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J. Biol. Chem., Vol. 279, Issue 45, 47379-47390, November 5, 2004

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Autocrine Transforming Growth Factor Regulates Cell Adhesion by Multiple Signaling via Specific Phosphorylation Sites of p70S6 Kinase in Colon Cancer Cells^*

Rajinder S. Sawhney, Michelle M. Cookson, Bhavya Sharma, Jennie Hauser, and Michael G. Brattain

From the Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263

Received for publication, February 24, 2004 , and in revised form, July 23, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Recently, we showed that autocrine transforming growth factor (TGF) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin ₂, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin ₂ and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861–19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin ₂ function in HCT116 cells. Selective MEK inhibitors attenuated TGF-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin ₂ expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGF regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The p70S6K (S6K)¹ enzyme is a member of the AGC (cAMP- and cGMP-dependent kinases and PKC) superfamily of kinases (1). The activation of S6K is complex and is associated with a series of phosphorylation events. Ribosomal S6K has multiple phosphorylation sites dispersed throughout the molecule. Classically, S6K is an activator of ribosomal protein S6, a component of the 40 S subunit of eukaryotic ribosomes (2–4). S6K is known to increase the translation of mRNA exhibiting unique 5'-terminal oligopyrimidine sequences in the 5'-untranslated region. Because these mRNAs encode ribosomal proteins, they contribute to the enhanced global translational capacity of cells (5). The S6K enzyme plays an important role in protein synthesis and cell growth control, since S6K knockout mice showed significantly reduced body sizes (6, 7). Recently, S6K has been shown to function as a dual pathway kinase, signaling cell survival as well as cell growth (8, 9). However, the role of S6K in cell adhesion, integrin expression, and cell motility is not clear.

Integrins are heterodimers of and subunits expressed in a cell- and/or tissue type-specific manner on the cell surface. Integrins play an important role in cell differentiation, adhesion, and motility (10). Integrin ₂₁ is formed by a noncovalent association of the ₂ subunit as a monogamous partner to the promiscuous ₁ subunit. Expression of integrin ₂ is regulated during normal cell differentiation and is altered during tumorigenesis (11). Its synthesis is considerably reduced in poorly differentiated cells, like HCT116 carcinoma cells, and downregulation of integrin ₂ in the colonic crypt may lead to a loss of cell-matrix binding and sloughing of cells (12). In human breast cancer, hepatocarcinoma, and rhabdomyosarcoma cells, the expression of integrin ₂ has been correlated with metastatic behavior (13–16). Although the mechanisms involved in the transformation of a cell from nonmetastatic to metastatic are poorly understood, inhibiting or eliminating cellular motility will disrupt metastatic disease.

We previously reported that in HCT116 cells, the biological responses of epidermal growth factor receptor (EGFR)-mediated functions including mitogenesis, integrin ₂ expression, cell adhesion, and cell micromotion depend on the degree of EGFR activation. For example, mitogenesis is saturated by autocrine levels of activation, whereas cell matrix functions occur at autocrine levels of activation and continue to increase with increasing EGFR activation (17). To further elucidate the mechanism(s) of integrin ₂ expression and its biological functions in HCT116 cells, we reported the critical role of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling downstream of the EGFR-mediated pathway (18). In the present report, we further elaborate downstream signaling mechanisms responsible for integrin ₂ functions in HCT116 cells. An important finding in this study is that autocrine TGF-induced S6K activation at specific phosphorylation sites in HCT116 cells is dependent upon multiple signaling pathways and ultimately mediates control of cell adhesion functions as a result of the activation by these pathways. One upstream pathway involves the ERK/MAPK signaling pathway that utilizes Thr-389, and the other is the PKC signaling pathway, which utilizes Thr-421/Ser-424 sites. Further, these sites are inhibited by the immunosuppressive macrolide rapamycin, a selective inhibitor of FKBP and rapamycin-associated protein (FRAP)/mammalian target of rapamycin (mTOR) signaling. The assignment of specific phosphorylation sites of S6K to cell adhesion functions was confirmed by ectopic expression of single site-specific dominant negative mutants to Thr-389, Thr-421, and Ser-424, respectively. The present data provide new insight into the complex signaling mechanism(s) by which autocrine TGF may contribute to the metastatic and invasive behavior of highly aggressive human colon cancer cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Methylthiazole tetrazolium (MTT), Me₂SO, collagen type IV (CN IV), polyclonal anti-actin antibody, and bovine serum albumin were purchased from Sigma, and bisindolylmaleimide (BIM-1) was purchased from LC Laboratories (Woburn, MA). MEK inhibitor U0126 was purchased from Promega Corp. (Madison, WI), whereas PD098059 and mTOR inhibitor rapamycin were procured from Calbiochem. The polyclonal antibody specific for integrin ₂ subunit (antibody 1936) was obtained from Chemicon International Inc. (Temecula, CA). Anti-ERK and p70S6k (Ser-411) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-p70S6k (Thr-389) and anti-p70S6k (Thr-421/Ser-424) were from Cell Signaling. Mouse and rabbit peroxide-conjugated AffiniPure goat IgG (H+L) secondary antibodies were from Jackson Laboratories (West Grove, PA). McCoy's 5A medium, transferrin, and insulin were obtained from Sigma, whereas epidermal growth factor (EGF) was purchased from BD Transduction Laboratories (San Diego, CA). N-Hydroxysuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-Biotin) was purchased from Pierce, and streptavidinagarose was procured from Novagen (Madison, WI). The empty vector pRK7 and the vector containing p70S6K hemagglutinin-tagged HA-S6K1 constructs T421A (threonine 421 mutated to alanine) and S424A (serine 424 mutated to alanine) were a generous gift of Dr. John Blenis and Diane Turcotte (Harvard Medical School, Boston, MA). The empty vector pRK5 and the vector containing Myc-tagged S6K1 construct T389A S6K (threonine 389 mutated to alanine) were a generous gift of Dr. Patrick B. Dennis (Wright State University, Dayton, OH) and Dr. George Thomas (Friedrich Miescher Institute, Basel, Switzerland). The plasmid pEGFP-N1 was obtained from BD Biosciences. FuGENE 6 transfection reagent was purchased from Roche Applied Science (Indianapolis, IN). An electric cell-substrate impedance sensing (ECIS) machine (model 1600R) and arrays of gold-film coated electrodes for cell motility (micromotion) experiments were purchased from Applied Biophysics Inc. (Troy, NY).

Plasmid Constructs and HCT116 Cell Transfection—The hemagglutinin-tagged constructs of p70S6K, HA-S6K1 T421A (Thr-421 Ala) and HA-S6K1 S424A (Ser-424 Ala), subcloned into the mammalian expression vector pRK7 have been described previously (19, 20). The Myc-tagged construct p70S6K-T389A (Thr-389 Ala) subcloned into the expression vector pRK5 has also been previously described (21, 22). These p70S6K constructs harboring neutral alanine residues were prepared by site-directed mutagenesis. The Thr and Ser residues were mutated to Ala as indicated. HCT116 cells were transfected with p70S6K mutant DNA using FuGENE 6 transfection reagent following the manufacturer's protocol as previously described (18). In brief, 1 x 10⁶ cells were seeded in 100-mm tissue culture dishes in serum-free medium 3 days before transfection and changed to supplemental McCoy's medium on the third day. HCT116 cells were transiently transfected either with empty vector or with various constructs at a 4:1 ratio of FuGENE to plasmid DNA. Forty-eight hours after transfection, cells were harvested by trypsinization and used in cell adhesion, cell micromotion, and cell lysates analyzed for integrin ₂ and S6K protein expression. The transfection efficiency using enhanced green fluorescent protein plasmid was about 50%.

Cell Culture and Adhesion Assay—These experiments were performed as described previously (17, 18). HCT116 cells were cultured at 37 °C in a humidified incubator with 5% CO₂ in a chemically defined serum-free medium consisting of either McCoy's 5A medium or supplemented with 4 µg/ml transferrin and 20 µg/ml insulin, either in the absence or presence of EGF (10 ng/ml), depending upon experimental design.

For adhesion assays, 96-well tissue culture plates were coated for 2 h at 37 °C with CN IV at concentrations of 0–0.25 µg/ml, blocked with 3% bovine serum albumin for 3 h, and then rinsed once with PBS. Subsequently, the MTT procedure was followed using HCT116 or S6K mutant-transfected HCT116 cells as described previously (17).

After trypsinization, cells were incubated at 37 °C with inhibitors for 2–3 h to determine cell adhesion functions. Cells were plated at 6 x 10⁴ cells/well on CN IV-coated plates and incubated for 90 min in the absence or presence of MEK inhibitors PD098059 and U0126 or Me₂SO as a control. Nonadherent cells were removed by washing three times with serum-free medium. The relative number of attached cells was determined by the MTT method. All inhibitors were dissolved in Me₂SO as stock solutions and diluted at the time of experiment.

Cell Lysates, Biotinylation, and Immunoblotting—The procedures were the same as described previously (17). Briefly, subconfluent cultures of HCT116 or S6K mutant-transfected HCT116 cells were treated either with Joklik's EDTA for 8 min or trypsinized for 3 min at room temperature, and cells were then scraped and pelleted by centrifugation in a clinical centrifuge for 3 min at 800 x g. The pellet was washed twice with cold PBS, and cells were biotinylated in suspension with N-hydroxysuccinimidyl-6-(biotinamido) hexanoate (NHS-LC-Biotin) (Pierce), 0.1 mg/ml in Me₂SO at room temperature for 1 h. The biotinylated cells for integrins were washed with PBS and lysed in buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1% Nonidet P-40, and a mixture of protease inhibitors), sheared through a 26-gauge needle, and centrifuged at 16,000 x g for 20 min at 4 °C in a microcentrifuge. Equal amounts of protein from treated and untreated (control) cell lysates were incubated with streptavidin agarose for 90 min at 4 °C. Agarose beads were pelleted by centrifugation at 4 °C and then washed five times with lysis buffer containing phenylmethylsulfonyl fluoride. The beads were boiled in 2x Laemmli buffer containing 4% -mercaptoethanol for 10 min, and supernatant was filtered through Bio-Rad columns and applied in 7.5% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Hybond). The membrane was blocked for 2–3 h with 5% nonfat dry milk in Triton/Tris-buffered saline (TTBS) and incubated overnight at 4 °C with appropriate primary antibody. For certain primary antibodies, the membranes were blocked with bovine serum albumin (5%) for 1 h at room temperature. After washing the membrane with TTBS, it was incubated for 1 h at room temperature with horseradish peroxidase-conjugated rabbit or mouse secondary antibody. The membrane was washed, and detection of specific binding was achieved by using enhanced chemiluminescence reagent (PerkinElmer Life Sciences). For Western blot analysis of cell signaling proteins, biotinylation of cells was omitted, and proteins were separated by 7% SDS-PAGE.

Cell Motility Measurements by the ECIS Technique—We have extensively used this real time, quantitative technique in our previous publications (17, 18). In the present system (model 1600R), HCT116 cells or S6K mutant transfectants were plated at 6 x 10⁴ cells/well on small gold electrodes (diameter 250 µm) at the bottom of tissue culture wells (area 0.5 cm²), and culture medium was used as the electrolyte (23, 24). Four hundred fifty microliters of medium were used per well. An array consists of eight individual small electrodes, allowing up to 16 individual experimental conditions to be monitored simultaneously. A constant current source applies a noninvasive 1-µA, 4000-Hz AC signal between the small electrode and a much larger counter electrode (0.15 cm²). Any variation of current due to cell movement is recorded. The in-phase and out-of-phase voltage across the electrode were recorded by the lock-in amplifier once every second to measure micromotion and once every 2 min for measuring cell attachment. The ECIS software (Applied BioPhysics, Troy, NY) calculated the resistance and capacitance values of the electrode over this period of time. Attachment and movement of the cells on the electrode changed the flow of the current, resulting in fluctuations in the electrode resistance and capacitance. These cellular movements were called micromotion (24) and were a measure of the motile ability of the cell being examined. As the cells moved on the electrode, the sensitive nature of the lock-in amplifier detected the fluctuations in the resistance and capacitance values (25). These fluctuations were then analyzed statistically using ECIS software to reveal the percentage variation in resistance, which in turn was a reflection of cellular micromotion on the electrode. Because large changes occur in resistance with cell movement, the results of experiments are presented as percentage variations in resistance rather than in capacitance. Since the measurements are electrical, they are quantitative and generate data that can readily be analyzed to provide sensitive measurements of changes in cell behavior.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
MEK Inhibitors Affect Autocrine Activation of S6K at Specific Phosphorylation Sites—Previously, we showed that in HCT116 cells, basal ERK/MAPK activation was under the control of autocrine TGF (18). We now determined whether the activation of S6K, a downstream enzyme mediated by the EGFR/ERK/MAPK signaling pathway in these cells, is controlled by autocrine TGF. Various phosphorylation sites of S6K were mapped for activation by autocrine TGF. We found that endogenously Thr-389, Thr-421, and Ser-424 sites were specifically activated by TGF-mediated multiple signaling pathways emanating from activated EGFR. Western blot analyses of cell lysates with specific phospho-primary antibodies did not show inhibition of the basal activation of S6K at the Ser-411 phosphorylation site by MEK inhibitors PD098059 or U0126 in the absence of exogenous growth factor (Fig. 1A). This demonstrates that endogenous S6K activation at Ser-411 is not under the control of basal ERK/MAPK signaling. However, it is noteworthy that when HCT116 cells are exposed to EGF (10 ng/ml), which saturates the EGFR, and then treated with MEK inhibitors, S6K activation at Ser-411 is inhibited in a concentration-dependent fashion (Fig. 1B). MEK inhibitor U0126 showed higher inhibitory effects on the activation of S6K at Ser-411 than did PD098059. Earlier we reported similar results in that MEK inhibitor U0126 was more potent in the inhibition of ERK activation (18). Mapping of the other phosphorylation sites activated by autocrine TGF revealed that U0126 inhibited the Thr-389 (Fig. 1C) but not the Thr-421 and Ser-424 sites of S6K (Fig. 1D). In the presence of EGF, however, U0126 inhibited Thr-421/Ser-424 phosphorylation sites of S6K similarly to the Ser-411 site (Fig. 1E). These results demonstrate that MEK inhibitors affect activation of S6K at specific phosphorylation sites as a function of whether S6K is activated by autocrine versus exogenous growth factor. The experiments described above also confirm that S6K is downstream of the ERK/MAPK signaling pathway.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Effect of MEK inhibitors on phosphorylation of S6K. HCT116 cells maintained in the absence or presence of EGF (10 ng/ml), as shown, were treated with different concentrations of U0126 (0–10 µM) and PD098059 (0–50 µM) for 48 h. Cells were lysed, and equal amounts of protein were analyzed by Western blot analysis as described under "Experimental Procedures," using antibodies against either phosphorylated S6K (upper panels) or total S6K (lower panels). In all experiments, before applying total antibodies, the membranes were stripped of the phosphoantibody with an SDS/-mercaptoethanol solution at 50 °C for 30 min. Routinely, 50 µg of protein were loaded per well. DMSO, Me2SO.

View larger version (37K): [in this window] [in a new window]   FIG. 2. Effect of EGF on the activation of S6K (Ser-411). HCT116 cells were maintained in the absence of EGF. Subconfluent cells (80%) were stimulated with EGF (10 ng/ml) for the indicated durations. Immunoblotting was performed as described under "Experimental Procedures" using antibodies against either phosphorylated Ser-411 of S6K (top panel), phosphorylated Tyr-204 of ERK1/2 (middle panel), or total ERK1/2 (bottom panel).

View larger version (36K): [in this window] [in a new window]   FIG. 3. Effect of rapamycin on phosphorylation of S6K. HCT116 cells maintained in the absence or presence of growth factor, as shown, were treated with the indicated concentrations of rapamycin for 24 h. Cells were lysed, and equal amounts of protein were analyzed by Western blot analysis as described under "Experimental Procedures," using antibodies either against phosphorylated S6K (upper panels) or total S6K (lower panels).

Rapamycin Inhibits Cell Adhesion and Integrin ₂ Expression—The data presented above show that autocrine TGF-mediated mTOR signaling contributes to the phosphorylation of Thr-389 and Thr-421/Ser-424 sites of S6K. To determine whether activation of these sites is linked with cell adhesion function, HCT116 cells were treated either with Me₂SO (control) or with rapamycin, and adhesion assays were performed as reported earlier (17, 18). Fig. 4A shows that rapamycin inhibited autocrine TGF-mediated cell adhesion on CN IV by 58% (after subtracting bovine serum albumin values). These results show that specific phosphorylation sites of S6K contribute to cell adhesion via mTOR signaling. Inhibition of cell adhesion by rapamycin indicated that integrin ₂, the receptor for CN IV, may be playing a role in cell adhesion. Therefore, we studied the effect of rapamycin on integrin ₂ expression. Fig. 4B shows that rapamycin does attenuate endogenous expression of integrin ₂ protein. Similarly, Fig. 4C shows inhibition of integrin ₂ by rapamycin in the presence of exogenous EGF. These experiments directly link some or all of the Thr-389 and Thr-421/Ser-424 sites of S6K to integrin ₂ functions in HCT116 cells.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Effect of rapamycin (Rap) on cell adhesion (A) and integrin 2 protein expression (B and C). A comparison of the adhesion to CN IV of HCT116 control and HCT116 cells treated with rapamycin is shown. Substrates were prepared by coating tissue culture 96-well plates with CN IV at a concentration of 0.10 µg/ml for 2 h at 37 °C. Cells in the absence of EGF were seeded at 6 x 104 cells/well onto coated plates and incubated for 90 min at 37 °C. The relative number of attached cells was determined by MTT assay as described under "Experimental Procedures." HCT116 cells in the absence (B) or presence (C) of EGF were treated with the indicated concentrations of rapamycin. Cells were biotinylated and lysed, and equal amounts of protein were analyzed by Western blot analysis. The upper panel shows the effect of rapamycin on the expression of integrin 2, whereas the lower panel shows levels of actin as a loading control. DMSO, Me2SO.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Specific phosphorylation site mutants T389A, T421A, and S424A inhibit basal levels of phospho-Thr-389, -Thr-421, and -Ser-424 of S6K. HCT116 cells were transiently transfected with individual mutants for 48 h. The cell lysates were immunoblotted as detailed under "Experimental Procedures" with phosphospecific antibodies for Thr-389 and Thr-421/Ser-424. Top panel, lane 1, phospho-Thr389 when cells were transfected with empty vector pRK5; lane 2, phospho-Thr-389 when cells were transfected with mutant T389A; lane 3, phospho-Thr-421/Ser-424 when cells were transfected with mutant T389A. The second panel shows total S6K for equal loading. Third panel, lane 1, phospho-Thr-421/Ser-424 when cells were transfected with empty vector pRK7; lane 2, phospho-Thr-421/Ser-424 when cells were transfected with mutant T421A; lane 3, phospho-Thr-421/Ser-424 when cells were transfected with mutant S424A; lanes 4 and 5, phospho-Thr-389 when cells were transfected with mutants T421A and S424A, respectively. The bottom panel shows total S6K for equal loading.

View larger version (48K): [in this window] [in a new window]   FIG. 6. Specific phosphorylation site mutants T389A, T421A, and S424A inhibit basal levels of cell adhesion (A) and integrin 2 protein expression (B and C). A comparison of the adhesion to CN IV of HCT116 cells transiently transfected either with empty vector (control) or with T389A, T421A, or S424A mutated DNA. Cell adhesion and integrin 2 expression experiments were performed as shown in the legend to Fig. 4.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Rapamycin inhibits cell micromotion in the absence of EGF. HCT116 cells (6 x 104) were plated in a medium devoid of EGF. Subconfluent cultures were either treated with Me2SO (DMSO; upper panel) or rapamycin (10 nM)(bottom panel). After attachment of cells for 3 h, micromotion was recorded.

View larger version (23K): [in this window] [in a new window]   FIG. 8. Specific phosphorylation site dominant negative mutants T389A (A), T421A (B), and S424A (C) inhibit basal levels of cell micromotion. HCT116 cells (6 x 104) were transiently transfected either with empty vector (control) or with individual mutants T389A, T421A, or S424A as shown in the figure and under "Experimental Procedures." The upper panels show micromotion when HCT116 cells were transfected with individual empty vectors, whereas the lower panels show micromotion when cells were transfected with individual dominant negative mutants. Cells growing on gold electrodes were monitored for micromotion.

View larger version (26K): [in this window] [in a new window]   FIG. 9. Effect of BIM-1 on phosphorylation of S6K. HCT116 cells maintained in the absence or presence of EGF, as shown, were treated with BIM-1 (10 µM) for 24 h. Cells were lysed, and equal amounts of protein were analyzed by Western blot analysis as described under "Experimental Procedures," using either antibodies against phosphorylated S6K (upper panels) or total S6K (lower panels).

View larger version (39K): [in this window] [in a new window]   FIG. 10. Effect of PKC inhibitor BIM-1 on expression of integrin 2 protein (A and B) and cell adhesion (C). HCT116 cells were treated with Me2SO (DMSO) or BIM-1 (10 µM) in the absence or presence of EGF as shown. Cells were lysed, and equal amounts of protein were analyzed by Western blot analysis using specific monoclonal antibody (antibody 1936) against integrin 2 (upper panels) as described under "Experimental Procedures." Actin was used as a loading control (lower panels). A comparison of the adhesion to CN IV of control and BIM-1 (10 µM)-treated HCT116 cells is shown. Substrates were prepared by coating tissue culture 96-well plates with different concentrations of CN IV (0.025–0.25 µg/ml) for 2 h at 37 °C. Cells in the absence of EGF were seeded at 6 x 104 cells/well onto coated plates and incubated for 90 min at 37 °C. The relative number of attached cells was determined by MTT assay as described under "Experimental Procedures."

That S6K phosphorylation sites Thr-421 and Ser-424 contribute to cell adhesion functions was confirmed by experiments where mutation of these sites to alanine and transfection of dominant negative constructs reduced basal cell adhesion in HCT116 cells (Fig. 6A).

BIM-1 Inhibits Endogenous Cell Micromotion in a Concentration-dependent Fashion—We used the sensitive, quantitative, and real time ECIS technique to investigate the effect of a selective PKC inhibitor on cell motility. This technique, in addition to cell micromotion, has also been used for studying cell morphology, cell-extracellular matrix interactions, and cancer metastasis (28). To determine the role of autocrine TGF-stimulated PKC signaling in cell micromotion, HCT116 cells (6 x 10⁴) were cultured on electrodes precoated with CN IV in EGF-free medium. Cells were either treated with Me₂SO (control) or with different concentrations of BIM-1 and allowed to attach for 3 h, and subsequently micromotion was recorded. Fig. 11A shows that in the control Me₂SO-treated cells, the percentage variation in resistance was recorded as 1.094% (upper panel). Treatment of the cells with 5 µM BIM-1 (middle panel) decreased the fluctuations, such that the percentage variation in resistance was now 0.849%, indicating a decrease in cell micromotion (22.4%) by BIM-1. A higher concentration of BIM-1 (10 µM) generated a further decrease in endogenous micromotion. The percentage variation recorded was 0.544%, showing a decrease of 50.25% in cell micromotion as compared with control Me₂SO-treated cells. These results demonstrate that the inhibitory effect of BIM-1 was concentration-dependent. These experiments show that the endogenous HCT116 cell micromotion is selectively under the control of PKC signaling mediated by autocrine TGF. Since BIM-1 inhibits the basal Thr-421/Ser-424 phosphorylation site of S6K, this site is involved in cell motility of HCT116 cells. The results were further confirmed by ectopic expression of T421A and S424A mutants (Fig. 8). Taking together the results of inhibitors and S6K dominant negative single site-specific mutants, we conclude that Thr-421 and Ser-424 of S6K control cell motility function via PKC signaling in metastatic HCT116 cells.

View larger version (34K): [in this window] [in a new window]   FIG. 11. BIM-1 inhibits cell micromotion in a dose-response fashion in the absence (A) or presence (B) of EGF. HCT116 cells (6 x 104) were plated in a medium either devoid of (A) or containing EGF (10 ng/ml) (B). Subconfluent cultures were either treated with Me2SO (DMSO; top panels) or with BIM-1 (5 µM (middle panels) or 10 µM (bottom panels)). Cells after attachment for 3 h were monitored for micromotion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Recently, we showed that the biological responses of EGFR-mediated functions differ in sensitivity with respect to varying degrees of EGFR activation in response to endogenous and exogenous ligands in HCT116 cells (17). These EGFR-mediated functions included DNA synthesis, integrin ₂ expression, cell adhesion, and motility. Subsequently, we reported that basal TGF/EGFR-mediated cell adhesion functions in HCT116 cells were under the control of autocrine-mediated ERK/MAPK signaling but that stimulation of the EGFR with exogenous ligands could further enhance integrin ₂ and cell motility (18). In the present work, we examined signaling downstream of ERK/MAPK and found a role for basal and exogenous S6K activation in HCT116 cells linking the ERK pathway and integrin ₂ function. We also examined the potential roles of PKC and mTOR in the activation of S6K. Our results show that in addition to ERK/MAPK activation, mTOR and PKC are important elements in the control of integrin ₂ function. Our data establish a novel cell motility function of S6K in a cancer cell. To our knowledge, this is the first report where the specific phosphorylation sites of S6K are assigned to the cell adhesion, integrin ₂, and cell motility by multiple signaling.

Recently, it was reported that MEK inhibitors, PD098059 and U0126, inhibited both ERK/MAPK and further downstream S6K pathways (29). The pharmacological agent U0126 was shown to reverse Ki-ras-mediated transformation of fibroblasts by blocking both MAPK and S6K pathways. PD098059 attenuated S6K activation moderately, whereas the U0126 inhibitory effect was more dramatic. However, the suppressing mechanism of activation of S6K by MEK inhibitors was not clear. To investigate the role of S6K in HCT116 cells, we focused on the analyses of the phosphorylation status of four key amino acid residues, Ser-411, Thr-389, Thr-421, and Ser-424. To study the mechanism of cell adhesion functions via S6K, we used several approaches. First, in continuing our earlier studies, we determined the effect of MEK inhibitors PD098059 and U0126 on the activation of S6K as evoked by autocrine TGF. We found that U0126 inhibited endogenous S6K activation at a specific phosphorylation site, Thr-389, thus indicating that this site of S6K is downstream of autocrine TGF-induced MEK signaling. At the level of autocrine EGFR activation, the MEK inhibitor did not have any effect on the other three phosphorylation sites, Ser-411, Thr-421, and Ser-424, of S6K. Previously, we showed that MEK inhibitors PD098059 and U0126 inhibit endogenous integrin ₂ expression, cell adhesion, cell micromotion, and ERK activation (18). Our present experiments with MEK inhibitors on S6K activation further define the mechanism by which the basal levels of integrin ₂ expression and its functions may be mediated by Thr-389 phosphorylation of S6K. This appears to be the first report of an autocrine EGFR activated site for control of integrin function by S6K. Our experiments using mutant approaches demonstrate that autocrine phosphorylation of Thr-389 via ERK/MAPK signaling is critical for these biological responses in HCT116 cells.

In HCT116 cells, the hyperactivation of ERK activity by saturation of the EGFR with exogenous EGF led to activation of S6K at three other specific phosphorylation sites. These sites were sensitive to MEK inhibitors as demonstrated by inhibition with the selective MEK inhibitors. Treatment of cells with exogenous EGF activated S6K at the Ser-411 phosphorylation site, in a MEK inhibitor-sensitive fashion. Fig. 2 shows mitogen-dependent phosphorylation kinetics indicating enhanced activation of S6K by EGF as compared with control cells. Moreover, the pattern of activation of S6K by EGF was the same as that of ERK activation. Our results indicate that higher threshold levels of ERK activation by exogenous stimulation of EGFR links to downstream activation of S6K at specific phosphorylation sites, whereas endogenous activated ERK provides a critical threshold for S6K at Thr-389, which mediates basal integrin ₂ functions. Thus, taken in the context of our previous findings regarding the hierarchical nature of EGFR functions that are saturated at low receptor versus high receptor occupancy, these results reflect a hierarchy of autocrine versus exogenous EGFR within a single molecule that affects the overall extent of integrin ₂ function.

MAPKs are activated by phosphorylation of a threonine/tyrosine sequence (TXY) in the activation loop of their catalytic domain (30). It is plausible that both threonine and tyrosine are not endogenously phosphorylated by autocrine TGF or that these amino acid residues are not fully phosphorylated. Further activation of ERK by exogenous EGF transduces S6K possibly through sequential phosphorylation of amino acid residues and makes it responsive to MEK inhibitors.

Our experiments support the concept that ERK/MAPK signaling may directly phosphorylate S6K in some cellular contexts. In any case, the results indicate that in HCT116 cells, the ERK/MAPK cascade is upstream of S6K. We show here that exogenous EGF further activates ERK, which in turn appears to phosphorylate S6K at several specific sites, whereas the endogenous activation of ERK is not sufficient to activate S6K at any phosphorylation site other than Thr-389. The carboxyl-terminal region of S6K contains an autoinhibitory domain having at least four serine-threonine sites in this region (Ser-411, Ser-418, Thr-421, and Ser-424). These sites may be hypophosphorylated in the absence of EGF in HCT116 cells. When cells are treated with the growth factor, these serine-threonine residues are most likely hyperphosphorylated for fully functional S6K. These sites are present in a consensus motif similar to those recognized in the MAPK. Our findings suggest that in HCT116 cells, a hierarchy of ERK/MAPK signaling is manifested, at least in part, by a hierarchy of S6K phosphorylation that is dependent on the extent of the EGFR activation.

Previously, we reported that in HCT116 cells, endogenous activation of ERK controlled integrin ₂ expression, cell adhesion, and cell motility (micromotion) (18). On the basis of our present data, we can further elaborate the mechanism of cell adhesion functions controlled by autocrine TGF via downstream S6K signaling. Since autocrine TGF specifically activates Thr-389, the linker domain of S6K, we now assign the Thr-389 phosphorylation site to the endogenous cell adhesion functions via ERK/MAPK cascade. The data presented here with overexpression of T389A mutant strongly support Thr-389 as the endogenous critical site of cell adhesion function via ERK/MAPK signaling in HCT116 cells (Figs. 6, A and B, and 8A). Since endogenous S6K activation at various sites is not fully responsive to MEK inhibitors, additional pathway(s) also appear to regulate the basal phosphorylation of S6K and affect its signaling.

Earlier reports suggested that it was unlikely that S6K played a direct role in the regulation of cell adhesion, because it is well known that S6K regulates mainly cell cycle progression (31). However, our report is the first that establishes a direct correlation between integrin function and EGFR/ERK/S6K signaling (18) (present data).

To further elucidate the mechanism of cell adhesion functions via S6K in HCT116 cells, we explored the possibility of contribution of the mTOR signaling. The mechanism(s) underlying mitogen modulation of FRAP/mTOR activity is not clear. The immunosuppressive macrolide rapamycin, the most potent inhibitor of mTOR, can inhibit S6K activation independent of phosphoinositide 3-kinase signaling (4, 31–33). Our experiments suggested, as in previous studies, that mTOR may respond to mitogenic signals (34). To understand the role of mTOR signaling via S6K, we treated HCT116 cells with different concentrations of rapamycin in the absence or presence of EGF and examined its effect on key phosphorylation sites by immunoblotting. Our data show that rapamycin specifically inhibits autocrine TGF-mediated activation of Thr-389 and Thr-421/Ser-424 sites. In addition to selective inhibitors, use of dominant negative mutants of specific sites provided an additional approach for probing regulatory signaling pathways that govern S6K activation and mechanisms that link the distinct phosphorylation sites to cell adhesion functions. In this approach, we used well established S6K DNA constructs in which Thr-389, Thr-421, and Ser-424 were mutated to neutral alanine residues (19–22). The transfectants were examined for cell adhesion, integrin ₂ protein expression, and cell micromotion. The results showed that mutants inhibited these biological responses, thus demonstrating direct cause and effect for the biological functions. These dominant negative mutant experiments confirm the results of experiments in which selective pharmacological inhibitors of different signaling pathways were used. Therefore, we conclude that phosphorylation sites Thr-389, Thr-421, and Ser-424 of S6K are specifically controlled by autocrine TGF in cell adhesion functions.

Due to the complex nature of S6K activation by different signaling cascades, we also studied the role of PKC signaling in HCT116 cells. We mapped different phosphorylation sites of S6K using BIM-1, a well known selective inhibitor of PKC activation (27). To determine whether endogenous activation of Ser-411 of S6K was under the control of PKC signaling, HCT116 cells were treated with BIM-1 (10 µM), and phospho-Ser-411 was analyzed by immunoblotting with specific antibodies. We did not observe any inhibition of Ser-411 by BIM-1. These results indicate that endogenous activation of Ser-411 was not under the control of PKC pathway. This site, however, responded to BIM-1 in the presence of EGF.

To further determine whether the specific phosphorylation site Thr-389 of S6K was under the control of PKC signaling, cell lysates prepared in the absence or presence of EGF or BIM-1 were analyzed by immunoblotting. BIM-1 did not inhibit the endogenous phospho-Thr-389 site. However, this site responded to BIM-1 when cells were treated with exogenous EGF (Fig. 9D). It was noteworthy that we observed inhibition of basal Thr-421/Ser-424 phosphorylation with BIM-1 in the absence as well as in the presence of exogenous EGF. These results demonstrate that, among the sites studied, only Thr-421/Ser-424 of S6K is activated by autocrine TGF via PKC signaling.

To determine whether there is a link between autocrine TGF-mediated phospho-Thr-421/Ser-424 sites of S6K and integrin ₂ expression due to PKC activation, cell lysates were analyzed for integrin ₂ by immunoblotting with specific antibodies. Fig. 10A shows that BIM-1 inhibits endogenous integrin ₂ expression. Therefore, autocrine TGF controls integrin ₂ protein expression, specifically by autoinhibitory domain Thr-421/Ser-424 activation via PKC signaling. These results were further confirmed by the ectopic expression of T421A and S424A mutants (Fig. 6C).

Our results furthermore demonstrate that integrin ₂ protein expression mediated by fully saturated EGFR is under the control of Ser-411, Thr-389, and Thr-421/Ser-424 via PKC signaling. Previously, we reported that integrin ₂ regulates cell adhesion function by the "inside-out" signaling mediated by autocrine TGF in HCT116 cells (18). Now we sought to determine whether under autocrine conditions cell adhesion function was linked with PKC signaling. Cells were treated with BIM-1, and MTT cell adhesion assays were performed using different concentrations of CN IV. These experiments revealed (Fig. 10C) that, depending upon the concentration of CN IV, BIM-1 inhibited basal cell adhesion (34–61%), thus showing that PKC activity is critical for the basal adhesiveness of HCT116 cells. These results establish a link between endogenous integrin ₂ expression and cell adhesion function under the control of PKC signaling. Our present data further delineate the mechanism and allow us to assign the Thr-421/Ser-424 phosphorylation sites of S6K to endogenous integrin ₂ protein expression and its functions controlled by PKC signaling in HCT116 cells. Importantly, findings from single point dominant negative mutant experiments that both T421A and S424A mutants inhibit cell adhesion function are consistent with the selective inhibitor results. Overall, based on the data presented in this paper, it is clear that various endogenous inputs from multiple signaling pathways (ERK/MAPK, mTOR, and PKC) contribute to the phosphorylation of distinct sites of S6K in HCT116 cells.

In the present study, we examined another cell adhesion function as well. To date, a role in cell motility function by S6K via PKC signaling has not been established. Our present data link the positive role of basal PKC activation by autocrine TGF to S6K in cell motility (micromotion) of HCT116 cells. Fig. 11A shows inhibition of HCT116 cell micromotion by PKC inhibitor BIM-1 in a concentration-dependent fashion as mediated by autocrine TGF. Under these conditions, BIM-1 specifically inhibited the Thr-421/Ser-424 phosphorylation sites of S6K activation; therefore, it is reasonable to assign the Thr-421/Ser-424 sites of S6K to cell micromotion via PKC signaling. These results were confirmed by transfection with dominant negative mutants T421A and S424A, which were overexpressed in micromotion experiments (Fig. 8, B and C). We demonstrate for the first time that the mechanism by which autocrine TGF controls cell motility via PKC signaling is linked to specific phosphorylated sites of S6K. Obviously, among the mapped phosphorylation sites, Ser-411 of S6K does not contribute to the motility function by autocrine TGF via PKC signaling cascade. However, in the presence of EGF, all four phosphorylation sites are activated and may contribute to cell motility via PKC signaling (Fig. 11B). Since motility is an intrinsic component of metastasis, it seems likely that phosphorylation sites Thr-421 and Ser-424 of S6K may be contributing to the metastatic capability of HCT116 cells seen in orthotopic implant models.

In this communication, we have focused on multiple cell signaling mechanisms regulating cell adhesion functions in human colon cancer cells. It is apparent from these results that MEK, mTOR, and PKC enzymes play critical roles in autocrine TGF-mediated integrin ₂ protein expression, cell adhesion, and cell motility functions through convergence on S6K. Data presented here demonstrate that autocrine TGF controls biological functions via activation of S6K at specific phosphorylation sites. To control cell functions, we found by several approaches that autocrine TGF utilizes discrete signaling pathways for the phosphorylation of S6K (ERK/MAPK, mTOR, and PKC) at specific sites but that when the EGFR is saturated, all S6K sites are activated by each of these pathways. These results are summarized in Table I. These results suggest that autocrine TGF elicits its action by multiple signaling cascades acting upon S6K. Our quantitative cell micromotion experiments establish a novel function of S6K in controlling cell motility. Thus, the present work, along with the emerging concept that mTOR signaling is an antitumor target (35), indicates that this pathway may well be an antimetastatic target as well.

View this table: [in this window] [in a new window]   TABLE I Activation of S6K by multiple signaling pathways Protein kinase inhibitors used in signaling pathways are shown in the left column. The middle column shows the effect of inhibitors on various phosphorylation sites of S6K activated by autocrine TGF. The right column shows the effect of inhibitors on phosphorylation sites of S6K activated by exogenous growth factor in HCT116 cells.

	FOOTNOTES

To whom correspondence may be addressed: Dept. of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Calton St., Buffalo, NY 14263. Tel.: 716-845-5874; Fax: 716-845-8857; E-mail: rajinder.sawhney{at}roswellpark.org. To whom correspondence may be addressed: Dept. of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton St., Buffalo, NY 14263. Tel.: 716-845-3044; Fax: 716-845-8857; E-mail: michael.brattain{at}roswellpark.org.

¹ The abbreviations used are: S6K, p70S6 kinase; BIM-1, bisindolylmaleimide; CN IV, collagen IV; ECIS, electrical cell-substrate impedance sensor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FRAP, FKBP and rapamycin-associated protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; mTOR, mammalian target of rapamycin; MTT, methylthiazole tetrazolium; PKC, protein kinase C; TGF, transforming growth factor .

	ACKNOWLEDGMENTS

 
We are indebted to Drs. John Blenis, Patrick Dennis, and George Thomas for providing the S6K mutants.

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