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1
Departments of Surgery [P. M. G., R. B., J. I. K.] and Pathology [M. M.], University of Texas Health Science Center, San Antonio, Texas, and South Texas Veterans Health Care System, Audie Murphy Veterans Administration Hospital, San Antonio, Texas [J. I. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which is downstream of PI3K, was activated in cells expressing active RhoA. In addition, expression of constitutively activated PKC in TRAMP cells enhanced proliferation and p70S6 kinase phosphorylation, whereas the inhibition of PKC activation resulted in activation of Akt and enhanced cell survival. Thus, the effects of RhoA on TRAMP cell proliferation and apoptosis may be mediated by PKC. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stimulation of cell proliferation takes place by the transmission of signals relayed by growth factors from membrane-bound receptor Tyr kinases to the nucleus through a series of protein cascades that require serial phosphorylation at Tyr or Ser/Thr sites (10) . The two best-known signaling cascades involved in the transmission of these signals are the MAPK3 and the PI3K pathways. Activation of receptor Tyr kinases results in the activation of p21Ras (11 , 12) . In turn, Ras activates Raf, which then activates MAPK kinase (MEK), resulting in p42/p44MAPK [also known as extracellular regulated kinase (ERK1/2)] phosphorylation (13) . PI3K is an enzyme that participates in a myriad of cellular processes and whose activity has been linked to cell growth and transformation, differentiation, motility, insulin action, and cell survival (14) . PI3K comprises a family of two-subunit (regulatory p85 and catalytic p110) lipid signaling enzymes that initiate signaling cascades by generating three distinct membrane lipids, including PIP3 (15) . PIP3 phosphorylates and activates PDK-1, which mediates most of the effects of PI3K (16) .
One downstream effector of PI3K, the ubiquitous mitogen-activated Ser/Thr kinase, p70S6 kinase, has emerged as an important regulator of cell growth. It participates in the translation of mRNAs that contain an oligopyrimidine tract at the transcriptional start site. The regulation of p70S6 kinase includes phosphorylation at multiple sites. Thr 229, which is located in the catalytic loop of p70S6 kinase, is phosphorylated in a PIP3-dependent manner by PDK1. p70S6 kinase is also activated in vivo in part, by a phosphatidylinositol kinase-related kinase, mTOR (17, 18, 19) . The immunosuppressant drug rapamycin inhibits mTOR by forming a stable complex with FK506-binding protein, which binds mTOR. As a result of this interaction, rapamycin induces dephosphorylation of several sites of p70S6 kinase, leading to its inactivation (17) . The cell survival protein, Akt/PKB, is another downstream effector of PI3K that has been extensively studied. PKB/Akt is controlled at the level of protein kinases (PDK1 and ILK), that by themselves are stimulated in the presence of PIP2 and PIP3 (20) . Activation of Akt has been implicated in protection from apoptosis in response to several signals (reviewed in Ref. 20 ).
A third class of protein kinases regulated by PI3K are the members of the PKC superfamily. The PKC superfamily is comprised of Ser/Thr protein kinases found in mammalian cells; these distinct isoforms of PKC were implicated in a multitude of cellular processes (21)
. PKCs are subdivided into three subfamilies according to their lipid-activation profiles: conventional PKCs (
, ßI, ßII, and 
) are activated by DAG and Ca2+; novel PKCs (
, 
, 
, and 
) do not respond to Ca2+ but require DAG; and atypical PKCs (, µ, and 
/
) are not activated by either DAG or Ca2+ (22)
. PIP3 activates all isoforms of PKCs (23)
. Phosphorylation of PKC is an important mechanism for regulating its activity (24)
.
PKC, an atypical family member, has received considerable attention as a downstream target of PI3K. PDK-1 phosphorylates and activates PKC in a PIP3-enhanced manner (25)
. Stimulation of cells by insulin and insulin-like growth factor leads to activation of PKC, which can be inhibited by chemical inhibitors of PI3K (26)
. The closely related PKC isoform is activated in cells by epidermal growth factor or platelet-derived growth factor by a PI3K-dependent mechanism (27)
. These findings implicate atypical PKCs as effectors of PI3K.
We previously reported the importance of RhoA in the growth of prostate tumor cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells; Ref. 3
). TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein (T19N cells) displayed few actin stress fibers and grew at a slower rate than control cells because of a lengthening of the G1 phase of the cell cycle. On the other hand, TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein (Q63L cells) displayed a contractile phenotype and grew at a faster rate than controls because of a shorter G1 phase of the cell cycle (3)
. In this article, we show that RhoA activation results in activation of a p70S6 kinase-dependent cell proliferation pathway. In contrast, inhibition of RhoA activation results in the activation of Akt and enhanced cell survival. RhoA activation resulted in phosphorylation (activation) of PKC, whereas inactivation of PKC induced Akt phosphorylation, which indicates a role for PKC in RhoA-dependent inactivation of Akt.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(Thr410) was a generous gift from Dr. Alex Toker, Harvard Medical School, Boston, MA (28)
. Total PKC was purchased from Upstate Biotechnology, Lake Placid, NY.
Cell Culture.
Transgenic mouse prostate (TRAMP) cancer cells were kindly supplied by Dr. Norman Greenberg, Baylor College of Medicine, Houston, TX, and are a clonal derivative of the TRAMP-C2 cell line (29)
. This cell line expresses cytokeratin and androgen receptor, but does not express E-cadherin or T-antigen oncoprotein. The cells were cultured in Dulbecco’s Modified Eagle Medium containing high glucose (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% fetal bovine serum (Life Technologies, Inc.), 5% NuSerum (Collaborative Biomedical Products, Becton Dickinson Labware, Bedford, MA), 5 µg/ml insulin (Sigma Chemical Co., St. Louis, MO), 25 units/ml penicillin-streptomycin (CellGro Mediatech, Herndon, VA) and 10 nM dihydrotestosterone (Sigma Chemical Co.).
Molecular Constructs and Transformation Assays.
TRAMP cell lines expressing mutant RhoA proteins were obtained by stable transfection with the following constructs: pZip-rhoA(T19N)-DNA (dominant negative) and pZip-rhoA(Q63L)-DNA (constitutively active), (kindly provided by Dr. Danny Manor, Cornell University, Cornell, NY). TRAMP cell lines expressing mutant PKC clones were obtained by stable transfection with the following constructs: FLAG-PKC (wild type), FLAG-PKC-myr (constitutively activated), and FLAG-PKC-K281W (dominant negative; kindly provided by Dr. Alex Toker, Harvard Medical School, Boston, MA). Cells were stably transfected using LipofectAMINE PLUS reagent (Life Technologies, Inc.). Cells expressing mutant proteins were selected by resistance to G-418 (Mediatech, Inc., Herndon, VA) to establish cell lines that stably express the mutant proteins.
Flow Cytometry.
TRAMP cells were grown under desired conditions in 100-mm dishes at 500,000 cells/dish. Cells to be processed for flow cytometry were trypsinized, resuspended in 2 ml of cell growth medium, and spun down in a tabletop centrifuge. The medium was aspirated and the pellet washed once in PBS. For staining with PI for DNA analysis, the cells were resuspended in 500 µl of 70% ethanol, and incubated 30 min at -20°C. The cells were repelleted, and washed twice in 1% BSA/PBS. The cells were suspended in 150 µl of PBS containing 50 µl of 1 mg/ml RNase A (Sigma Chemical Co.) and 100 µl of 100 µg/ml PI (Sigma Chemical Co.) and were incubated overnight at 4°C. The next day, the fixed cells were analyzed by flow cytometry. For detection of apoptosis using Annexin V (FITC-conjugated), cells were resuspended in 1x binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2] with 5 µl of 0.2 µg/ml Annexin V and 10 µl of 0.25 mg/ml PI (Santa Cruz Biotechnology, Santa Cruz, CA). After 15-min incubation at room temperature, an additional 400 µl of 1x binding buffer was added to the cells, and the cells were passed through a nylon mesh to remove cell clumps. The cells were immediately analyzed by flow cytometry. Flow cytometry was conducted on FACStar Plus (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cells were illuminated with 200 mW of 488 nm light produced by an argon-ion laser. Fluorescence was read through a 630/22 nm band-pass filter (for PI) or a 530/30 nm band-pass filter (for Annexin V-FITC). Cells stained with Annexin V alone were considered apoptotic, whereas those stained with both Annexin V and PI or with PI alone were considered necrotic. Viable cells did not stain with either dye. Data were collected on 20,000 cells, as determined by forward and right-angle light scatter, and stored as frequency histograms; data used for cell cycle analysis were further analyzed using MODFIT (Verity software, Topsham, ME).
Western Blotting.
TRAMP cells were grown on 100-mm dishes at 1,000,000 cells/dish under required conditions. Whole-cell extracts were prepared by washing the cells twice in PBS and lysing cells in 250 µl of radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 10% NP40; protease inhibitors: 0.1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml each phenathroline, leupeptin, aprotinin, and pepstatin A]. Proteins were quantitated using a bicinchoninic acid (BCA) assay (Pierce, Rockford IL) and fractionated on SDS-polyacrylamide gels. Most proteins could be detected using a 10% acryamide:bis 29:1 gel, except for p70S6 kinase, which was detected using a 10% 118:1 acrylamide:bis gel. Electrophoresis was performed at 45 mA for 
45 min using mini-vertical electrophoresis cells (Mini-PROTEAN II Electrophoresis Cell, Bio-Rad, Hercules, CA). The gels were electroblotted for 1.5 h at 200 mA using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) onto 0.2-µm polyvinylidene difluoride membrane (Micron Separations, Inc., Westborough, MA). The blots were stained with primary antibodies. The staining was detected by enhanced chemiluminescence (Pierce, Rockford, IL) after incubation with a peroxidase-labeled secondary antibody [Donkey antimouse IgG ( Chemicon, Temecula, CA); goat antirabbit IgG, Fc-specific (Jackson Immunoresearch Laboratories, West Grove, PA)].
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RESULTS |
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illustrates the rate of proliferation of control, Q63L, and T19N cell lines using the percentage of cells in the S phase of the cell cycle as the proliferative index. T19N cells displayed a 50% decrease in proliferation rate compared with parent cells (Fig. 1)
and were further growth-inhibited after treatment with both PD 98059 (26% decrease as compared with untreated T19N cells), and LY 294002 (38% decrease as compared with untreated T19N cells). In contrast, treatment of TRAMP and Q63L cells with LY 294002 displayed a 50% decrease in the percentage of cells in S phase of the cell cycle; a much smaller effect (6–12% decrease) was observed with PD 98059 (Fig. 1)
. Decreased cell proliferation was attributable to G0-G1 arrest (Fig. 1)
. This suggested the activation of a PI3K-dependent proliferation pathway in cells expressing active RhoA. To verify the results obtained with LY 294002, we stably transfected TRAMP cells with a plasmid expressing a dominant-negative mutant of the p85 (catalytic) subunit of PI3K (
p85). The p85 transfected cells (T-p85) expressed decreased Tyr phosphorylation of p85PI3K, and decreased phosphorylation of p70S6 kinase and Akt, two prominent downstream targets of PI3K (Fig. 2A)
. Expression of p85 in TRAMP cells reduced the proliferation rate of these cells by about 75% (Fig. 2B)
.
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). T19N cells were significantly protected from apoptosis induced by serum deprivation compared with TRAMP and Q63L cells (5% apoptotic T19N cells versus 10% TRAMP and 18.6% apoptotic Q63L cells). Inhibition of the PI3K signaling pathway with LY 294002 resulted in increased apoptosis in T19N cells after serum withdrawal (24.7 versus 5% in untreated cells). PD 98059 induced apoptosis in T19N cells, but not in the cells expressing active RhoA (Fig. 3)
.
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). The T19N cells, on the other hand, displayed reduced expression and phosphorylation of p70S6 kinase (Fig. 4A)
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, inhibited p70S6 kinase phosphorylation as well (Fig. 4B)
. p70S6 kinase phosphorylation was not inhibited by PD 98059 treatment (not shown). Fig. 4B
also shows overexpression and enhanced phosphorylation of p70S6 kinase in Q63L, but not in T19N cells.
To determine whether p70S6 kinase plays a role in RhoA-mediated TRAMP cell proliferation, we inhibited p70S6 kinase activation in these cells with rapamycin. Activation of the mTOR is necessary for p70S6 kinase phosphorylation (activation) along with PI3K (32)
. The immunosuppressant rapamycin (100 nM), a specific inhibitor of mTOR, inhibited p70S6 kinase activation identical to LY 294002 (not shown) and inhibited cell cycle progression by 50% both in the presence and absence of active RhoA (Fig. 4C)
. This demonstrates the essential role of p70S6 kinase activity for TRAMP cell proliferation. On the other hand, rapamycin did not significantly affect the apoptotic rate of TRAMP cells (not shown).
RhoA Negatively Regulates the Cell Survival Protein Akt.
The PI3K-dependent protein kinase Akt (also known as PKB) has been identified as a prominent antiapoptotic protein. Akt was examined by immunoblotting with a phospho-specific antibody against Akt (Ser473). Although the T19N cells demonstrated a high level of activation of Akt, Akt phosphorylation in the TRAMP and Q63L cells was suppressed, which indicated that the expression of active RhoA suppresses the activation of a cell survival pathway (Fig. 5A)
. Activation of Akt was dependent on PI3K activation as demonstrated by the reduced phosphorylation of Akt with LY 294002 treatment, but not with PD 98059 treatment (Fig. 5B)
. The activation of p70S6 kinase in the presence of active RhoA, and the activation of Akt in its absence, suggests that PI3K is activated both in the presence and the absence of active RhoA.
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., another downstream effector of PI3K (28)
. Immunoblots of TRAMP, Q63L (expressing constitutively active RhoA), and T19N (expressing dominant-negative RhoA) cells were stained with a phospho-specific antibody to PKC (Thr410). As can be seen in Fig. 6A
, and T19N cells expressed decreased levels of phosphorylated PKC compared with TRAMP and Q63L cells. This suggests that RhoA activation stimulates PKC phosphorylation, a state required for its activation (33
, 34)
. Next, we determined the proliferative and apoptotic effects of PKC on TRAMP cells. TRAMP cells were transfected with constitutively active (myristoylated), wild-type, and dominant-negative (K281W) PKC plasmids (Fig. 6B)
, and examined by flow cytometry for cell cycle progression and apoptosis. As can be seen in Fig. 6C
, cells expressing dominant-negative PKC showed reduced proliferative rates and were protected from apoptotic cell death, similar to T19N cells. On the other hand, expression of constitutively active PKC resulted in enhanced proliferation and increased apoptosis, similar to Q63L cells.
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. acts upstream of both p70S6 kinase and Akt activation (35, 36, 37)
. We, therefore, determined whether PKC influenced the activation of these proteins in TRAMP cells. Akt phosphorylation was inhibited by the expression of myristolated PKC but was enhanced by the expression of dominant negative PKC (Fig. 7A)
. On the other hand, expression of constitutively active PKC in TRAMP cells induced enhanced phosphorylation of p70S6 kinase (Fig. 7B)
, which indicated that PKC causes the same changes in phosphorylation of Akt and p70S6 kinase as does RhoA. These data suggest that the effects of RhoA may be mediated by PKC.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Data presented in this paper suggest that the effects of RhoA on TRAMP cell proliferation and apoptosis are mediated by PKC. In mammalian cells, signal transduction leading to cell proliferation and survival may proceed via the MAPK or the PI3K pathways (10) . Data presented in this report show that in cells expressing active RhoA (TRAMP and Q63L cells), the PI3K pathway plays a greater role than does the MAPK pathway in cell proliferation and survival. This is indicated by the data showing that the PI3K inhibitor LY 294002, but not the MAPK inhibitor PD 98059, induced growth arrest and apoptosis in these cells. Again, p70S6 kinase, which mediates proliferation downstream of PI3K, was overexpressed in the highly proliferative RhoA-active cell lines (TRAMP and Q63L). Additional data supporting a major role for p70S6 kinase in cell proliferation, in both the presence and the absence of active RhoA, came from the inhibition of cell growth by the immunosuppressant rapamycin, which prevented the activation of p70S6 kinase. These data suggest that in the absence of active RhoA, both the p70S6 kinase- and the MAPK-dependent proliferative pathways are activated in TRAMP cells. However, the expression of active RhoA in these cells causes the enhanced activation of the p70S6 kinase-dependent proliferation pathway, leading to increased proliferative rates.
Another novel observation reported here is that, unlike p70S6 kinase, in the absence of RhoA activation, Akt is phosphorylated (activated). This is accompanied by enhanced cell survival in response to serum deprivation in cells expressing inactive RhoA. This suggests the suppression of Akt activation and cell survival by active RhoA. This is in contrast to a recent report indicating that dominant-negative RhoA inhibited TGFß-induced phosphorylation of Akt at Ser473, whereas constitutively active RhoA increased the basal phosphorylation of Akt in NMuMG mammary epithelial cells (38) . In support of our observations, however, stable transfectants of NIH3T3 cells, expressing a plasmid containing K-ras mutated at codon 12, exhibited enhanced resistance to apoptosis accompanied by higher AKT/protein kinase B activation and RhoA underexpression (39) . This suggests that in certain cells, the presence of active RhoA may be associated with Akt inactivation. Again, earlier studies suggested that p70S6 kinase activation was mediated by Akt, because p70S6 kinase was stimulated by active mutants of Akt in cotransfection assays (40 , 41) . However, it now appears that Akt mediates p70S6 kinase activation only as a function of constitutive membrane localization (17) . Our data also suggest that p70S6 kinase and Akt are on parallel pathways downstream of PI3K because in cells expressing active RhoA, p70S6 kinase was activated and Akt was inactivated, whereas the opposite was true in cells expressing inactive RhoA.
To determine a cause for this difference in protein activation in the presence or absence of active RhoA, we examined PKC, which was previously shown to stimulate p70S6 kinase activity (37)
but down-regulate Akt (35
, 36)
. PKC, PKB/Akt, and p70S6 kinase are all substrates for PDK1. Whereas both p70S6 kinase and Akt can be phosphorylated by PDK1, p70S6 kinase can also be phosphorylated by PKC by direct binding (37)
, although Akt can be dephosphorylated by the same PKC isoform by direct binding as well (35)
. In our cells, PKC was stimulated in the presence of active RhoA and inactivated in its absence. Similar to RhoA, PKC activation also stimulated TRAMP cell proliferation, whereas inhibition of its activity inhibited TRAMP cell growth. Similarly, the activation of PKC stimulated apoptosis, whereas the inhibition of its activity stimulated cell survival (inhibited apoptosis). Again, like RhoA, inactivation of PKC activated Akt, although less active Akt was observed in cells expressing active PKC. Thus, our data suggest that PKC mediates the effects of RhoA on cell proliferation and apoptosis, and especially the inactivation of Akt in RhoA-active cells.
Because p70S6 kinase, one downstream effector of PI3K, is activated in cells expressing active RhoA, whereas Akt, another downstream effector of PI3K, is activated in cells expressing inactive RhoA, it is likely that PI3K is activated both in the presence and the absence of active RhoA. Therefore, RhoA probably affects PKC directly. In fact, it has been shown that pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases (RhoA, RhoB, RhoC), blocks membrane translocation of PKC but not that of PKC (42)
. Plasma membrane translocation is required for the activation of all PKC isoforms (42)
. In addition, it was recently shown in a cell-free system that the level of PKC activity was potentiated 5-fold over basal levels by the addition of RhoA, whereas the level of PKC resulted in only a slight increase (see Fig. 1A
in Ref. 43
). Thus, it is likely that RhoA activates PKC directly, and that PKC in turn activates p70S6 kinase and inactivates Akt. This effect would be additive to that of PDK1 on Akt and p70S6 kinase. This is suggested by the evidence that the inhibition of PI3K with LY 294002 inhibits p70S6 kinase and cell proliferation, as well as Akt and cell survival. A scheme depicting the possible signaling pathway is depicted in Fig. 8
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 J. I. K. was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs and by a grant from the San Antonio Cancer Institute. J. I. K. is a Career Scientist with the Department of Veterans Affairs. P. M. G. was supported by NIH Training Grant 2T32HL07446-16 and by an institutional research grant from the University of Texas Health Science Center-San Antonio. 
2 To whom requests for reprints should be addressed, at Department of Surgery, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229. Phone: (210) 567-1522; Fax: (210) 567-4664; E-mail: ghosh{at}uthscsa.edu
3 The abbreviations used are: MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; mTOR, mammalian target of rapamycin; PKC, protein kinase C; PI, propidium iodide; PDK-1, 3-phosphoinositide-dependent kinase-1; DAG, diacylglycerol.
Received 10/23/01. Accepted 2/27/02.
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REFERENCES |
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by mechanisms that are both dependent and independent of phosphorylation of activation loop (T410) and autophosphorylation (T560) sites. Biochemistry, 40: 249-255, 2001.[Medline]
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