down-regulated when cells are induced to proliferate [65]. Conversely, some p21 Cip1 proteins were detected during quiescence. The levels of p21 Cip1 are slightly increased during mitogenic stimulation and then declined before the cells enter into S phase. To examine whether there are any changes in p27 Kip1 and p21 Cip1 expression upon cytoskeleton disruption, Western blot analysis using anti-p27 Kip1 and antip21 Cip1 antibodies was conducted. Similar to those reported previously, high levels of p27 Kip1 could be detected readily in quiescent cells (Fig. 9A). The levels of p27 Kip1 appeared to persist in cells stimulated by FBS for 6 h, but were greatly reduced in cells stimulated for 14 h. Addition of H 2CB to serum-stimulated cells for 6 or 14 h did not affect the expression pattern of p27 Kip1. In a similar Western blot analysis, we detected some p21 Cip1 expression in quiescent cells (Fig. 9B). p21 Cip1 levels were slightly increased when cells were stimulated by FBS for 4 or 8 h, and then decreased when cells were stimulated for 14 h. Treatment of serumstimulated cells with H 2CB for 4, 8, or 14 h did not affect the expression pattern of p21 Cip1. These results indicate that expression of both p27 Kip1 and p21 Cip1 inhibitors during growth stimulation is not affected by the loss of normal actin structure. H 2CB treatment affects the phosphorylation pattern of cdk2. Activation of the cyclin E– cdk2 complex requires CAK-mediated phosphorylation of a single thre-

FIG. 9. Expression patterns of p27 kip1 and p21 cip1 inhibitors were not affected by disruption of the actin cytoskeleton. Quiescent cells were stimulated by FBS with or without simultaneous addition of H 2CB. Stimulated cells were harvested at the indicated time. Equal amounts of protein extracts were immunoblotted with an antibody to p27 kip1 (A) or p21 cip1 (B).

onine residue Thr 160 in cdk2 [65]. This phosphorylation can be detected as an increase in the electrophoretic mobility of cdk2 in a 15% SDS–polyacrylamide gel [18]. To examine whether there was any change in cdk2 phosphorylation upon H 2CB treatment, cell extracts (400 ␮g) prepared in the same manner as those for in vitro kinase assay were immunoprecipitated and then subjected to immunoblot analysis with an anti-cdk2 antibody. As shown in Fig. 10, the cdk2 protein expressed in cycling cells migrated as a doublet in the gel. In quiescent cells the slower migrating form of cdk2 appeared to be the predominant species, whereas in cells stimulated with serum for 14 h most of the cdk2 protein became phosphorylated, which existed as the faster migrating form. H 2CB treatment of stimulated cells resulted in the accumulation of the slow-migrating form of cdk2, similar to that observed in quiescent cells (Fig. 10). These results suggest that in addition to the inhibition of cyclin E expression, reduction of the cdk2-associated kinase activity upon cytoskeleton disruption is in part attributed to the decrease in cdk2 phosphorylation. Disruption of the actin cytoskeleton affects nuclear compartmentation of cdk2 during G 1-to-S progression. In addition to the inhibition of cyclin E expression upon H 2CB treatment, there are several possible reasons

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FIG. 10. Disruption of the actin cytoskeleton in serum-stimulated Swiss 3T3 cells affect cdk2 phosphorylation and subcellular localization. (A) Analysis of cdk2 phosphorylation pattern. Cell extracts prepared as described for the cdk2 kinase assay were immunoprecipitated with an anti-cdk2 antibody. The immunoprecipitate was electrophoresed in a 15% SDS–polyacrylamide gel and then subjected to immunoblot analysis with the anti-cdk2 antibody. A doublet of cdk2 bands was detected with the fast-migrating form corresponding to the phosphorylated molecule [23]. (B) Subcellular localization of cdk2 and cyclin E. Quiescent cells or serum-stimulated cells with or without H 2CB treatment were separated into cytosolic and nuclear fractions as described under Materials and Methods. An equal amount of protein (50 ␮g) from each fraction was electrophoresed onto an SDS–polyacrylamide gel and Western blotted with an anti-cdk2 or anti-cyclin E antibody.

that could account for the decrease in cdk2 phosphorylation in cells without the actin cytoskeleton. Phosphorylation of cdks is catalyzed by the cdk-activating kinase (CAK), which is a heterotrimer composed of cdk7, cyclin H, and MAT1 [65]. It is possible that expression of the CAK subunits may be affected by cytoskeleton disruption or proper localization of the three CAK subunits may depend on the presence of normal actin structure. Alternatively, since subcellular distribution of certain cyclins and cdks are regulated by cell-cycle-dependent events [11, 14, 55, 57, 77], the actin cytoskeleton may be important for proper subcellular localization of cdk2 during G 1-to-S progression. To examine these possibilities, we first studied the expression of the three CAK subunits by Western blot analysis. We found that expression of Mat1, cyclin H,

and cdk7 subunits appeared to be relatively constant among quiescent cells and cells stimulated by FBS for 3–16 h (data not shown), consistent with the observation of constitutive cyclin H– cdk7 kinase activity during the cell cycle [62]. Disruption of the actin cytoskeleton by H 2CB treatment in serum-stimulated cells also did not affect the expression levels of all three CAK subunits. In addition, fractionation experiments showed that all three CAK subunits were localized in both cytosolic and nuclear compartments, and their distribution pattern was not changed in serum-stimulated cells with or without H 2CB treatment (data not shown). These results indicate that disruption of the actin cytoskeleton does not affect the expression and subcellular localization of CAK subunits. Next, we examined the subcellular localization of

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FIG. 11. Disruption of the actin cytoskeleton affects nuclear accumulation of p107 during G 1-to-S progression. Quiescent cells were stimulated with serum in the presence or absence of H 2CB for 14 h. Cell fractionation followed by Western blot analysis using an anti-pRb (top) or anti-p107 antibody (bottom) was performed as described in Fig. 10.

cdk2 in quiescent cells or serum-stimulated cells with or without H 2CB treatment for 14 h. Analogous to that shown in Fig. 10A, phosphorylation of cdk2 resulted in an increase in the electrophoretic mobility of the protein and in quiescent cells cdk2 existed as a slowmigrating, nonphosphorylated form, which was primarily detected in the cytosolic fraction (Fig. 10B). In serum-stimulated cells cdk2 was found in both the cytosolic and the nuclear fractions; both the fast-migrating, phosphorylated cdk2 and the nonphosphorylated species were detected in the cytosolic fraction, while the majority of cdk2 in the nuclear fraction existed as a phosphorylated protein. In contrast, disruption of the actin cytoskeleton by H 2CB in serum-stimulated cells prevented cdk2 phosphorylation; only the slow-migrating, nonphosphorylated cdk2 was seen in H 2CB-treated cells and the majority of them remained localized in the cytoplasm (Fig. 10B). As a comparison, we examined the subcellular localization of cyclin E protein. Consistent to that observed in Fig. 7A, the level of the cyclin E protein is induced in serum-stimulated cells and the majority of the cyclin E protein was found in the nucleus (Fig. 10B). Cytoskeleton disruption by H2CB in serum-stimulated cells inhibited cyclin E protein induction and only a very small amount of cyclin E was detected in the cytosolic fraction. Taken together, these results indicate that the presence of normal actin structure is important for proper subcellular localization of cdk2 during G 1-to-S progression. Nuclear accumulation of p107 is also affected by disruption of the actin cytoskeleton. pRb and p107 change their intracellular localization during G 1-to-S

progression. Since disruption of the actin cytoskeleton could prevent cdk2 from entering the nucleus during G 1-to-S progression, we also carried out similar fractionation analysis to examine whether H 2CB treatment had any effect on the intracellular localization of pRb and p107. Consistent with previous observation, pRb existed as a hypophosphorylated form and was found mostly in the nuclear fraction in quiescent Swiss 3T3 cells (Fig. 11). Upon serum stimulation for 14 h, pRb became hyperphosphorylated, existing as a fast-migrating form in the gel, and was released from the nucleus into the cytoplasm. Cytoskeleton disruption by H 2CB inhibited pRb hyperphosphorylation and the majority of the hypophosphorylated pRb remained in the nucleus. Similar to that found in Fig. 5B, only a small amount of p107 protein was detected in quiescent cells, and the protein existed predominantly as a slow-migrating, hypophosphorylated species which was present in both the cytosolic and the nuclear fractions (Fig. 11). Upon serum stimulation, p107 levels rose, and the protein became hyperphosphorylated and accumulated in the nucleus. Interestingly, treatment with H 2CB of serumstimulated cells prevented the migration of p107 protein to the nucleus as well as blocked its phosphorylation (Fig. 11). These results, together with those reported previously [1, 39, 47, 48, 50, 73], indicate that the changes in subcellular localization of pRb and p107 are linked to their phosphorylation states. In addition, our results further suggest that the actin cytoskeleton may be important for nuclear accumulation of p107 during G 1-to-S progression.

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DISCUSSION

The actin cytoskeleton has been shown to be involved in the regulation of cell proliferation and/or cell signaling induced by growth factors. We have examined the molecular mechanism underlying the actin cytoskeleton-dependent growth arrest. By using the cytochalasin B derivative H 2CB that selectively disrupts the actin cytoskeleton, we have found that during growth stimulation of normal Swiss 3T3 fibroblasts the most actin-sensitive period extends from 8 to 12 h, which corresponds to mid to late G 1 phase. Only during this time period, disruption of normal actin structure blocks the initiation of DNA synthesis in growth-factor-stimulated cells. These results are consistent with those previously reported [5, 8, 31, 32, 43, 52, 70] and further extend the notion that the actin cytoskeleton is important for the transmission of some growth signals appearing at mid to late G 1 phase. The progression through G 1 phase and entry into S phase correlates with hyperphosphorylation of pRb [38, 65]. In G 0/G 1 phase, pRb is hypophosphorylated and acts as a proliferation inhibitor. It is generally believed that pRb exerts its growth-inhibitory function by binding a number of cellular proteins, among those are the family of E2F transcription factors. Upon mitogen stimulation, pRb becomes hyperphosphorylated during mid to late G 1. Hyperphosphorylation of pRb releases E2F from the pRb–E2F complex, enabling free E2F to activate genes required for the progression into S phase. These observations suggest that phosphorylation of pRb represents a critical key event in regulating cell cycle progression. Consistently, we also observed hyperphosphorylation of pRb in cells stimulated by FBS or EGF. The actin cytoskeleton-dependent G 1 arrest in normal fibroblasts also appears to link to pRb phosphorylation. Upon disruption of normal actin structure, growth factors fail to induce pRb hyperphosphorylation. These results suggest that one or more of the kinases, which can phosphorylate pRb, are linked to the actin cytoskeleton-dependent G 1 arrest. Phosphorylation of the Rb protein occurs at multiple sites that fit the cdk consensus [38] and, among cdks which are capable of performing this phosphorylation in vitro [65], cyclin-D-associated cdk4 and cyclin-Eassociated cdk2 are the two major kinases responsible for progression through G 1 phase in fibroblasts. Both cdk4 and cdk2 are expressed constitutively during the cell cycle. However, they are inactive in the absence of their cyclin partners. When quiescent cells are stimulated to enter the cell cycle, the expression of both cyclins D and E is induced, thereby activating the cdks that phosphorylate pRb [26, 35, 45, 54]. Cyclin D1 induction is first detected at early G 1, while cdk4 activation appears at mid G 1 [45, 54]. We also observed cyclin D1 induction in serum-stimulated

49

Swiss 3T3 cells. However, disruption of normal actin structure did not affect cyclin D1 induction, cdk4 expression, or cdk4-associated kinase activity in stimulated cells, suggesting that the actin cytoskeleton is not important for the regulatory events occurring at early G 1. It has been shown that mitogen-induced expression of cyclin D1 is regulated by the p42/p44 MAP kinases [36, 74]. Consistently, we found that activation of the p42/p44 MAP kinases was not affected by the disruption of the actin cytoskeleton in serum-stimulated cells. As discussed before, the cyclin D1– cdk4 complex is capable of phosphorylating pRb in vitro. Curiously, without the actin cytoskeleton growth-factor-stimulated cells did not show pRb hyperphosphorylation and could not enter S phase. These results support the notion that cyclin D1 induction and activation of the cdk4-associated kinase activity alone at G 1 do not lead to pRb hyperphosphorylation and are not sufficient to induce DNA synthesis. However, it is possible that partial pRb phosphorylation may be achieved by cyclin D1– cdk4 complexes, but that this action does not change the mobility of pRb in the gel (Fig. 5) [16, 40]. It has been shown that cyclin E appears to be induced at later stages of G 1 phase than cyclin D1 in mitogen-stimulated cells and the cyclin E– cdk2 kinase activity reaches a maximum level during G 1-to-S transition [15]. Interestingly, we found that the mid to late G 1 phase was the most actin-dependent period. Disruption of the actin cytoskeleton in growth-stimulated cells lead to inhibition of cyclin E induction as well as a large reduction of the cyclin E– cdk2 kinase activity at late G 1 phase. Recently, Harbour et al. [25] presented evidence that phosphorylation of pRb by G 1 cdks leads to successive intramolecular interactions that initially block histone deacetylase binding to the pocket and thus active transcription repression and then disrupt pocket structure, preventing pRb from binding and inactivating E2F. These intramolecular interactions provide a molecular basis for sequential phosphorylation of pRb by cdk4/6 and cdk2. Cdk4/6 is activated at early G 1, blocking active repression by pRb. However, it is not until mid to late G 1, when cyclin E is expressed and cdk2 is activated, that pRb is prevented from binding and inactivating E2F. Along with this model, without the presence of active cyclin E– cdk2 complexes as we observed in serum-stimulated cells treated with H 2CB, pRb hyperphosphorylation could not ensue, consequently blocking the cells from entering S phase. In addition to cyclin E association, activation of cdk2 requires the phosphorylation at Thr 160 by CAK [65]. Although disruption of the actin cytoskeleton affects the pattern of cdk2 phosphorylation, it has no effect on expression of the three CAK subunits and their subcellular localization. Since CAK is localized within the nucleus [20, 34, 55, 69], it is possible that proper nu-

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clear localization of cdk2 at mid to late G1 may be a key requirement for its activation by CAK. Disruption of normal actin structure in growth-stimulated cells leads to abnormal intracellular localization of cdk2 (Fig. 10) and consequently impedes CAK-dependent phosphorylation. Alternatively, without cyclin E induction and association, cdk2 may not be phosphorylated by CAK. The pRb-related p107 protein is also capable to inhibit cell growth and its phosphorylation is associated with the loss of the growth suppressive activity [7, 30, 67]. The p107 protein exists at very low levels in quiescent Swiss 3T3 cells and upon serum stimulation its expression is induced with concomitant phosphorylation during mid G 1 to S phase. Interestingly, disruption of the actin cytoskeleton in growth-stimulated cells also leads to inhibition of p107 phosphorylation but not its induction. It has been shown that the cyclin-D1associated cdk4 kinase can phosphorylate p107 [7, 22]. Although the cdk4 activity was not affected by cytoskeleton disruption in serum-stimulated cells, the majority of p107 appeared to remain in the cytoplasm (Fig. 11). These results indicate that without translocation into the nucleus the p107 protein could not be phosphorylated by cdk4, and they further suggest that such regulation of subcellular localization and phosphorylation of p107 is also important for G1-to-S progression. Upon growth stimulation, pRb becomes hyperphosphorylated and is released into the cytoplasm. Disruption of the actin cytoskeleton in growth-stimulated cells affects pRb hyperphosphorylation and thus its release from the nucleus. We have also shown that the actin cytoskeletondependent inhibition of cyclin E expression is mediated at least in part at the RNA level. The promoter of the cyclin E genes contains E2F-binding sites and the cyclin E gene is regulated by E2F [51]. Currently, six E2F members, termed E2F-1 to -6, have been identified. E2F expression is cell cycle-regulated [49] and the transcriptional potential of individual E2F species appears to be dependent upon their nuclear localization [1, 39, 50, 73]. In cycling cells, the majority of E2F complexes containing p107, other pRb-related p130, and the free E2F remain in the cytoplasm, while pRb– E2F complexes constitute most of the nuclear E2F activity. The pRb–E2F complexes are present at high levels during G 1, but disappear once the cells have passed the restriction point. It is reasonable to speculate that disruption of the actin cytoskeleton also affects proper localization of some E2F complexes in mitogen-stimulated cells. For example, it has been shown that E2F-4 lacks a nuclear localization signal and nuclear translocation of E2F-4 may be facilitated by binding to p107 [1, 39]. Since disruption of the actin cytoskeleton in growth-stimulated cells prevents p107 migration into the nucleus, it is likely that such abnormal subcellular localization of these G 1 regulators may

result in inhibition of transcription induction of certain E2F-responsive genes. In addition, it should be mentioned that cyclin E– cdk2 complexes also contribute to pRb phosphorylation and inactivation. The increase in cyclin E– cdk2 activity at mid to late G 1 in mitogenstimulated cells would contribute to further activation of E2F and thus to enhanced transcription of the cyclin E gene [25]. Conversely, serum-stimulated cells with H 2CB treatment show a great reduction of the cyclin E– cdk2 activity. Such a reduction would further inhibit cyclin E induction at a later time of the cell cycle. The actin cytoskeleton is critical for many cell functions, including cell locomotion, cytokinesis, intracellular transport, establishment of cell polarity, and the elaboration of cell surface projections that increase cell surface area and sense the environment [3, 28]. In addition, during the process of cell transformation the actin cytoskeleton undergoes architectural changes [61]. Consequently, transformed cells can grow in suspension without attachment to substratum, a phenomenon known as anchorage-independence of growth that is closely correlated with tumorigenicity in vivo [66]. Interestingly, cell anchorage that involves interaction of integrins with the extracellular matrix and the actin cytoskeleton also appears to depend on cyclin E– cdk2 but not cyclin D– cdk4 kinase activity [17]. In untransformed human diploid fibroblasts, the cyclin E– cdk2 complex was found to be activated at late G 1 when cells were grown under attached conditions but not in suspension; in contrast, the complex was active in transformed fibroblasts regardless of attachment. The cdk activities are tightly regulated by association with a number of cdk inhibitors [65]. Among them, the p21 Cip1 and p27 Kip1 inhibitors interact directly with both cyclin D– cdk4 and cyclin E– cdk2 complexes and prevent the activation of kinases by CAK. The lack of cyclin E– cdk2 activity in cells grown in suspension was found to be due to the decrease in cdk2 phosphorylation and increased expression of some cdk2 inhibitors [17]. Although we observed the decrease in cdk2 phosphorylation in growth-stimulated Swiss 3T3 cells upon cytoskeleton disruption, no change in the expression patterns of p21 Cip1 and p27 Kip1 was found. It is possible that anchorage-dependent growth involves multiple integrin-mediated signaling pathways [29], while our system examined only the role of the actin cytoskeleton in growth-factor-mediated signaling. Under our H 2CB treatment condition normal Swiss 3T3 cells remain attached to the substratum and only the stress fibers but not the cortical actin structure was disrupted. In addition, the adhesion-mediated MAP kinase pathway appeared to remain intact in these normal fibroblasts treated with H 2CB. A similar observation was also reported using a low dose of cytochalasin D. Aplin and Juliano [2] showed that cytochalasin D treatment did not alter the ability of cells to attach to fibronectin.

ACTIN-DEPENDENT CYCLIN E/cdk2 REGULATION AND p107 LOCALIZATION

Treated cells lost their stress fibers but retained some degree of cortical actin structure and were able to spread and respond to growth-factor-mediated signaling, leading to MAP kinase activation. Alternatively, differences in the cell types and culture conditions used may be considered. Nonetheless, these results support the notion that cyclin E– cdk2 complex is the ultimate target of the cell anchorage-dependent regulatory pathway and further implicate the importance of the actin cytoskeleton in growth signaling. In summary, we have shown that the actin cytoskeleton-dependent G 1 arrest is linked to the cyclin– cdk pathway. Disruption of the actin cytoskeleton leads to inhibition of cyclin E induction, cdk2 phosphorylation and subcellular localization, as well as nuclear accumulation of p107 in growth-stimulated cells. Since actin filaments have been shown to interact with several proteins involved in growth signaling [6, 19, 24, 27, 56, 60, 70 –72], it is tempted to postulate that the actin cytoskeleton may act directly or indirectly on certain G 1 regulators to affect their proper subcellular localization. Further actin-binding and immunofluorescence experiments are currently in progress and should provide us with a complete understanding of the molecular mechanisms underlying the actin cytoskeleton-dependent G 1 arrest. We greatly appreciate Ed Harlow for the anti-pRb antibody, William Kaelin, Jr., for the pGST–Rb plasmid, and Andy Oberyszyn and Cindy McAllister for flow cytometer analysis. We sincerely thank Phil Nowicki and Joseph Lin for critical reading of the manuscript. This work was supported by grants to L.S.C. from American Cancer Society (GMC-89165), National Cancer Institute-supported Ohio State University Comprehensive Cancer Center (CA16058 and OSUCCC1102), and Children’s Hospital Research Foundation, and grants from the Russian Fund of Fundamental Investigations (9804-49973 to G.R. and 97-04-47784 to R.B.).

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