From the Molecular and Cellular Pharmacology Program, Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706
Received for publication, November 7, 2002 , and in revised form, April 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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PIP2 is generated from phosphatidylinositol by sequential phosphorylation of the 4- and 5-hydroxyl groups by phosphatidylinositol 4-kinase and phosphatidylinositol phosphate kinases (PIPK), respectively (910). PI 3-kinase (PI3K) utilizes PIP2 as substrate resulting in the production of PI 3,4,5-trisphosphate (PIP3). PIP3 is an activator of the small G-protein Rac leading to membrane ruffle formation. Inhibition of PI3K by both molecular and pharmacological approaches blocks membrane ruffling and inhibits cell migration (11). Thus, PIP2 and PIPK activity are intrinsically linked to membrane ruffling and cell migration, but the mechanism for PIPK involvement has not been defined.
Two PIPK subfamilies, each consisting of ,
, and
isoforms, have been identified and characterized both biochemically and molecularly (10). Type II PIPKs (PIPKIIs) are distinct, in their substrate specificity and function, from the type I PIPK (PIPKI) enzymes. Overexpression of PIPKI, but not PIPKII isoforms, in fibroblasts results in decreased stress fiber formation and formation of highly dynamic actin structures termed actin foci and comets (1214). In fibroblasts and macrophages, overexpression of kinase-dead PIPKI
and PIPKI
mutants do not induce actin foci and comets, indicating that PIP2 production is a necessary for remodeling of the actin cytoskeleton (12, 15).
When overexpressed, all PIPKI isoforms target to the plasma membrane; thus, it has been difficult to define the specific PIPKI isoform involved in membrane ruffling. In contrast, PIPKII are primarily cytosolic upon overexpression. Interestingly, Kunz et al. (16) have shown that the activation loop (amino acids 374396 in PIPKII) is responsible for determination of substrate specificity and subcellular targeting of the overexpressed PIPKs. When the activation loops of the type I and type II PIPKs are swapped, the substrate specificity is also swapped, where a type II PIPK can then utilize PI4P as substrate and localize to the plasma membrane. Interestingly, the type II PIPK with a type I PIPK activation loop has PIPK activity at the plasma membrane but very poorly induces actin remodeling (16, 17). From these studies, simple PIP2 production at the plasma membrane is not sufficient to induce actin remodeling. Therefore, other features of the PIPKI isoforms are required for actin remodeling. There is growing evidence that each type I PIPK isoform has distinct subcellular localization and therefore distinct function. PIPKI
661 is targeted specifically to focal adhesions, modulates talin assembly, and regulates formation of focal adhesions (18). The targeting of PIPKI
661 to focal adhesions provides an example of a mechanism by which PIP2 is generated at specific subcellular sites. Determining the specific PIPKI isoform that is involved in membrane ruffling will provide a mechanism for spatial PIP2 production in regulation of actin dynamics.
Signaling pathways that coordinate changes in the actin cytoskeleton include the Rho family of small G-proteins (1920). In migrating cells, Rac1 and RhoA activities are required for the formation of lamellipodia and membrane ruffles, stress fibers, and focal adhesions, which are each actin-dependent cellular structures that are dynamically reorganized during migration (21). The Rho family of small G-proteins requires stimulation with agonists such as PDGF as well as PIP2 signaling to modulate actin assembly (2224). RhoA, Rac1, and Arf6 have been shown to regulate PIPK activity (2529). Stimulation of cells via G-protein-coupled receptors leads to PIPKI translocation to the plasma membrane and is dependent upon Rac and Rho signaling (30). Interestingly, recent reports have shown that Rac is also able to activate phosphatidylinositol 4-kinase activity, providing substrate to the PIPKIs (31). Therefore, these data suggest that Rac is potentially involved in stimulating PI4P and PIP2 synthesis at the plasma membrane. However, the mechanism by which PIPKI isoforms function in actin remodeling and Rac-dependent membrane ruffling is not well defined.
In this report, the role of PIPKI signaling in the regulation of actin remodeling leading to the formation of actin foci and membrane ruffles is investigated. PIPKI is identified as the specific PIPKI isoform that localizes to membrane ruffles. The results show that following PDGF stimulation, endogenous PIPKI
is targeted to membrane ruffles. In addition, PIPKI
activity coordinates with Rac signaling and is required for the formation of membrane ruffles.
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EXPERIMENTAL PROCEDURES |
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Construction of RacV12K186E MutantsWT Rac in a pGEX 2T prokaryotic expression vector was used as the template for PCR amplification of a mutant Rac fragment. Forward primer GGT GCG GCA CCA CTG TCC CAA CAC and reverse mutagenic primer (EcoRI) GAA TTC TTA CAA CAG CAG GCA TTT TCT CTC CCT TTC C were used. The digested and purified PCR product was then subcloned into each of the Rac pGEX vectors (WT, V12, N17) with NcoI and EcoRI. For construction of the mammalian expression vector, mutant Rac genes were subcloned from the pGEX vector into a Myc-tagged pCMV3b plasmid (Stratagene) using BamHI and EcoRI cloning sites.
Affinity Purification of PIPKI AntibodiesPolyclonal antibodies were generated as described (32, 33). PIPKI antibodies were affinity-purified with denatured PIPKI
immobilized to Sepharose. His-tagged PIPKI
was expressed in E. coli BL21(
DE3) (Novagen) for 3 h and purified with His resin (Novagen) under denaturing conditions according to the manufacturer's protocol.
Cell Culture, Transfection, and Indirect Immunofluorescence MG-63 osteosarcoma fibroblasts (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (Mediatech Inc.) supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics. Transfection and immunofluorescence was performed as described (16). Briefly, PIPKI and Rac were cotransfected at a ratio of 1:3 (PIPKI/Rac) or as indicated with LipofectAMINE Plus (Invitrogen). The cells were then processed for immunofluorescence by fixation in 4% paraformaldehyde, permeabilized, and washed (15). Primary antibodies (PIPKI polyclonal antibodies or anti-Myc 9E10) were diluted in 3% IgG free bovine serum albumin (Jackson Immunoresearch). Secondary antibodies were from Jackson Immunoresearch. Actin was stained with Texas Red-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR). The coverslips were mounted in Vectashield (Vector Laboratories) mounting medium.
Confocal Microscopy and Fluorescence ImagingCells were examined using a x 60 plan oil immersion lens (numerical aperture 1.4) on a Bio-Rad MR 1000 laser-scanning confocal microscope mounted transversely to an inverted Nikon Diaphot 200 (Keck Biological Imaging Laboratory; available on the World Wide Web at keck.bioimaging.wis-c.edu). Images were processed as described (18). Fluorescent images were also captured using a Zeiss Axiovert 135 microscope with a CoolSNAP CCD camera (RS Photometrics) with the CoolSNAP software. Images were then further processed in Photoshop 6.0.
Quantification of Actin RemodelingActin foci formations in cells microinjected with PIPKI were quantified by counting cells as positive for actin foci when large increases in the formation of these actin structures were present compared with control surrounding cells. Cells with few foci (10 or less) were counted as negative. Cells were counted in both the PDGF and +PDGF treatment. The same criteria were followed when counting cells after treatment with LY294002 or Me2SO control. Membrane ruffling was quantified by counting the cells overexpressing PIPKI
compared with control neighboring cells. In all cotransfection experiments, PIPKI
was transfected at a ratio of 1:3 (PIPKI
/Rac) unless otherwise indicated.
PIPKI-overexpressing cells were divided into three categories: cells with no ruffles, peripheral ruffles, and increased membrane ruffles. Peripheral ruffles were defined as cells with a single membrane ruffle around the edge of the cell similar to membrane ruffles induced by expression of dominant active Rac in cells (see example in Fig. 4). Cells with peripheral membrane ruffles were not counted as representing an increase in membrane ruffling. An increase in membrane ruffles was identified as cells with small yet numerous ruffles over the surface of the cell, sometimes forming circular dorsal ruffles. All quantifications were determined from two or three independent experiments. Error bars were determined by S.E. and calculated using Sigma Plot.
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ImmunoblottingMG-63 cell lysates were made by washing the cells with cold phosphate-buffered saline on ice and then lysed in radioimmune precipitation buffer (1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, 2 mM EDTA, pH 7.2, with the addition of protease inhibitors aprotinin, leupeptin, and phenyl-methylsulfonyl fluoride). Protein concentration was determined with the BCA method (Pierce). Lysates were separated on a 10% polyacrylamide denaturing gel and transferred to polyvinylidene difluoride (Millipore Corp.). Chemiluminescent substrate (Kirkegaard & Perry Laboratories) was used for visualization on x-ray film (Eastman Kodak Co.).
MicroinjectionMG-63 fibroblasts were plated on glass bottom dishes (Bioptechs) and grown for 24 h. The cells were serum-starved with serum-free Dulbecco's modified Eagle's medium with 25 mM Hepes for 72 h prior to microinjection. PIPKI constructs were prepared in microinjection buffer (5 mM potassium glutamate and 150 mM KCl) at a final concentration of 50 µg/ml (34). The cells were allowed to express protein for 3 h prior to fixation. For PDGF stimulation, 10 ng/ml PDGF-BB (R & D Systems, Inc.) was added to the cells for 10 min and then fixed as previously described. In experiments with LY294002, 25 µM LY294002 (Sigma) was added to the cells for 30 min, and PDGF was then added for 10 min followed by immunofluorescence. Microinjection of recombinant Rac protein was injected into cells expressing PIPKI cDNA for 3 h. Glutathione S-transferase Rac was purified and prepared as described (35). Rac protein was microinjected at 500 µg/ml with nonspecific mouse IgG at 500 µg/ml, and the cells were allowed to recover for 30 min and then treated as indicated prior to fixation. Microinjections were performed on a Zeiss Axiovert 135 microscope (Zeiss, Thornwood, NY) with an Eppendorf microinjection unit (Micro-injector model 5242, Micromanipulator model 5170 with a heated stage from Bioptechs).
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RESULTS |
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Endogenous PIPKI was detected at membrane ruffles, diffuse in the cytosol, and within the nucleus by immunofluorescent staining. In contrast, PIPKI
was detected primarily in cytosolic vesicular structures that are perinuclear, and staining was not observed in membrane ruffles (Fig. 1C). These results support the hypothesis that endogenous PIPKI
and PIPKI
isoforms generate PIP2 at specific subcellular sites and that PIPKI
may be responsible for synthesis of PIP2 at membrane ruffles.
PIPKI Translocates to Membrane Ruffles following PDGF StimulationPDGF stimulation of serum-starved cells, including MG-63 fibroblasts, resulted in rapid reorganization of actin to form membrane ruffles (3941). Since PIP2 modulates actin dynamics and is reported to be concentrated at membrane ruffles, we investigated the effects of PDGF stimulation on the subcellular localization of endogenous PIPKI
and PIPKI
. MG-63 fibroblasts were serum-starved for 72 h and then stimulated with PDGF-BB (10 ng/ml) to induce membrane ruffling. As shown in Fig. 2, serum-starved MG-63 fibroblasts contain stress fibers and cortical actin bands but lack membrane ruffles. To define endogenous PIPKI localization, cells were fixed and analyzed with and without PDGF stimulation. Only PIPKI
accumulated at PDGF-induced membrane ruffles (Fig. 2A). In the serum-starved cells, PIPKI
is primarily cytosolic and nuclear. Localization of PIPKI
remains unchanged after stimulation (Fig. 2B). Costaining with an antibody to PIP2 (AM212) clearly showed an increase in PIP2 and colocalization with PIPKI
at membrane ruffles upon PDGF stimulation (Fig. 2C). PIP2 has also been detected at membrane ruffles with a phosphatidylinositol 4,5-bisphosphate-specific PH domain of PLC
(36, 42). These data show that endogenous PIPKI
localizes to membrane ruffles and correlates with the localized synthesis of PIP2.
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PIP2 synthesis was inhibited to assess the significance of PIP2 production in membrane ruffling. PIPKI kinase-dead (PIPKI
kn) is able to act as a dominant negative in the inhibition of phagocytosis in macrophages. PIPKI
kn has <1% of wild-type activity but retains its localization at the plasma membrane (15). To determine whether PIPKI
kn was able to inhibit PDGF-stimulated membrane ruffle formation, PIPKI
kn was transiently transfected into MG-63 fibroblasts and expressed for 15 h without serum. The formation of membrane ruffles was stimulated with PDGF for 15 min and analyzed by immunofluorescence to detect actin and PIPKI
kn. PIPKI
kn effectively blocked PDGF-stimulated membrane ruffling (Fig. 3). The data indicate that PIPKI
is responsible for PIP2 synthesis at membrane ruffles and supports a key role for PIP2 in membrane ruffle formation.
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Endogenous PIPKI Translocates to Rac-induced Membrane RufflesMembrane ruffle formation requires the activation of Rac1 and downstream effectors for regulation of actin assembly. Recent studies have also shown that cotransfection of dominant active Rac1 or Arf6 with epitope-tagged murine PIPKI
or PIPKI
, respectively, results in translocation of overexpressed PIPKI to the plasma membrane and to membrane ruffles (29, 30). Together, these data suggest that when overexpressed, both PIPKI isoforms can be targeted to membrane ruffles. We next wanted to define which endogenous PIPKI isoform is targeted to Rac-induced membrane ruffles. Transient overexpression of dominant active Rac1 (Myc-RacL61) in MG-63 fibroblasts results in the formation of membrane ruffles around the cell periphery. Endogenous PIPKI
, but not endogenous PIPKI
, translocated to Rac-induced membrane ruffles (Fig. 4). These data demonstrated that PIPKI
localized to both PDGF- and Rac-induced membrane ruffles.
PIPKI-induced Actin Remodeling Is PDGF-dependentWe next investigated how PIPKI
and PIP2 contributed to membrane ruffle assembly. Previous studies have shown that transient overexpression of all murine PIPKI isoforms induces actin remodeling, forming actin foci and actin comets (1214). In serum-stimulated cells, overexpressing human PIPKI
resulted in similar actin remodeling. In MG-63 cells, these actin structures are primarily foci that localize to the plasma membrane when imaged by confocal microscopy (data not shown). Recent studies have illustrated that hydrolysis of PIP2 results in a decrease in cortical actin dynamics, providing evidence for the importance of PIP2 in inducing actin remodeling (43). The requirement for PIP2 synthesis in the formation of actin remodeling is further supported by overexpression of PIPKI
kn, which decreases membrane ruffle formation and does not induce actin foci or comets (Fig. 3, data not shown).
PDGF stimulation of serum-starved MG-63 cells resulted in translocation of endogenous PIPKI to membrane ruffles. If PIPKI
is required for the formation of ruffles, overexpression of PIPKI
followed by PDGF stimulation could enhance membrane ruffling. To assess this hypothesis, MG-63 cells were serum-starved for 72 h, and then PIPKI
WT expression constructs were microinjected and expressed for 6 h prior to stimulation with 10 ng/ml PDGF-BB for 10 min. The microinjected cells were fixed and stained for PIPKI
and actin. In serum-starved cells, PIPKI
overexpression had little effect on actin organization when compared side-by-side with control noninjected cells (Fig. 5A). In the serum-free condition, 28 ± 8% of microinjected cells overexpressing PIPKI
induced actin remodeling, but upon stimulation with PDGF, 74 ± 11% of microinjected cells underwent massive actin remodeling. Actin remodeling is manifested as a rapid loss of actin stress fibers with a gain of actin foci. Epitope-tagged PIPKI
also relocalized to sites of actin remodeling in PDGF-stimulated cells (Fig. 5A). Overexpression of PIPKI
in serum-starved cells resulted in specific targeting to the plasma membrane but diffuse staining on the membrane, consistent with our previous data (16). Upon stimulation with PDGF PIPKI
reorganized in the plane of the membrane to puncta that partially colocalize with actin foci.
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Different expression levels of PIPKI resulted in similar actin remodeling. Low to moderate expression levels (
35-fold increase) were ideal to observe the subcellular localization of PIPKI
, and these cells showed the largest PDGF-dependent actin remodeling. As would be expected, a fraction of cells expressing high levels of PIPKI
reorganized actin independent of PDGF. PDGF stimulation of PIPKI
-overexpressing cells did not lead to the formation of membrane ruffles. However, PDGF stimulation induced the formation of membrane ruffles in noninjected control cells (Fig. 5A).
To verify that PIP2 was produced at sites of actin remodeling, a GFP-PLC PH domain was used to detect local changes in PIP2 (36, 44). The GFP-PLC
PH domain expression vector was coexpressed with PIPKI
by microinjection. In the serum-free condition, the PLC
PH domain is primarily cytosolic, consistent with previous reports, whereas PIPKI
is predominantly plasma membrane-associated when overexpressed (16, 44). Upon stimulation with PDGF, the PLC
PH domain rapidly reorganized and colocalized with PIPKI
at actin foci (Fig. 5B). These data indicate that PIP2 is generated upon PDGF stimulation at sites where both PIPKI
WT and actin are assembled. The observation that PIPKI
-overexpressing cells did not ruffle initiated our hypothesis that other signaling partners are required to coordinate PIPKI
-induced actin remodeling, leading to membrane ruffle formation.
PIPKI-induced Actin Remodeling Is Independent of PI3K To explore the role of other signaling molecules in actin foci formation, the role of PI3K was investigated. PI3K is well characterized in PDGF signaling. PI3K utilizes PIP2 as a substrate to form PIP3, thus regulating actin remodeling by activation of the small G-protein Rac1, leading to membrane ruffle assembly (11). RacGEFs bind PIP3 and activate GDP-bound Rac by exchanging GDP for GTP (4547). First, to determine whether PI3K activity is required for PIPKI
-induced actin remodeling, PIPKI
was microinjected into serum-starved MG-63 cells as described above. Prior to PDGF stimulation, cells were treated with 25 µM LY294002, a potent and selective PI3K inhibitor, and then stimulated with PDGF (48). With inhibition of PI3K, 64 ± 5% of PIPKI
-overexpressing cells form actin foci (Fig. 6, A and B). These data demonstrate that PI3K inhibition does not have a significant effect on PIPKI
-induced actin foci. Importantly, inhibition of PI3K completely blocked PDGF-stimulated membrane ruffling on adjacent noninjected cells, illustrating that LY294002 was effective at inhibiting PI3K as reported previously (Fig. 6A) (49). These data support a model where PIPKI
and the localized production of PIP2 induce actin remodeling independent of PI3K. In support of direct modulation by PIP2, we used a PLC inhibitor, U73122
[GenBank]
, to block PIP2 hydrolysis (44). With this inhibitor, the extent of actin foci generation by PIPKI
and PDGF stimulation was enhanced, supporting PIP2 modulation of actin foci assembly (data not shown).
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Rac and PIPKI Signaling Pathways Are Both Required for Membrane RufflingAs illustrated above, ectopic expression of PIPKI
induced actin remodeling upon PDGF stimulation. However, cells overexpressing PIPKI
were not able to form membrane ruffles in response to PDGF. These data demonstrate that PIPKI
alone is not sufficient to form membrane ruffles, suggesting that additional signaling partners are required to induce membrane ruffle formation.
The formation of membrane ruffles requires the regulated remodeling of actin filaments and the localized activation of Rac (2). PIPKI associates with Rac, resulting in enhanced PIPKI
activity (26). Potentially, both Rac and PIPKI
activities are required for the coordination of actin remodeling, resulting in membrane ruffle assembly. This was investigated using two approaches. First, PIPKI
expression vectors were microinjected into serum-starved MG-63 fibroblasts as described previously, followed by microinjection of recombinant RacV12 protein, a dominant active form of Rac (35). These cells were then stimulated with PDGF. As above, overexpression of PIPKI
alone resulted in the formation of actin foci and lacked membrane ruffles in response to PDGF stimulation (Fig. 7). Control cells microinjected with RacV12 alone resulted in the formation of membrane ruffles around the cell periphery (21). Strikingly, when recombinant RacV12 protein was microinjected into PIPKI
-expressing cells, these cells had an increase in membrane ruffles compared with control cells microinjected with RacV12 alone (Fig. 7, PIPKI
+ RacV12). Both cells as indicated expressed both PIPKI
and RacV12; however, the expression levels of PIPKI
WT in the cell on the right were 4-fold higher than in the cell on the left. In addition, the cell on the left is overexpressing PIPKI
WT 3-fold higher than endogenous PIPKI
. The relative expression levels were determined by quantification of pixel intensity (data not shown). Furthermore, overexpressed PIPKI
is localized to membrane ruffles (Fig. 7, Merge). When cells expressing both PIPKI
and dominant active Rac are imaged in real time, dynamic membrane ruffles over the cell surface are observed (data not shown).
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To confirm that PIPKI activity is necessary for RacV12-induced membrane ruffles, PIPKI
kn expression vector was microinjected and expressed for 2 h followed by injection of RacV12. Expression of PIPKI
kn alone did not induce actin remodeling compared with control surrounding cells (Fig. 7, PIPKI
kn). However, the expression of PIPKI
kn followed by injection of recombinant RacV12 (Fig. 7, PIPKI
kn + RacV12) blocked RacV12-induced ruffles in a PIPKI
kn dose-dependent manner (data not shown). The combined data support a synergism for PIPKI
and Rac in membrane ruffling stimulated by PDGF.
To further establish a role for both PIPKI and Rac in membrane ruffle assembly, a transient transfection assay was used to confirm the microinjection phenotypes and to quantify membrane ruffling induced by coexpression of PIPKI
and Rac1. In these studies, the dominant active RacL61 construct was used. RacL61 has been used interchangeably with RacV12, and in parallel studies these dominant Rac mutants induce indistinguishable phenotypes (50). In this assay, MG-63 cells were cotransfected with expression constructs at a ratio of 1:3 (PIPKI
/Rac) and then serum-starved for 20 to 24 h. These cells were stimulated with 10 ng/ml PDGF for 10 min and then stained for actin and PIPKI
. PIPKI
or Rac were also transiently expressed alone, and the actin phenotypes were assessed following PDGF stimulation (Fig. 8A). Control cells expressing RacL61 alone resulted in cells with modest membrane ruffles around the cell periphery with and without PDGF stimulation. With PDGF stimulation, 10 ± 2% of control RacL61-transfected cells showed an increased membrane ruffling (Fig. 8C). Transient overexpression of PIPKI
alone consistently induced actin foci, and the cells possessed an elongated morphology (Fig. 8, A and C).
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Dominant active RacL61 was coexpressed with PIPKI to determine whether both activities are required for increased membrane ruffling. When RacL61 was cotransfected with PIPKI
, the cell morphology was vividly transformed compared with nontransfected and control cells with PIPKI
or RacL61 alone (Fig. 8). Coexpression of RacL61 and PIPKI
caused transfected cells to become spread with dramatic membrane ruffles (Fig. 8B). Cells expressing both PIPKI
and RacL61 had phenotypes similar to cells in the above microinjection experiments with PIPKI
and RacV12, except the increased membrane ruffles were more pronounced (Fig. 8B). Before PDGF stimulation, 12% of PIPKI
and RacL61 cotransfected cells showed increased membrane ruffling, whereas PDGF stimulation resulted in a 2-fold increase in the percentage of cotransfected cells with increased membrane ruffling shown in Fig. 8B and quantified in Fig. 9B. Quantification of membrane ruffles is described under "Experimental Procedures."
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The enhancement of membrane ruffling was further investigated by cotransfecting constant amounts of PIPKI with increasing RacL61 concentrations at ratios of PIPKI
to RacL61 of 1:1, 1:2, and 1:4. The number of transfected cells with enhanced membrane ruffling rose as the ratio of RacL61 was increased. Consistent with this observation, the transfected cells with membrane ruffles increased, and the number of cotransfected cells with actin foci decreased with a greater ratio of active Rac to PIPKI
(Fig. 8C). Thus, activated Rac in a dose-dependent fashion induced formation of membrane ruffles and a loss of actin foci in PIPKI
-expressing cells.
As Rac modulates membrane ruffling its role in the formation of actin foci was investigated. PIPKI was microinjected into serum-starved MG-63 cells and expressed for 2 h. Recombinant, dominant negative Rac (RacN17) purified from E. coli was microinjected into PIPKI
-overexpressing cells. After 30 min, the cells were stimulated with PDGF and fixed for immunofluorescence. Actin foci induced by PDGF and PIPKI
were unaffected by dominant negative RacN17. The lack of an effect of RacN17 on PIPKI
-induced actin foci was repeated and verified by transient transfection with similar results as obtained by microinjection (Fig. 8B). Control cells with RacN17 alone had a decreased number of membrane ruffles and showed an increase in actin foci (Fig. 8A). These data demonstrate that PIPKI
directly induced actin foci independent of Rac signaling.
PIPKI interacts with Rac1 in both GDP- and GTP-bound states (26). This was elegantly mapped in Rac to a region outside of the effector domain in the C terminus, and mutagenesis of this region resulted in a diminished interaction with PIPKI (26, 51). Since an interaction between Rac1 and PIPKI
may facilitate membrane ruffle formation, we tested this requirement using the Rac loss of interaction mutant (RacV12K186E). Expression of RacV12K186E alone with PDGF stimulation did induce modest ruffles (Fig. 8A). PDGF stimulation of PIPKI
- and RacV12K186E-coexpressing cells resulted in clearly diminished membrane ruffle formation compared with dominant active RacL61 (Fig. 8B). Many RacV12K186E-expressing cells contained actin foci with few membrane ruffles. These data confirm that the association of PIPKI
with activated Rac is important for efficient formation of membrane ruffles.
PIPKI kn and RacL61 were coexpressed to assess PIP2 synthesis requirements in enhanced membrane ruffling following PDGF stimulation. PIPKI
kn and RacL61 coexpression had little effect on Rac induced membrane ruffles; however, there was a dramatic inhibition of the enhanced membrane ruffling phenotype (compare Fig. 8B with Fig. 9A). The transient transfection data differ slightly from the microinjection experiments, where microinjection resulted in a complete loss of membrane ruffling, whereas transient transfection only inhibited the enhanced membrane ruffling. For PIPKI
kn to induce dominant negative effects, it must be present in excess concentrations compared with wild-type PIPKI
. In microinjection experiments, PIPKI
kn inhibited membrane ruffles in a dose-dependent manner, which also required RacV12 to be present at low levels (data not shown). The presence of membrane ruffles in the transfection experiments may be due to prolonged and elevated RacL61 expression, or remaining ruffles could result from pathways independent of PIPKI
, as addressed below.
PIPKI may facilitate parallel pathways by increasing local PIP2 levels in coordination with local increases in PIP3. PI3K is also required for membrane ruffling and participates in Rac activation (11). PDGF may activate endogenous PI3K and Rac1, contributing to membrane ruffle formation. To determine whether endogenous PI3K played a role in enhanced membrane ruffling by PIPKI
and Rac1, PIPKI
- and RacL61-cotransfected cells were treated with 25 µM LY294002. In MG-63 cells, the highly specific PI3K inhibitor LY294002 potently inhibited PDGF-induced ruffling (data not shown) (49). LY294002-treated cells expressing RacL61 alone had no change in ruffling (Fig. 9A). This is consistent with Rac being downstream of PI3K (11). Thus, inhibition of PI3K does not affect membrane ruffling induced by dominant active Rac. The effects of PIPKI
kn and LY294002 on membrane ruffling were quantified by counting transfected cells that possessed enhanced membrane ruffles as positive. LY294002 treatment resulted in a 2-fold decrease in the percentage of PIPKI
WT- and RacL61-cotransfected cells that have enhanced ruffling after PDGF stimulation. Inhibition of enhanced membrane ruffles with LY294002 resulted in the same level of inhibition as PIPKI
kn when coexpressed with RacL61 (Fig. 9C). The cellular morphology of PIPKI
WT- and RacL61-cotransfected cells treated with LY294002 versus cells cotransfected with PIPKI
kn and RacL61 are similar, where a significant number of cells contain ruffles but the percentage of transfected cells with enhanced membrane ruffling is decreased as illustrated in Fig. 9C. Finally, when PIPKI
kn and RacL61 were coexpressed and treated with LY294002, there was a nearly complete block of the enhanced membrane ruffle formation (Fig. 9C). These data demonstrate that PIPKI
, Rac1, and PI3K are functioning in concert in the formation of membrane ruffles. In addition, these data support a direct role for PIP2 as a lipid second messenger that directly modulates actin remodeling and serves as a substrate for PI3K, facilitating Rac signaling during the formation of membrane ruffles.
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DISCUSSION |
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We propose the following model. PDGF stimulation increases local PIP2 synthesis, specifically through PIPKI. The increased PIP2 induces actin remodeling, resulting in actin foci and actin comets. PIP2 may facilitate actin remodeling by uncapping barbed ends and stimulating de novo actin polymerization through the Arp2/3 complex. In addition to local rises in PIP2 following PDGF stimulation, PIP3 levels increase activating Rac. Subsequent activation of Rac effectors induces actin remodeling and actin assembly necessary for membrane ruffling. Thus, simultaneous and synchronized activation of PIPKI
and Rac signaling appear required for membrane ruffle formation.
For PIP2 to act as a second messenger, its localized synthesis must be regulated. Many PIP2 effectors are located at diverse cellular compartments. This suggests that PIP2 synthesis may be modulated in these cellular compartments. PIP2 plays a role in distinct cellular processes including the regulation of ion channels, vesicular trafficking, and nuclear events and the modulation of the actin cytoskeleton (10, 52). All of these events are likely to be modulated by spatial and temporal PIP2 synthesis. A complex cadre of proteins that facilitate actin filament polymerization, depolymerization, severing, and targeting controls assembly of actin in a migratory cell. The majority of these proteins are PIP2 effectors, and their regulation by PIP2 may be conserved in all eukaryotes. In yeast, the PIPKI homologue Mss4 generates PIP2, and actin assembly is modulated by PIP2 and by the enzymes that metabolize PIP2 (5355). In mammalian cells, PIP2 modulates actin assembly, and this is illustrated by the PIPKI initiation of actin remodeling. The specific roles played by each of the PIPKI isoforms in actin assembly have remained vague. Nevertheless, in both yeast and mammals, PIP2 has been proposed to function as a dynamic second messenger undergoing spatial and temporal concentration changes.
The ability of the PIPKI isoforms to regulate spatial PIP2 production occurs by two mechanisms. First, the difference in substrate specificity between the PIPKI and PIPKII enzymes in part determines their subcellular targeting (16). Second, association of PIPKI with targeting molecules at the plasma membrane and in other compartments is critical. One example is PIPKI 661 localization to focal adhesions (18). PIPKI
661 is targeted to focal adhesions by an association with talin. PIPKI
661 activity is increased specifically at focal adhesions by focal adhesion kinase signaling and regulates focal adhesion dynamics. Here we have shown that PIPKI
localizes to membrane ruffles, whereas the highly homologous PIPKI
targets to vesicular structures in the perinuclear region. The requirement for PIP2 synthesis at sites of dynamic actin reorganization is also demonstrated by the recruitment of PIPKI
and the production of PIP2 at the phagocytic cup, an actin-dependent structure formed during macrophage phagocytosis (56). Furthermore, inhibition of kinase activity in macrophages results in a significant decrease in phagocytosis (15). This is consistent with our data, in which inhibition of PIP2 by PIPKI
kn blocks membrane ruffling. Together, these data support a model in which PIPKI
regulates actin dynamics by local production of PIP2.
Actin assembly and membrane ruffle formation requires multiple phosphoinositides. Phosphatidylinositol 4-kinase produces PI4P, which serves as substrate for PIPKIs, and then PI3K utilizes PIP2 to produce PIP3. Rac has also been shown to be important in regulating the production of both PIP2 and, recently, PI4P (31). One function of PI3K is to activate Rac (11, 57). Activated Rac has been shown to be concentrated in membrane ruffles (58). PI3K also participates in PDGF-stimulated membrane ruffling (22). PI3K and Rac are not required for the assembly of actin foci by PIPKI and PIP2 but are required for membrane ruffling. Inhibition of either PIPKI
or PI3K results in partial inhibition of Rac-induced ruffling. However, a loss of both PIPKI
and PI3K activity blocks ruffling, supporting a role for both phosphoinositide kinases in ruffle formation. These data support a model where PI3K signaling and Rac signaling coordinate the actin assembly induced by PIPKI
. Rac induces both PI4P and PIP2 production at the plasma membrane. PIPKI
induces actin foci assembly in the absence of both PI3K and Rac, but PI3K and Rac are required to coordinate the actin assembly such that membrane ruffles are formed. This cooperation between PIPKI
, PI3K, and Rac is consistent with data demonstrating that both PIPKI and PI3K activities associate with Rac. Recently, Tolias et al. (26) showed that PIPKI enhances the formation of actin barbed ends in platelet extracts that were induced by Rac. Rac-dependent actin assembly is also blocked by the addition of PIPKI kinase negative. These data provide biochemical evidence for the coordination of Rac and PIPKI in the induction of actin assembly (26). This is also consistent with the role of PIPKI in lower eukaryotes. In yeast, loss of Mss4 results in a disorganized actin cytoskeleton. The Mss4 null phenotype is rescued by the small G-protein Rho2 (54). These data are consistent with a conserved role for both PIPKI isoforms and small G-proteins in coordination of actin dynamics in eukaryotic cells.
Small G-proteins have been shown to regulate type I PIPKs. Rac, Rho, and Arf6 have all been shown to stimulate PIPKI activity in vitro (2530). Coexpression of Arf6 and murine PIPKI (human PIPKI
) results in the formation of membrane ruffles in HeLa cells (29). In our system, dominant active Arf6 is unable to induce membrane ruffles upon overexpression and coexpression with PIPKI
(data not shown). Arf6-induced membrane ruffles may be specific only for human PIPKI
and not for PIPKI
.
Deciphering the role of phosphoinositide signaling in actin remodeling and modification of other signaling pathways will provide insight into the mechanism of cell migration and other cellular events requiring the actin cytoskeleton. Understanding the role of each PIPKI isoform and potentially splice variants will begin to unravel the mechanism of spatial and temporal PIP2 synthesis. Identification of the signaling molecules and effectors that interact specifically with PIPKI will provide further understanding of how PIPKI regulates actin remodeling at the leading edge.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Pharmacology, 1300 University Ave., University of Wisconsin Medical School, Madison, WI 53706. Tel.: 608-262-3753; Fax: 608-262-1257; E-mail: raanders{at}facstaff.wisc.edu.
1 The abbreviations used are: PIP2, phosphatidylinositol 4,5-bisphosphate; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PIPK, phosphatidylinositol phosphate kinase; PIPKI, type I phosphatidylinositol phosphate kinase; PIPKII, type II phosphatidylinositol phosphate kinase; PDGF, platelet-derived growth factor; kn, kinase-negative; GFP, green fluorescent protein; PH, pleckstrin homology; PLC, phospholipase C.
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ACKNOWLEDGMENTS |
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REFERENCES |
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