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The Lipid Products of Phosphoinositide 3-Kinase Increase Cell Motility through Protein Kinase C

The Lipid Products of Phosphoinositide 3-Kinase Increase Cell Motility through Protein Kinase C THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 10, Issue of March 7, pp. 6465–6470, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Lipid Products of Phosphoinositide 3-Kinase Increase Cell Motility through Protein Kinase C* (Received for publication, September 24, 1996, and in revised form, December 16, 1996) Melanie P. Derman‡§, Alex Toker§¶, John H. Hartwigi, Katherine Spokes‡, J. R. Falck**, Ching-Shih Chen‡‡, Lewis C. Cantley§¶, and Lloyd G. Cantley‡ From the Department of Medicine, Divisions of ‡Nephrology and §Signal Transduction, Beth Israel Deaconess Medical Center and the ¶Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02215, the iDivisions of Experimental Medicine and Hematology-Oncology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02215, the **Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75325 and the ‡‡Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506-0286 Phosphoinositide 3-kinase has been implicated as an phosphoinositide 3-kinase PI 3-kinase-impair PDGF-depend- activator of cell motility in a variety of recent studies, ent chemotaxis (1, 5, 6), and selective activation of the PI yet the role of its lipid product, phosphatidylinositol 3-kinase is sufficient to initiate motility (7). The lipid products 1,4,5-trisphosphate (PtdIns-3,4,5-P ), has yet to be eluci- 3 of PI 3-kinase, PtdIns-3,4-P , and PtdIns-3,4,5-P are elevated 2 3 dated. In this study, three independent preparations of acutely in response to PDGF (8) and are thought to act as PtdIns-3,4,5-P were found to increase the motility of second messengers (9–11). Although the in vivo function of NIH 3T3 cells when examined utilizing a microchemo- these lipids has not been demonstrated, they activate calcium- taxis chamber. Dipalmitoyl L-a-phosphatidyl-D-myo-ino- independent protein kinase C family members in a stereospe- sitol 3,4,5-triphosphate (Di-C -PtdIns-3,4,5-P ) also pro- 16 3 cific manner (12–16). Thus, we investigated the possibility that duced actin reorganization and membrane ruffling. PtdIns-3,4,5-P stimulates cell motility via activation of a PKC Cells pretreated with 12-O-tetradecanoylphorbol-13-ac- family member. etate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility in- MATERIALS AND METHODS duced by Di-C -PtdIns-3,4,5-P . These results are con- 16 3 Cell Culture and Reagents—The majority of experiments were per- sistent with previous observations that PtdIns-3,4,5-P formed with NIH 3T3 fibroblasts, using PDGF as the positive control. activates Ca -independent PKC isoforms in vitro and Selected experiments were repeated with mIMCD-3 cells, a murine in vivo and provide the first demonstration of an in vivo renal tubular epithelial cell line that expresses the c-met receptor and role for the lipid products of the phosphoinositide 3-ki- exhibits striking chemotaxis to a gradient of HGF (17–19). All cells were nase. PtdIns-3,4,5-P appears to directly initiate cellular 3 cultured in Dulbecco’s modified Eagle’s medium with 5% fetal calf motility via activation of a PKC family member. serum using standard techniques. PDGF (Upstate Biotechnology, Inc., Lake Placid, NY) and HGF (Institute of Immunology, Tokyo, Japan) were used in concentrations of 10 and 40 ng/ml, respectively, based on previous dose response curves for maximal chemotaxis (19). Initiation of cellular motility has been demonstrated with PtdIns-4,5-P was obtained from Upstate Biologicals, and phosphati- multiple growth factors, including platelet-derived growth fac- dylserine (PtdSer) was from Avanti Polar Lipids. Diacylglycerol (DAG) tor (PDGF) (1), hepatocyte growth factor (HGF) (2), and insu- and horseradish anti-mouse conjugate were purchased from Boehringer lin (3). The mechanisms whereby cells undergo chemotaxis Mannheim. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was obtained (directional cell movement) and chemokinesis (random cell from Life Technologies, Inc., and wortmannin was from Sigma. Cal- phostin C was obtained from Calbiochem, and P81 phosphocellulose movement) are complex, requiring dissolution of cell-cell con- paper was purchased from Whatman. Thin layer chromatography tacts (such as tight junctions in epithelial cells) and cell-surface plates (Silica Gel 60) were obtained from EM Separations. contacts, formation of lamellipodia, actin filament severing and Preparation of PtdIns-3,4,5-P from PtdIns-4,5-P —Phosphoinositide 3 2 nucleation, and finally contraction of the actin filament net- 3-kinase was purified from rat liver cytosol as described previously (20) work leading to movement of the cell body (4). An understand- and used immediately for the preparation of PtdIns-3,4,5-P . Lipid ing of the signaling pathways required to orchestrate these substrates were prepared by drying under a stream of nitrogen. PtdSer (10 mg/ml) was added to the PtdIns-4,5-P (2 mg/ml) as a carrier, and cellular events should provide critical new insights into numer- 2 the mixture was sonicated in 10 mM Hepes, pH 7.0, 1 mM EGTA for 10 ous biological events such as cell migration and organization min using a bath sonicator. This mixture was then incubated with during organ development and wound healing, tumor cell me- phosphoinositide 3-kinase at 37 °C in the presence of 50 mM [g- P]ATP tastasis, and progression of arterial atherosclerotic plaques. (3000 Ci/mmol), 5 mM MgCl ,50mM Hepes, pH 7.5, for 60 min. The Mutations in the PDGF receptor that eliminate binding of reaction (200 ml) was stopped by the addition of 65 mlof5 N HCl, and lipids were extracted in 400 ml of ChCl /MeOH (1:1). Lipids were dried and stored at 270 °C until needed. [ P]PtdIns-3,4,5-P was quantified * This work was supported in part by National Institutes of Health by thin layer chromatography (n-propanol, 2 M acetic acid extract (65: Grant DK48871. The costs of publication of this article were defrayed in 35)) and radiation detection on a Bio-Rad molecular imager. Based on part by the payment of page charges. This article must therefore be the specific activity of the [g- P]ATP, 20% of the PtdIns-4,5-P was hereby marked “advertisement” in accordance with 18 U.S.C. Section converted to PtdIns-3,4,5-P . 1734 solely to indicate this fact. Preparation of Synthetic PtdIns-3,4,5-P —Dipalmitoyl L-a-phos- § To whom correspondence should be addressed: Div. of Nephrology, phatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C -PtdIns-3,4,5-P ) (21) 16 3 Dana 517, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. and dioctanoyl-L-a-phosphatidyl-D-myo-inositol-3,4,5-trisphosphate Tel.: 617-667-2147; Fax: 617-667-5276. were synthesized as described previously (13, 22). The abbreviations used are: PDGF, platelet-derived growth factor; Chemotaxis Assay—Chemotaxis was evaluated using a modified Boy- HGF, hepatocyte growth factor; PI, phosphoinositide; PtdIns, phos- den chamber assay with a 48-well microchemotaxis chamber as de- phatidylinositol; PtdSer, phosphatidylserine; DAG, diacylglycerol; TPA, 12-O-tetradecanoylphorbol-13-acetate. scribed previously (Neuro Probe Inc., Cabin John, MD) (19, 23). Lipids This paper is available on line at http://www-jbc.stanford.edu/jbc/ 6465 This is an Open Access article under the CC BY license. 6466 PI 3-Kinase Mediates Motility via Protein Kinase C FIG.1. PtdIns-3,4,5-P enhances motility of NIH 3T3 fibroblasts (A and B) and IMCD epithelial cells (C). Cell motility was evaluated using a modified Boyden chamber assay with a 48-well microchemotaxis chamber. A, the lower section of the Boyden chamber was filled with media alone or media containing PDGF, PtdSer/PtdIns-4,5-P lipid substrate, or enzymatically generated PtdIns-3,4,5-P . B and C, either chemically 2 3 PI 3-Kinase Mediates Motility via Protein Kinase C 6467 were dried in a stream of nitrogen and then sonicated for 5 min in serum-free media. The lower section of the Boyden chamber was filled with media alone or media containing either PDGF (10 ng/ml) or PtdSer/PtdIns-4,5-P (100 mM/25 mM) or PtdSer/PtdIns-4,5-P /PtdIns- 2 2 3,4,5-P (100 mM/25 mM/5 mM). A polycarbonate filter (Nucleopore Corp., Pleasanton, CA) coated with rat tail collagen type I (Collaborative Biomedical, Bedford, MA) was placed over the lower compartment, and 1.5 3 10 cells were added to the upper compartment. In some experi- ments, wortmannin was diluted 1:1000 in serum-free media immedi- ately prior to use and added at the appropriate concentration to both the upper and lower chambers at time 0. Control experiments were performed with Me SO vehicle alone. After4hof incubation at 37 °C, filters were removed, the cells were fixed and stained with Diff-Quik (Baxter Healthcare Corp., Miami, FL), and the upper surface was wiped with a cotton applicator to remove nonchemotaxing cells. For each well, cells that had passed through the pores were counted, and the mean value of cells/mm was calculated. Electrophoresis and Western Blotting—Confluent plates of cells were serum-starved overnight in the presence of either 0.3% Me SO or 300 nM TPA followed by a wash with phosphate-buffered saline and lysis in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris, 1 mM MgCl ,1mM CaCl , 10% glycerol, 1% Nonidet P-40, 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, pH 7.5). The suspension was centri- FIG.2. Dose response curve for 3T3 fibroblast chemotaxis to fuged for 10 min at 12,000 3 g. Equal aliquots of supernatant deter- Di-C -PtdIns-3,4,5-P and PtdIns-4,5-P . n 5 4–6 for each point. 16 3 2 mined by protein assay (Bio-Rad) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon (Millipore). Expres- sion of protein kinase Ce was detected by a monoclonal antibody specific of the lipid products. This resulted in conversion of 20% of the for this enzyme (Transduction Laboratories) and quantified using a PtdIns-4,5-P to PtdIns-3,4,5-P to give a final mixture of 100 2 3 Molecular Dynamics PhosphorImager. mM PtdSer, 25 mM PtdIns-4,5-P ,5 mM PtdIns-3,4,5-P . The In Vivo Labeling of Lipids—In vivo levels of D3 phosphoinositides in 2 3 response to stimuli were measured as described previously (8). Briefly, addition of this PtdSer/PtdIns-4,5-P /PtdIns-3,4,5-P mixture 2 3 3T3 cells maintained in Dulbecco’s modified Eagle’s medium, 5% fetal to the bottom well of the chemotaxis chamber resulted in a calf serum were grown to 80% confluence and then placed in Dulbecco’s 10-fold increase in motility of NIH 3T3 fibroblasts compared modified Eagle’s medium, 0.1% fetal calf serum for 12–16 h. For label- with vehicle control and a 3-fold increase compared with the ing purposes, monolayers were placed in phosphate-free Dulbecco’s PtdSer/PtdIns-4,5-P mixture alone (Fig. 1A). The small but modified Eagle’s medium in the absence of serum for 1 h, followed by 2 reproducible motility response to 100 mM PtdSer, 25 mM PtdIns- mCi/ml of [ P]orthophosphate for 3 h. Cells were then stimulated with PDGF (20 ng/ml), Di-C -PtdIns-3,4,5-P , or vehicle control for 10 min. 4,5-P may be due to either activation of PKC isoforms by the 16 3 2 Following stimulation, cells were washed twice with ice-cold phosphate- high concentrations of PtdIns-4,5-P (13) or impurities in these buffered saline and lysed in 750 ml of methanol, 1 M HCl (1:1). 20 mgof lipids not seen when lower concentrations of lipids were inves- crude brain phosphoinositides (Sigma) were added as carrier. Lipids tigated individually (see “Discussion”). were extracted by the addition of 380 ml of chloroform, and the organic To more directly examine the isolated effects of PtdIns-3,4,5- phase was washed twice with 400 ml of methanol, 0.1 M EDTA. Phos- P , we utilized two synthetically prepared sources of PtdIns- pholipids were then deacylated and prepared for Sepharose A ex- change-high pressure liquid chromatography analysis as described 3,4,5-P (Fig. 1, B and C). 5 mM Di-C -PtdIns-3,4,5-P , which 3 16 3 previously (8). forms micelles when sonicated in the absence of carrier lipids, Membrane Ruffling—F-actin was localized in coverslip adherent induced a 7-fold increase in cell motility over base line in 3T3 cells. Quiescent 3T3-fibroblasts or cells exposed to 40 ng/ml of PDGF, 5 cells (control, 10.0 6 1.7 cells/mm , n 5 17; Di-C -PtdIns- mM Di-C -PtdIns-3,4,5-P ,or5 mM PtdIns-4,5-P for 10–60 min were 16 3 2 3,4,5-P , 70.2 6 7.9, n 5 22; Fig. 1B) and a 4-fold increase in fixed by the addition of an equal volume of 3.7% formaldehyde in IMCD cells (control, 7.4.0 6 1.1 cells/mm , n 5 27; Di-C - phosphate-buffered saline at 37 °C for 30 min. Fixed cells were perme- abilized with 0.1 volume of 1% Triton X-100 containing 2 mM tetra- PtdIns-3,4,5-P , 34.5.2 6 2.5, n 5 36; Fig. 1C). When Di-C - 3 16 methylrhodamine B isothiocyanate-phalloidin at 37 °C for 60 min (24), PtdIns-3,4,5-P was added to both compartments of the chemo- washed three times with phosphate-buffered saline for 5 min each, and taxis chamber, a significant but somewhat smaller number of magnified in a Zeiss IM45 Inverted microscope. cells was found to migrate through the pores, indicating an Statistical Analysis—Results were averaged, and statistical rele- increase in both chemokinesis and chemotaxis (control, 8.4 6 vance was determined by Student’s t test. Data are presented as 0.9 cells/mm , n 5 10; Di-C -PtdIns-3,4,5-P on the bottom mean 6 S.E. 16 3 only, 39.0 6 2.6, n 5 11; Di-C -PtdIns-3,4,5-P on both the top 16 3 RESULTS and bottom, 30.8 6 1.9, n 5 12 (p 5 0.016)). Enzymatically Generated and Synthetic PtdIns-3,4,5-P Ini- Di-C -PtdIns-3,4,5-P , a short chain PtdIns-3,4,5-P that is 3 8 3 3 tiate Cell Motility and Ruffling—Since exogenously added lipid soluble as a monomer in water, was also tested in 3T3 cells and vesicles and micelles are known to fuse with the plasma mem- found to initiate chemotaxis although to a lesser extent (con- brane of live cells, we investigated the role of PtdIns-3,4,5-P in trol, 10.0 6 1.7; Di-C -PtdIns-3,4,5-P , 29.4 6 4.2, n 5 27, p 5 3 8 3 cell motility by directly adding this lipid to the cells in a Boyden 0.001; Fig. 1B). There was no chemotactic effect when cells chamber. Three independent preparations of PtdIns-3,4,5-P were exposed to 5 mM PtdIns-4,5-P (3T3 cells, 11.7 6 2.8 3 2 2 2 were employed to evaluate the motility response of this puta- cells/mm , n 5 23; IMCD cells, 4.5 6 1.0 cells/mm , n 5 18) or tive second messenger (Fig. 1). PtdIns-3,4,5-P was enzymati- 50 mM PtdSer (3T3 cells, 17.8 6 3.7 cells/mm , n 5 12; IMCD cally generated by adding purified PI 3-kinase to a 1:4 mixture cells, 10.5 6 1.5 cells/mm , n 5 6). Concentrations of Di-C - of PtdIns-4,5-P and PtdSer followed by chloroform extraction PtdIns-3,4,5-P from 1 nM to 100 mM were evaluated (Fig. 2). 5 2 3 synthesized (dioctanoyl)-PtdIns-3,4,5-trisphosphate (Di-C -PI-3,4,5-P ;5 mM), chemically synthesized (dipalmitoyl)-PtdIns-3,4,5-trisphosphate 8 3 (Di-C -PI-3,4,5-P ;5 mM), commercial PtdIns-4,5-bisphosphate (PI-4,5-P ;5 mM), or PDGF (10 ng/ml) were added to the lower chamber when 16 3 2 indicated. In some experiments, 10 nM wortmannin (wort.) was added. The number of chambers assayed for each condition is indicated by n.*, p , 0.001 compared with PtdIns-4,5-P control. 2 6468 PI 3-Kinase Mediates Motility via Protein Kinase C FIG.3. Membrane ruffling by cells exposed to Di-C -PtdIns-3,4,5-P 16 3 mimics the response seen with PDGF. The top panels show that quies- cent cells and cells exposed to 5 mM PtdIns-4,5-P (PIP ) are no different, 2 2 whereas significant membrane ruffling can be seen in the cells exposed to either 40 ng/ml PDGF or 5 mM PtdIns-3,4,5-P (PIP )at10(middle panels) and 30 min (bottom panels). mM was chosen for further experiments, since this was the lowest dose that consistently resulted in a chemotactic response. The polymerization of cytoplasmic actin that follows receptor stimulation and leads to membrane ruffling and lamellipodia formation is felt to be downstream of the PI 3-kinase (6, 25). To test this hypothesis, we evaluated actin filament reorganiza- tion and membrane ruffling following the addition of Di-C - FIG.4. TPA down-regulates PKCe in 3T3 cells. NIH 3T3 cells PtdIns-3,4,5-P (Fig. 3). The synthetic form of PtdIns-3,4,5-P were treated for 12 h with either vehicle control (2) or 300 nM TPA (1) 3 3 followed by SDS-polyacrylamide gel electrophoresis and immunoblot- stimulated membrane ruffling in 3T3 fibroblasts to the same ting with an antibody specific for PKCe. Densitometric analysis of the extent as PDGF. PtdIns-4,5-P had no effect on quiescent cells. blot revealed 35.9 6 4.6 densitometric units for control PKCe versus Exogenously Added PtdIns-3,4,5-P Does Not Activate En- 8.2 6 0.9 for cells pretreated with TPA (experiment performed in dogenous PI 3-Kinase and Is Not Inhibited by Wortmannin—It triplicate; p 5 0.001). was conceivable that a contaminant or a breakdown product of PtdIns-3,4,5-P might initiate the observed effects via activa- NIH 3T3 cells following the addition of extracellular Di-C - 3 16 tion of a cell surface receptor (as has been shown for lysophos- PtdIns-3,4,5-P . This approach was chosen because the recep- phatidic acid). Although this seemed unlikely, since PtdIns- tors known to initiate chemotaxis (PDGF receptor, insulin re- 3,4,5-P made by three different procedures stimulated cell ceptor, c-met receptor, lysophosphatidic acid receptor) have motility and comparable concentrations of PtdIns-4,5-P and/or also been found to activate the PI 3-kinase (8, 26–28). While PtdSer failed to stimulate cell motility, we searched for evi- stimulation with PDGF produced a dramatic rise in intracel- 32 32 dence that exogenously added PtdIns-3,4,5-P might act via cell lular [ P]PtdIns-3,4-P and [ P]PtdIns-3,4,5-P (2.3- and 17- 3 2 3 surface receptor activation by examining intracellular produc- fold), no increase in either of these lipids was seen in cells 32 32 tion of PtdIns-3,4-P and PtdIns-3,4,5-P in PO -labeled treated with 5 mM Di-C -PtdIns-3,4,5-P . 2 3 4 16 3 PI 3-Kinase Mediates Motility via Protein Kinase C 6469 FIG.5. Down-regulation of PKC in- hibits Di-C -PtdIns-3,4,5-P stimu- 16 3 lated cell motility. A, 16-h pretreatment with 300 nM TPA caused complete inhibi- tion of the motility response to Di-C - PtdIns-3,4,5-P and DAG as compared with vehicle control. PDGF-mediated cell movement was inhibited by 70%. n 5 12. B, 30-min pretreatment of NIH 3T3 cells with 100 nM calphostin C caused a 91% inhibition of Di-C -PtdIns-3,4,5-P -medi- 16 3 ated cell motility. n 5 6. *, p , 0.001 versus control; **, p , 0.01 versus stimulated. The PI 3-kinase inhibitor wortmannin binds irreversibly to DAG, 116.7 6 13.4, p , 0.001). Of note, 5 mM DAG fails to the catalytic subunit of the enzyme and prevents production of induce chemotaxis, while 5 mM Di-C -PtdIns-3,4,5-P does, 16 3 the D3 phosphorylated lipid products of the enzyme. 10 nM indicating that the PtdIns-3,4,5-P effect is not due to hydrol- wortmannin, the lowest dose that produces reliable inhibition ysis to DAG. When DAG and Di-C -PtdIns-3,4,5-P were both 16 3 of the PI 3-kinase in vivo in 3T3 fibroblasts and mIMCD-3 cells present in the bottom well, the chemotactic rate was similar to (19), caused a 60% inhibition of PDGF- and HGF-dependent that seen with DAG alone (Di-C -PtdIns-3,4,5-P , 55.5 6 4.5 16 3 cell motility but had no effect on Di-C -PtdIns-3,4,5-P -stim- cells/m ; DAG, 116.7 6 13.4; Di-C -PtdIns-3,4,5-P with DAG, 16 3 16 3 ulated cell movement in 3T3 cells or in IMCD cells (Fig. 1, B 127.3 6 19.5, n 5 12), suggesting that these two stimuli were and C). These results demonstrate that wortmannin at a dose acting via the same signaling pathway. that inhibits PDGF receptor-mediated activation of the PI 3-ki- To examine this possibility, the TPA-activable PKC family nase does not prevent PtdIns-3,4,5-P -initiated motility, further members were down-regulated by overnight preincubation of supporting the hypothesis that these lipids are inserting into the NIH 3T3 cells with 300 nM TPA. Under these conditions, there membrane and directly initiating downstream signaling events. was a 78% decline in the concentration of PKCe by Western 100 nM wortmannin, a concentration where effects on several analysis (Fig. 4), an effect comparable with that seen in human other kinases have been observed, caused essentially complete dermal fibroblasts (34). PKCe was chosen because it shows the inhibition of both PDGF and Di-C -PtdIns-3,4,5-P -stimulated greatest activation to PtdIns-3,4,5-P in vitro and in vivo. The 16 3 3 cell motility (data not shown). In light of the observation by migratory response to PtdIns-3,4,5-P was completely elimi- Kundra et al. (1) that selective activation of phospholipase Cg nated in NIH 3T3 cells pretreated with TPA (Fig. 5A), while by the PDGF receptor resulted in a substantial chemotactic PDGF-mediated cell movement was inhibited by 70%, a finding response, even in the absence of PI 3-kinase activation, this similar to that seen with exposure to 10 nM wortmannin. In result suggests that other targets of wortmannin that are likely addition, the specific PKC inhibitor calphostin C was tested (35, to be inhibited at the higher concentration, such as myosin 36). NIH 3T3 cells exposed to 100 nM calphostin C (IC 5 75–100 light chain kinase (29) or PtdIns 4-kinase (30), may be critical nM) for 30 min demonstrated a 90% reduction in PtdIns-3,4,5-P - for cell motility as well. mediated cell movement (Fig. 5B). These results suggest that Inhibition of PKC Prevents PtdIns-3,4,5-P -mediated Cell activation of the PI 3-kinase mediates cell motility via the local Motility—It was previously shown that activation of PKC by generation of PtdIns-3,4,5-P and subsequent activation of PKC. DAG or TPA can stimulate chemotaxis (31–33). Therefore, we DISCUSSION examined the role of activation of PKC in PtdIns-3,4,5-P - mediated chemotaxis. 100 mM DAG produced a consistent in- The PI 3-kinase has been clearly implicated in cell motility crease in motility of NIH 3T3 cells (control, 1.3 6 0.2 cells/mm ; by several laboratories (1, 6, 7, 19), yet the actual mechanism of 6470 PI 3-Kinase Mediates Motility via Protein Kinase C REFERENCES this effect is poorly understood. The p85 subunit of the PI 3-kinase has a BCR homology domain, which is capable of 1. Kundra, V., Escobedo, J. A., Kazlauskas, A., Kim, H. K., Rhee, S. G., Williams, L. T., and Zetter, B. R. 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Hartwig, J., Bokoch, G., Carpenter, C., Janmey, P., Taylor, L., Toker, A., and PtdIns-3,4,5-P , while both down-regulation of PKC by over- Stossel, T. (1995) Cell 82, 643–653 night treatment with TPA and inhibition of PKC with calphos- 26. Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1411–1415 tin C completely blocked the PtdIns-3,4,5-P response. The 27. Graziani, A., Gramaglia, D., Cantley, L. C., and Comoglio, P. M. (1991) J. Biol. present data cannot distinguish which PKC family member or Chem. 266, 22087–22090 members are directly involved, although in vitro data suggest 28. Vemuri, G. S., and Rittenhouse, S. E. (1994) Biochem. Biophys. Res. Commun. 202, 1619–1623 that the PKCe is strongly up-regulated by these lipids. The 29. Nakanishi, S., Kakita, S., Takahashi, I., Kawahara, K., Tsukuda, E., Sano, T., observation that high concentrations of PtdIns-4,5-P caused a Yamada, K., Yoshida, M., Kase, H., and Matsuda, Y. (1992) J. Biol. Chem. modest increase in cell motility (Figs. 1A and 2) is consistent 267, 2157–2163 30. Nakanishi, S., Catt, K., and Balla, P. (1995) Proc. Natl. Acad. Sci. 92, with this hypothesis, since this polyphosphoinositide has also 5317–5321 been found to weakly activate PKC in vitro (13). 31. Daviet, I., Herbert, J. M., and Maffrand, J. P. (1990) FEBS Lett. 259, 315–317 In addition to PKC activation, there are several other targets 32. Adelmann-Grill, B. C., Wach, F., Behr, J., and Krieg, T. (1989) Eur. J. Cell S6k Biol. 50, 128–131 for the lipid products of the PI 3-kinase. pp70 was the first 33. de la Fuente, M., Delgado, M., del Rio, M., Garrido, E., Leceta, J., Hernanz, A., target shown to be downstream of PI 3-kinase (42), and re- and Gomariz, R. P. (1994) Peptides 15, 1157–1163 cently, Akt (41, 43) and Rac (38) have also been implicated. The 34. Reynolds, N. J., Baldassare, J. J., Henderson, P. A., Suler, J. L., Ballas, L. M., Burns, D. J., Moomaw, C. R., and Fisher, G. J. (1994) J. Invest. Dermatol. latter is of particular interest, since microinjection of constitu- 103, 364–369 tively active forms of rac leads to membrane ruffling (39). The 35. Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T. (1989) Biochem. recent availability of synthetic forms of these phosphoinosi- Biophys. Res. Commun. 159, 548–553 tides should help identify their targets and determine the path- 36. Rotenberg, S. A., Huang, M. H., Zhu, J., Su, L., and Riedel, H. (1995) Proc. Annu. Meet. Am. Assoc. Cancer Res. 36, 2589 (abstr.) ways that lead to the motile response. 37. Nobes, C. D., Hawkins, P., Stephens, L., and Hall, A. (1995) J. Cell. Sci. 108, Previous results from our laboratory and others have shown 225–233 38. Hawkins, P. T., Eguinoa, A., Qiu, R.-G., Stokoe, D., Cooke, F. T., Walters, R., that activation of the PI 3-kinase is essential for PDGF and Wennstro¨m, S., Claesson-Welsh, L., Evans, T., Symons, M., and Stephens, HGF-dependent cell movement. The present experiments dem- L. (1995) Curr. Biol. 5, 393–403 onstrate that the lipid products of the PI 3-kinase act directly 39. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. as second messengers in cell motility and provide the first (1992) Cell 70, 401–410 40. Ridley, A. J., Comoglio, P. M., and Hall, A. (1995) Mol. Cell. Biol. 15, indication that PKC family members are required for the mo- 1110–1122 tility effects of this lipid in vivo. 41. Burgering, B., and Coffer, P. (1995) Nature 376, 599–602 42. Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J., and Kahn, C. R. (1994) Mol. Cell. Biol. 14, 4902–4911 43. Franke, T. F., Yang, S., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., P. Janmey, unpublished results. Kaplan, D. R., and Tsichlis, P. N. 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The Lipid Products of Phosphoinositide 3-Kinase Increase Cell Motility through Protein Kinase C

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 10, Issue of March 7, pp. 6465–6470, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Lipid Products of Phosphoinositide 3-Kinase Increase Cell Motility through Protein Kinase C* (Received for publication, September 24, 1996, and in revised form, December 16, 1996) Melanie P. Derman‡§, Alex Toker§¶, John H. Hartwigi, Katherine Spokes‡, J. R. Falck**, Ching-Shih Chen‡‡, Lewis C. Cantley§¶, and Lloyd G. Cantley‡ From the Department of Medicine, Divisions of ‡Nephrology and §Signal Transduction, Beth Israel Deaconess Medical Center and the ¶Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02215, the iDivisions of Experimental Medicine and Hematology-Oncology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02215, the **Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75325 and the ‡‡Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506-0286 Phosphoinositide 3-kinase has been implicated as an phosphoinositide 3-kinase PI 3-kinase-impair PDGF-depend- activator of cell motility in a variety of recent studies, ent chemotaxis (1, 5, 6), and selective activation of the PI yet the role of its lipid product, phosphatidylinositol 3-kinase is sufficient to initiate motility (7). The lipid products 1,4,5-trisphosphate (PtdIns-3,4,5-P ), has yet to be eluci- 3 of PI 3-kinase, PtdIns-3,4-P , and PtdIns-3,4,5-P are elevated 2 3 dated. In this study, three independent preparations of acutely in response to PDGF (8) and are thought to act as PtdIns-3,4,5-P were found to increase the motility of second messengers (9–11). Although the in vivo function of NIH 3T3 cells when examined utilizing a microchemo- these lipids has not been demonstrated, they activate calcium- taxis chamber. Dipalmitoyl L-a-phosphatidyl-D-myo-ino- independent protein kinase C family members in a stereospe- sitol 3,4,5-triphosphate (Di-C -PtdIns-3,4,5-P ) also pro- 16 3 cific manner (12–16). Thus, we investigated the possibility that duced actin reorganization and membrane ruffling. PtdIns-3,4,5-P stimulates cell motility via activation of a PKC Cells pretreated with 12-O-tetradecanoylphorbol-13-ac- family member. etate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility in- MATERIALS AND METHODS duced by Di-C -PtdIns-3,4,5-P . These results are con- 16 3 Cell Culture and Reagents—The majority of experiments were per- sistent with previous observations that PtdIns-3,4,5-P formed with NIH 3T3 fibroblasts, using PDGF as the positive control. activates Ca -independent PKC isoforms in vitro and Selected experiments were repeated with mIMCD-3 cells, a murine in vivo and provide the first demonstration of an in vivo renal tubular epithelial cell line that expresses the c-met receptor and role for the lipid products of the phosphoinositide 3-ki- exhibits striking chemotaxis to a gradient of HGF (17–19). All cells were nase. PtdIns-3,4,5-P appears to directly initiate cellular 3 cultured in Dulbecco’s modified Eagle’s medium with 5% fetal calf motility via activation of a PKC family member. serum using standard techniques. PDGF (Upstate Biotechnology, Inc., Lake Placid, NY) and HGF (Institute of Immunology, Tokyo, Japan) were used in concentrations of 10 and 40 ng/ml, respectively, based on previous dose response curves for maximal chemotaxis (19). Initiation of cellular motility has been demonstrated with PtdIns-4,5-P was obtained from Upstate Biologicals, and phosphati- multiple growth factors, including platelet-derived growth fac- dylserine (PtdSer) was from Avanti Polar Lipids. Diacylglycerol (DAG) tor (PDGF) (1), hepatocyte growth factor (HGF) (2), and insu- and horseradish anti-mouse conjugate were purchased from Boehringer lin (3). The mechanisms whereby cells undergo chemotaxis Mannheim. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was obtained (directional cell movement) and chemokinesis (random cell from Life Technologies, Inc., and wortmannin was from Sigma. Cal- phostin C was obtained from Calbiochem, and P81 phosphocellulose movement) are complex, requiring dissolution of cell-cell con- paper was purchased from Whatman. Thin layer chromatography tacts (such as tight junctions in epithelial cells) and cell-surface plates (Silica Gel 60) were obtained from EM Separations. contacts, formation of lamellipodia, actin filament severing and Preparation of PtdIns-3,4,5-P from PtdIns-4,5-P —Phosphoinositide 3 2 nucleation, and finally contraction of the actin filament net- 3-kinase was purified from rat liver cytosol as described previously (20) work leading to movement of the cell body (4). An understand- and used immediately for the preparation of PtdIns-3,4,5-P . Lipid ing of the signaling pathways required to orchestrate these substrates were prepared by drying under a stream of nitrogen. PtdSer (10 mg/ml) was added to the PtdIns-4,5-P (2 mg/ml) as a carrier, and cellular events should provide critical new insights into numer- 2 the mixture was sonicated in 10 mM Hepes, pH 7.0, 1 mM EGTA for 10 ous biological events such as cell migration and organization min using a bath sonicator. This mixture was then incubated with during organ development and wound healing, tumor cell me- phosphoinositide 3-kinase at 37 °C in the presence of 50 mM [g- P]ATP tastasis, and progression of arterial atherosclerotic plaques. (3000 Ci/mmol), 5 mM MgCl ,50mM Hepes, pH 7.5, for 60 min. The Mutations in the PDGF receptor that eliminate binding of reaction (200 ml) was stopped by the addition of 65 mlof5 N HCl, and lipids were extracted in 400 ml of ChCl /MeOH (1:1). Lipids were dried and stored at 270 °C until needed. [ P]PtdIns-3,4,5-P was quantified * This work was supported in part by National Institutes of Health by thin layer chromatography (n-propanol, 2 M acetic acid extract (65: Grant DK48871. The costs of publication of this article were defrayed in 35)) and radiation detection on a Bio-Rad molecular imager. Based on part by the payment of page charges. This article must therefore be the specific activity of the [g- P]ATP, 20% of the PtdIns-4,5-P was hereby marked “advertisement” in accordance with 18 U.S.C. Section converted to PtdIns-3,4,5-P . 1734 solely to indicate this fact. Preparation of Synthetic PtdIns-3,4,5-P —Dipalmitoyl L-a-phos- § To whom correspondence should be addressed: Div. of Nephrology, phatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C -PtdIns-3,4,5-P ) (21) 16 3 Dana 517, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. and dioctanoyl-L-a-phosphatidyl-D-myo-inositol-3,4,5-trisphosphate Tel.: 617-667-2147; Fax: 617-667-5276. were synthesized as described previously (13, 22). The abbreviations used are: PDGF, platelet-derived growth factor; Chemotaxis Assay—Chemotaxis was evaluated using a modified Boy- HGF, hepatocyte growth factor; PI, phosphoinositide; PtdIns, phos- den chamber assay with a 48-well microchemotaxis chamber as de- phatidylinositol; PtdSer, phosphatidylserine; DAG, diacylglycerol; TPA, 12-O-tetradecanoylphorbol-13-acetate. scribed previously (Neuro Probe Inc., Cabin John, MD) (19, 23). Lipids This paper is available on line at http://www-jbc.stanford.edu/jbc/ 6465 This is an Open Access article under the CC BY license. 6466 PI 3-Kinase Mediates Motility via Protein Kinase C FIG.1. PtdIns-3,4,5-P enhances motility of NIH 3T3 fibroblasts (A and B) and IMCD epithelial cells (C). Cell motility was evaluated using a modified Boyden chamber assay with a 48-well microchemotaxis chamber. A, the lower section of the Boyden chamber was filled with media alone or media containing PDGF, PtdSer/PtdIns-4,5-P lipid substrate, or enzymatically generated PtdIns-3,4,5-P . B and C, either chemically 2 3 PI 3-Kinase Mediates Motility via Protein Kinase C 6467 were dried in a stream of nitrogen and then sonicated for 5 min in serum-free media. The lower section of the Boyden chamber was filled with media alone or media containing either PDGF (10 ng/ml) or PtdSer/PtdIns-4,5-P (100 mM/25 mM) or PtdSer/PtdIns-4,5-P /PtdIns- 2 2 3,4,5-P (100 mM/25 mM/5 mM). A polycarbonate filter (Nucleopore Corp., Pleasanton, CA) coated with rat tail collagen type I (Collaborative Biomedical, Bedford, MA) was placed over the lower compartment, and 1.5 3 10 cells were added to the upper compartment. In some experi- ments, wortmannin was diluted 1:1000 in serum-free media immedi- ately prior to use and added at the appropriate concentration to both the upper and lower chambers at time 0. Control experiments were performed with Me SO vehicle alone. After4hof incubation at 37 °C, filters were removed, the cells were fixed and stained with Diff-Quik (Baxter Healthcare Corp., Miami, FL), and the upper surface was wiped with a cotton applicator to remove nonchemotaxing cells. For each well, cells that had passed through the pores were counted, and the mean value of cells/mm was calculated. Electrophoresis and Western Blotting—Confluent plates of cells were serum-starved overnight in the presence of either 0.3% Me SO or 300 nM TPA followed by a wash with phosphate-buffered saline and lysis in ice-cold lysis buffer (137 mM NaCl, 20 mM Tris, 1 mM MgCl ,1mM CaCl , 10% glycerol, 1% Nonidet P-40, 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, pH 7.5). The suspension was centri- FIG.2. Dose response curve for 3T3 fibroblast chemotaxis to fuged for 10 min at 12,000 3 g. Equal aliquots of supernatant deter- Di-C -PtdIns-3,4,5-P and PtdIns-4,5-P . n 5 4–6 for each point. 16 3 2 mined by protein assay (Bio-Rad) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon (Millipore). Expres- sion of protein kinase Ce was detected by a monoclonal antibody specific of the lipid products. This resulted in conversion of 20% of the for this enzyme (Transduction Laboratories) and quantified using a PtdIns-4,5-P to PtdIns-3,4,5-P to give a final mixture of 100 2 3 Molecular Dynamics PhosphorImager. mM PtdSer, 25 mM PtdIns-4,5-P ,5 mM PtdIns-3,4,5-P . The In Vivo Labeling of Lipids—In vivo levels of D3 phosphoinositides in 2 3 response to stimuli were measured as described previously (8). Briefly, addition of this PtdSer/PtdIns-4,5-P /PtdIns-3,4,5-P mixture 2 3 3T3 cells maintained in Dulbecco’s modified Eagle’s medium, 5% fetal to the bottom well of the chemotaxis chamber resulted in a calf serum were grown to 80% confluence and then placed in Dulbecco’s 10-fold increase in motility of NIH 3T3 fibroblasts compared modified Eagle’s medium, 0.1% fetal calf serum for 12–16 h. For label- with vehicle control and a 3-fold increase compared with the ing purposes, monolayers were placed in phosphate-free Dulbecco’s PtdSer/PtdIns-4,5-P mixture alone (Fig. 1A). The small but modified Eagle’s medium in the absence of serum for 1 h, followed by 2 reproducible motility response to 100 mM PtdSer, 25 mM PtdIns- mCi/ml of [ P]orthophosphate for 3 h. Cells were then stimulated with PDGF (20 ng/ml), Di-C -PtdIns-3,4,5-P , or vehicle control for 10 min. 4,5-P may be due to either activation of PKC isoforms by the 16 3 2 Following stimulation, cells were washed twice with ice-cold phosphate- high concentrations of PtdIns-4,5-P (13) or impurities in these buffered saline and lysed in 750 ml of methanol, 1 M HCl (1:1). 20 mgof lipids not seen when lower concentrations of lipids were inves- crude brain phosphoinositides (Sigma) were added as carrier. Lipids tigated individually (see “Discussion”). were extracted by the addition of 380 ml of chloroform, and the organic To more directly examine the isolated effects of PtdIns-3,4,5- phase was washed twice with 400 ml of methanol, 0.1 M EDTA. Phos- P , we utilized two synthetically prepared sources of PtdIns- pholipids were then deacylated and prepared for Sepharose A ex- change-high pressure liquid chromatography analysis as described 3,4,5-P (Fig. 1, B and C). 5 mM Di-C -PtdIns-3,4,5-P , which 3 16 3 previously (8). forms micelles when sonicated in the absence of carrier lipids, Membrane Ruffling—F-actin was localized in coverslip adherent induced a 7-fold increase in cell motility over base line in 3T3 cells. Quiescent 3T3-fibroblasts or cells exposed to 40 ng/ml of PDGF, 5 cells (control, 10.0 6 1.7 cells/mm , n 5 17; Di-C -PtdIns- mM Di-C -PtdIns-3,4,5-P ,or5 mM PtdIns-4,5-P for 10–60 min were 16 3 2 3,4,5-P , 70.2 6 7.9, n 5 22; Fig. 1B) and a 4-fold increase in fixed by the addition of an equal volume of 3.7% formaldehyde in IMCD cells (control, 7.4.0 6 1.1 cells/mm , n 5 27; Di-C - phosphate-buffered saline at 37 °C for 30 min. Fixed cells were perme- abilized with 0.1 volume of 1% Triton X-100 containing 2 mM tetra- PtdIns-3,4,5-P , 34.5.2 6 2.5, n 5 36; Fig. 1C). When Di-C - 3 16 methylrhodamine B isothiocyanate-phalloidin at 37 °C for 60 min (24), PtdIns-3,4,5-P was added to both compartments of the chemo- washed three times with phosphate-buffered saline for 5 min each, and taxis chamber, a significant but somewhat smaller number of magnified in a Zeiss IM45 Inverted microscope. cells was found to migrate through the pores, indicating an Statistical Analysis—Results were averaged, and statistical rele- increase in both chemokinesis and chemotaxis (control, 8.4 6 vance was determined by Student’s t test. Data are presented as 0.9 cells/mm , n 5 10; Di-C -PtdIns-3,4,5-P on the bottom mean 6 S.E. 16 3 only, 39.0 6 2.6, n 5 11; Di-C -PtdIns-3,4,5-P on both the top 16 3 RESULTS and bottom, 30.8 6 1.9, n 5 12 (p 5 0.016)). Enzymatically Generated and Synthetic PtdIns-3,4,5-P Ini- Di-C -PtdIns-3,4,5-P , a short chain PtdIns-3,4,5-P that is 3 8 3 3 tiate Cell Motility and Ruffling—Since exogenously added lipid soluble as a monomer in water, was also tested in 3T3 cells and vesicles and micelles are known to fuse with the plasma mem- found to initiate chemotaxis although to a lesser extent (con- brane of live cells, we investigated the role of PtdIns-3,4,5-P in trol, 10.0 6 1.7; Di-C -PtdIns-3,4,5-P , 29.4 6 4.2, n 5 27, p 5 3 8 3 cell motility by directly adding this lipid to the cells in a Boyden 0.001; Fig. 1B). There was no chemotactic effect when cells chamber. Three independent preparations of PtdIns-3,4,5-P were exposed to 5 mM PtdIns-4,5-P (3T3 cells, 11.7 6 2.8 3 2 2 2 were employed to evaluate the motility response of this puta- cells/mm , n 5 23; IMCD cells, 4.5 6 1.0 cells/mm , n 5 18) or tive second messenger (Fig. 1). PtdIns-3,4,5-P was enzymati- 50 mM PtdSer (3T3 cells, 17.8 6 3.7 cells/mm , n 5 12; IMCD cally generated by adding purified PI 3-kinase to a 1:4 mixture cells, 10.5 6 1.5 cells/mm , n 5 6). Concentrations of Di-C - of PtdIns-4,5-P and PtdSer followed by chloroform extraction PtdIns-3,4,5-P from 1 nM to 100 mM were evaluated (Fig. 2). 5 2 3 synthesized (dioctanoyl)-PtdIns-3,4,5-trisphosphate (Di-C -PI-3,4,5-P ;5 mM), chemically synthesized (dipalmitoyl)-PtdIns-3,4,5-trisphosphate 8 3 (Di-C -PI-3,4,5-P ;5 mM), commercial PtdIns-4,5-bisphosphate (PI-4,5-P ;5 mM), or PDGF (10 ng/ml) were added to the lower chamber when 16 3 2 indicated. In some experiments, 10 nM wortmannin (wort.) was added. The number of chambers assayed for each condition is indicated by n.*, p , 0.001 compared with PtdIns-4,5-P control. 2 6468 PI 3-Kinase Mediates Motility via Protein Kinase C FIG.3. Membrane ruffling by cells exposed to Di-C -PtdIns-3,4,5-P 16 3 mimics the response seen with PDGF. The top panels show that quies- cent cells and cells exposed to 5 mM PtdIns-4,5-P (PIP ) are no different, 2 2 whereas significant membrane ruffling can be seen in the cells exposed to either 40 ng/ml PDGF or 5 mM PtdIns-3,4,5-P (PIP )at10(middle panels) and 30 min (bottom panels). mM was chosen for further experiments, since this was the lowest dose that consistently resulted in a chemotactic response. The polymerization of cytoplasmic actin that follows receptor stimulation and leads to membrane ruffling and lamellipodia formation is felt to be downstream of the PI 3-kinase (6, 25). To test this hypothesis, we evaluated actin filament reorganiza- tion and membrane ruffling following the addition of Di-C - FIG.4. TPA down-regulates PKCe in 3T3 cells. NIH 3T3 cells PtdIns-3,4,5-P (Fig. 3). The synthetic form of PtdIns-3,4,5-P were treated for 12 h with either vehicle control (2) or 300 nM TPA (1) 3 3 followed by SDS-polyacrylamide gel electrophoresis and immunoblot- stimulated membrane ruffling in 3T3 fibroblasts to the same ting with an antibody specific for PKCe. Densitometric analysis of the extent as PDGF. PtdIns-4,5-P had no effect on quiescent cells. blot revealed 35.9 6 4.6 densitometric units for control PKCe versus Exogenously Added PtdIns-3,4,5-P Does Not Activate En- 8.2 6 0.9 for cells pretreated with TPA (experiment performed in dogenous PI 3-Kinase and Is Not Inhibited by Wortmannin—It triplicate; p 5 0.001). was conceivable that a contaminant or a breakdown product of PtdIns-3,4,5-P might initiate the observed effects via activa- NIH 3T3 cells following the addition of extracellular Di-C - 3 16 tion of a cell surface receptor (as has been shown for lysophos- PtdIns-3,4,5-P . This approach was chosen because the recep- phatidic acid). Although this seemed unlikely, since PtdIns- tors known to initiate chemotaxis (PDGF receptor, insulin re- 3,4,5-P made by three different procedures stimulated cell ceptor, c-met receptor, lysophosphatidic acid receptor) have motility and comparable concentrations of PtdIns-4,5-P and/or also been found to activate the PI 3-kinase (8, 26–28). While PtdSer failed to stimulate cell motility, we searched for evi- stimulation with PDGF produced a dramatic rise in intracel- 32 32 dence that exogenously added PtdIns-3,4,5-P might act via cell lular [ P]PtdIns-3,4-P and [ P]PtdIns-3,4,5-P (2.3- and 17- 3 2 3 surface receptor activation by examining intracellular produc- fold), no increase in either of these lipids was seen in cells 32 32 tion of PtdIns-3,4-P and PtdIns-3,4,5-P in PO -labeled treated with 5 mM Di-C -PtdIns-3,4,5-P . 2 3 4 16 3 PI 3-Kinase Mediates Motility via Protein Kinase C 6469 FIG.5. Down-regulation of PKC in- hibits Di-C -PtdIns-3,4,5-P stimu- 16 3 lated cell motility. A, 16-h pretreatment with 300 nM TPA caused complete inhibi- tion of the motility response to Di-C - PtdIns-3,4,5-P and DAG as compared with vehicle control. PDGF-mediated cell movement was inhibited by 70%. n 5 12. B, 30-min pretreatment of NIH 3T3 cells with 100 nM calphostin C caused a 91% inhibition of Di-C -PtdIns-3,4,5-P -medi- 16 3 ated cell motility. n 5 6. *, p , 0.001 versus control; **, p , 0.01 versus stimulated. The PI 3-kinase inhibitor wortmannin binds irreversibly to DAG, 116.7 6 13.4, p , 0.001). Of note, 5 mM DAG fails to the catalytic subunit of the enzyme and prevents production of induce chemotaxis, while 5 mM Di-C -PtdIns-3,4,5-P does, 16 3 the D3 phosphorylated lipid products of the enzyme. 10 nM indicating that the PtdIns-3,4,5-P effect is not due to hydrol- wortmannin, the lowest dose that produces reliable inhibition ysis to DAG. When DAG and Di-C -PtdIns-3,4,5-P were both 16 3 of the PI 3-kinase in vivo in 3T3 fibroblasts and mIMCD-3 cells present in the bottom well, the chemotactic rate was similar to (19), caused a 60% inhibition of PDGF- and HGF-dependent that seen with DAG alone (Di-C -PtdIns-3,4,5-P , 55.5 6 4.5 16 3 cell motility but had no effect on Di-C -PtdIns-3,4,5-P -stim- cells/m ; DAG, 116.7 6 13.4; Di-C -PtdIns-3,4,5-P with DAG, 16 3 16 3 ulated cell movement in 3T3 cells or in IMCD cells (Fig. 1, B 127.3 6 19.5, n 5 12), suggesting that these two stimuli were and C). These results demonstrate that wortmannin at a dose acting via the same signaling pathway. that inhibits PDGF receptor-mediated activation of the PI 3-ki- To examine this possibility, the TPA-activable PKC family nase does not prevent PtdIns-3,4,5-P -initiated motility, further members were down-regulated by overnight preincubation of supporting the hypothesis that these lipids are inserting into the NIH 3T3 cells with 300 nM TPA. Under these conditions, there membrane and directly initiating downstream signaling events. was a 78% decline in the concentration of PKCe by Western 100 nM wortmannin, a concentration where effects on several analysis (Fig. 4), an effect comparable with that seen in human other kinases have been observed, caused essentially complete dermal fibroblasts (34). PKCe was chosen because it shows the inhibition of both PDGF and Di-C -PtdIns-3,4,5-P -stimulated greatest activation to PtdIns-3,4,5-P in vitro and in vivo. The 16 3 3 cell motility (data not shown). In light of the observation by migratory response to PtdIns-3,4,5-P was completely elimi- Kundra et al. (1) that selective activation of phospholipase Cg nated in NIH 3T3 cells pretreated with TPA (Fig. 5A), while by the PDGF receptor resulted in a substantial chemotactic PDGF-mediated cell movement was inhibited by 70%, a finding response, even in the absence of PI 3-kinase activation, this similar to that seen with exposure to 10 nM wortmannin. In result suggests that other targets of wortmannin that are likely addition, the specific PKC inhibitor calphostin C was tested (35, to be inhibited at the higher concentration, such as myosin 36). NIH 3T3 cells exposed to 100 nM calphostin C (IC 5 75–100 light chain kinase (29) or PtdIns 4-kinase (30), may be critical nM) for 30 min demonstrated a 90% reduction in PtdIns-3,4,5-P - for cell motility as well. mediated cell movement (Fig. 5B). These results suggest that Inhibition of PKC Prevents PtdIns-3,4,5-P -mediated Cell activation of the PI 3-kinase mediates cell motility via the local Motility—It was previously shown that activation of PKC by generation of PtdIns-3,4,5-P and subsequent activation of PKC. DAG or TPA can stimulate chemotaxis (31–33). Therefore, we DISCUSSION examined the role of activation of PKC in PtdIns-3,4,5-P - mediated chemotaxis. 100 mM DAG produced a consistent in- The PI 3-kinase has been clearly implicated in cell motility crease in motility of NIH 3T3 cells (control, 1.3 6 0.2 cells/mm ; by several laboratories (1, 6, 7, 19), yet the actual mechanism of 6470 PI 3-Kinase Mediates Motility via Protein Kinase C REFERENCES this effect is poorly understood. The p85 subunit of the PI 3-kinase has a BCR homology domain, which is capable of 1. Kundra, V., Escobedo, J. A., Kazlauskas, A., Kim, H. K., Rhee, S. G., Williams, L. T., and Zetter, B. R. 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(1995) Cell 81, 727–736

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