©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rho Family GTPases Bind to Phosphoinositide Kinases (*)

(Received for publication, April 7, 1995; and in revised form, June 6, 1995 )

Kimberley F. Tolias (1) (3)(§) Lewis C. Cantley (1) (3) Christopher L. Carpenter (3) (2)

From the  (1)Departments of Cell Biology and (2)Medicine, Harvard Medical School and the (3)Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Rho family GTPases appear to play an important role in the regulation of the actin cytoskeleton, but the mechanism of regulation is unknown. Since phosphoinositide 3-kinase and phosphatidylinositol 4,5-bisphosphate have also been implicated in actin reorganization, we investigated the possibility that Rho family members interact with phosphoinositide kinases. We found that both GTP- and GDP-bound Rac1 associate with phosphatidylinositol-4-phosphate 5-kinase in vitro and in vivo. Phosphoinositide 3-kinase also bound to Rac1 and Cdc42Hs, and these interactions were GTP-dependent. Stimulation of Swiss 3T3 cells with platelet-derived growth factor induced the association of PI 3-kinase with Rac in immunoprecipitates. PI 3-kinase activity was also detected in Cdc42 immunoprecipitates from COS7 cells. These results suggest that phosphoinositide kinases are involved in Rho family signal transduction pathways and raise the possibility that the effects of Rho family members on the actin cytoskeleton are mediated in part by phosphoinositide kinases.


INTRODUCTION

The actin cytoskeleton plays a critical role in a number of cellular processes including motility, chemotaxis, and cell division (1, 2, 3, 4) . To function properly, these processes require precise spatial and temporal control of actin filament organization and assembly. Members of the Rho family of small GTP-binding proteins have been implicated in this regulation. Rac1 appears to mediate growth factor-induced membrane ruffling caused by actin reorganization at the plasma membrane, and RhoA promotes the formation of actin stress fibers and focal adhesions(5, 6) . Cdc42Sc has also been shown to control actin organization necessary for bud site assembly in Saccharomyces cerevisiae(7, 8) . The mechanism by which Rho family members mediate their effects on the actin cytoskeleton is unclear. Two potential biochemical targets for Rho family members have been identified. Manser et al.(9) demonstrated that the tyrosine kinase p120 complexes specifically with GTP-bound Cdc42Hs in vitro. Similarly, the serine/threonine kinase p65 binds to both Cdc42 and Rac1 in a GTP-dependent manner in vitro(10) . These proteins, however, have not been shown to associate with Rho family members in vivo, and a connection between these kinases and the actin cytoskeleton has not yet been established.

Phosphoinositide kinases and their products have also been implicated in the regulation of the cytoskeleton. Studies using platelet-derived growth factor (PDGF)()receptor mutants and the PI 3-kinase inhibitor wortmannin have demonstrated that PI 3-kinase is involved in growth factor-induced membrane ruffling (11, 12, 13) . In addition, PtdIns-4,5-P, the product of PtdIns-4-P 5-kinase, has been shown in vitro to bind actin regulatory proteins such as profilin and gelsolin and promote actin filament polymerization(2) .

Since both Rho family members and phosphoinositide kinases are involved in the regulation of the actin cytoskeleton, it is possible that these proteins are acting in the same signaling pathway. Evidence for a connection between Rho family members and phosphoinositide kinases is accumulating in the literature. For instance, Cdc42 has recently been shown to bind and weakly activate PI 3-kinase in vitro(14) . In addition, Rho may regulate PI 3-kinase in platelet lysates since C3 ADP-ribosyltransferase, a specific inhibitor of Rho, partially blocks GTPS stimulation of PI 3-kinase(15) . GTP-bound Rho also appears to enhance PtdIns-4,5-P synthesis in fibroblasts(16) . However, to our knowledge there is no evidence in the literature for binding of Rho family members to any enzymatic activity in vivo. We have therefore investigated the possibility that Rho family members interact specifically with phosphoinositide kinases in vivo and in vitro.


EXPERIMENTAL PROCEDURES

Materials

Antibodies against human Rac1 and Cdc42 were obtained from Santa Cruz Biotechnology, Inc. Phosphoinositides and glutathione-Sepharose 4B beads were purchased from Sigma. [-P]ATP was purchased from DuPont NEN.

Preparation and Nucleotide Loading of GST Fusion Proteins

Glutathione S-transferase fusion proteins of Rac1, RhoA, and Cdc42 (kindly provided by Dr. Larry Feig, Department of Biochemistry, Tufts University) were expressed in bacteria and purified with glutathione-Sepharose beads as described previously(17) . The fusion proteins were stored in 5 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM DTT with 50% glycerol at -70 °C. The proteins were determined to be active based on their ability to bind GTP in a filter binding assay (5) . The fusion proteins were loaded with nucleotide by incubating proteins in 20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT with a 10-fold excess of either GTPS or GDPS for 15 min at 30 °C. Following incubation, MgCl was added to a final concentration of 5 mM.

Association of Phosphoinositide Kinase Activity from Rat Liver Homogenate and Cell Lysate with Rho Family Members

Rat1 cells maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum were washed twice with PBS and lysed in lysis buffer (50 mM Hepes (pH 7.4), 150 mM NaCl, 5 mM MgCl, 1% Nonidet P-40, 10% glycerol, 1 mM DTT, 1 µg/ml each of leupeptin, [4-(2-aminoethyl)-benzenesulfonyl fluoride, HCl], and pepstatin, 0.5 mM ZnCl, and 0.5 mM orthovanadate). Crude cell extract was clarified by centrifugation (14,000 rpm for 10 min). Rat liver homogenate was prepared essentially as described(18) . Bovine serum albumin was added to both rat liver homogenate and fibroblast lysate at a final concentration of 2 mg/ml.

GST and GST fusion proteins (10 µg of each) were loaded with nucleotide as described above and then incubated with homogenate or fibroblast lysate for 2 h at 4 °C with constant rocking. The beads were washed twice with 1-ml volumes of ice-cold PBS, 1% Nonidet P-40 and twice with 1 ml of TNM (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 5 mM MgCl). Lipid kinase assays were then performed on the beads using PtdIns-4-P or crude brain phosphoinositides as lipid substrates as described previously(18) . Lipids were extracted and separated by thin layer chromatography(19, 20) . Phosphoinositides were visualized by autoradiography and quantitated using a molecular imager (Bio-Rad). The effects of spermine and phosphatidic acid on PtdIns-P kinase activities were assayed as described(21, 22) . For the determination of the effect of spermine on PtdIns-P kinase activities, the concentration of MgCl was decreased to 2 mM.

High Pressure Liquid Chromatography Analysis

The PtdIns-P kinase associated with GST-Rac1 was assayed as described above. The product was separated by thin layer chromatography and then deacylated and analyzed by HPLC as described(18) . Standards were made from tritiated PtdIns-4-P and PtdIns-4,5-P (DuPont NEN), which were deacylated and included with the P-labeled product in the HPLC run.

Immunoprecipitation Experiments

For growth factor stimulation experiments, confluent Swiss 3T3 cells were serum-starved with Dulbecco's modified Eagle's medium plus 0.1% fetal calf serum for 24 h and stimulated with 40 ng/ml PDGF for 10 min. Cells were lysed and clarified as described above, and Cdc42 or Rac was immunoprecipitated(23) . In some cases, antibodies were preincubated with 10-fold (by weight) excess peptide antigen for 2 h at 25 °C. The immune complexes were washed twice with 1-ml volumes of ice-cold PBS, 1% Nonidet P-40 and twice with 1-ml volumes of TNM (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 5 mM MgCl). After washes, selected samples were incubated with 100 nM wortmannin for 20 min at 25 °C. PI kinase assays were then performed on the immunoprecipitates and analyzed as described above.


RESULTS AND DISCUSSION

Association of Rho Family Members with Phosphoinositide Kinases in Vitro

To determine whether phosphoinositide kinases might directly associate with Rho family GTPases, we used GST fusion proteins of Rac1, RhoA, and Cdc42 expressed in bacteria. The purified proteins, bound to GSH beads, were incubated with rat liver cytosol and assayed for PI kinase activities using crude brain phosphoinositides. As shown in Fig. 1, a PtdIns-P kinase activity associated with both the GTP- and GDP-bound forms of Rac1 (lanes2 and 3), resulting in the synthesis of phosphatidylinositol bisphosphate. A small amount of PtdIns-P kinase activity also associated with RhoA (lanes4 and 5) and Cdc42 (lanes6 and 7). The PtdIns-P kinase activity complexed with Rho and Cdc42 was 10- and 15-fold less, respectively, than that associated with Rac, as quantified by a molecular imager. Negligible activity was found in the GST control (lane1). Recently, Rho has been shown to stimulate PtdIns-4,5-P synthesis in fibroblast cells(16) . Since we do not detect a significant association between Rho and a PtdIns-P kinase, our results suggest that Rho stimulates PtdIns-P kinase activity indirectly.


Figure 1: Association of PI kinases with members of the Rho family of small G proteins. GST fusion proteins of Rac1, RhoA, Cdc42, or GST only bound to glutathione-Sepharose beads were loaded with GTPS or GDPS and then incubated with rat liver cytosol. The beads were washed and assayed for PI kinase activities using crude brain phosphoinositides as described under ``Experimental Procedures.'' P-Labeled phosphoinositides generated by purified PI 3-kinase (PI3K) are included as standards (lane8). The migration positions of the phosphoinositide standards are indicated. PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol trisphosphate.



In contrast to the association of PtdIns-P kinase with Rac1, PI 3-kinase associated with both Rac1 and Cdc42 in a GTPdependent fashion (Fig. 1, lanes2 and 6). The presence of PI 3-kinase is indicated by the synthesis of PtdIns-P in the phosphoinositide kinase assays. Again, a negligible amount of PI 3-kinase bound to GST alone or to RhoA. Similar results were obtained when the association experiments were done using Rat 1a fibroblast lysates as the source of PI kinases (data not shown). We also detected a GTP-dependent association of Rac and Cdc42 with PI 3-kinase by Western blotting proteins associated with GST fusions of Rac, Rho, and Cdc42 with p85 antibodies (data not shown).

Characterization of the Rac-associated PtdIns-P Kinase

The two known isomers of PtdIns-P, PtdIns-3,4-P and PtdIns-4,5-P, are synthesized by phosphorylation of PtdIns-4-P by PI-3 kinase and PtdIns-4-P 5-kinase, respectively. To determine the type of PtdIns-P kinase that associates with Rac, the PtdIns-P product was deacylated and analyzed by HPLC. The product was identified as PtdIns-4,5-P based on comigration with a H-labeled PtdIns-4,5-P standard (Fig. 2). The Rac-associated PtdIns-P kinase is therefore a PtdIns-4-P 5-kinase. At least two forms of PtdIns-4-P 5-kinase exist in mammalian cells. The best characterized form is a 53-kDa protein, termed type II, that has been purified to homogeneity from human red blood cell membranes and cytosol and from bovine brain cytosol(21, 24, 25) . A second class, termed type I, is found in most tissues(21, 22) . The two enzymes can be distinguished by the ability of spermine and phosphatidic acid to stimulate type I PtdIns-4-P 5-kinases (but not type II)(21, 22) . When the effects of these compounds were tested on the Rac-associated PtdIns-4-P 5-kinase, we found that the PtdIns-4-P 5-kinase was significantly stimulated by both spermine and phosphatidic acid (17- and 18-fold, respectively), indicating that it is a member of the type I family of PtdIns-4-P 5-kinases (Fig. 3).


Figure 2: The Rac1-associated PtdIns-P kinase is a PtdIns-4-P 5-kinase. GST-Rac1 bound to glutathione-Sepharose beads was loaded with GTPS and incubated with Rat 1a cell lysate. The product of the associated PtdIns-P kinase was deacylated and then analyzed by HPLC. Migration positions of glycero-PtdIns-3,4-P, glycero-PtdIns-4,5-P, and inositol (Ins)-1,4,5-P standards are indicated.




Figure 3: Characterization of the Rac-associated PtdIns-4-P 5-kinase. GST-Rac1 loaded with GTPS or GST alone was incubated with Rat-1a cell lysates, washed, and assayed for PtdIns-P (PIP) kinase activity with or without 2 mM spermine or 80 µM phosphatidic acid (PA) as described under ``Experimental Procedures.'' A representative experiment from three independent experiments is presented.



Association of Phosphoinositide Kinases with Rac Immunoprecipitates

To determine whether the complexes that form between Rac and phosphoinositide kinases in vitro also occur in vivo, Rac1 immunoprecipitates were assayed for PI kinase activities. Rac immunoprecipitates from serum-starved Swiss 3T3 cells contained a significant amount of PtdIns-4-P 5-kinase activity (Fig. 4A, lane2). PDGF stimulation did not noticeably affect this activity (Fig. 4A, lane 3). The associated PtdIns-P kinase was identified as a type I PtdIns-4-P 5-kinase based on its activation by spermine and phosphatidic acid, consistent with it being the same enzyme that complexed with recombinant Rac (data not shown). The presence of the PtdIns-4-P 5-kinase in Rac immunoprecipitates was blocked by preincubating Rac antibodies with the peptide antigen, indicating that the interaction is specific (Fig. 4A, lane4). Negligible PI kinase activity was detected in control immunoprecipitates with non-immune serum (Fig. 4A, lane1).


Figure 4: Association of PtdIns-4-P 5-kinase and PI 3-kinase with Rac1 immunoprecipitates. A, Rac1 was immunoprecipitated from quiescent (lane2) or PDGF-stimulated (lane3) Swiss 3T3 cells and assayed for PtdIns-P (PIP) kinase activity as described under ``Experimental Procedures.'' Immunoprecipitations using non-immune (NI) serum and Rac1 antibody pretreated with the peptide antigen were done as negative controls (lanes1 and 4, respectively). PIP, phosphatidylinositol bisphosphate. B, Rac1 was immunoprecipitated from quiescent (lane2) and PDGF-stimulated (lane3) Swiss 3T3 cells and assayed for associating PI 3-kinase activity. Control immunoprecipitations were performed as described in A. To confirm the presence of PI 3-kinase, Rac immunoprecipitates from PDGF-stimulated cells were treated with wortmannin (Wort., lane5).



In contrast to the PtdIns-4-P 5-kinase, no significant PI 3-kinase activity was detected in Rac immunoprecipitates from serum-starved cells (Fig. 4B, lane2). Addition of PDGF to cells induced the association of PI 3-kinase with Rac immunoprecipitates (Fig. 4B, lane3). The presence of PI 3-kinase activity was blocked by preincubating Rac antibodies with the peptide antigen (Fig. 4B, lane4). Treatment of Rac immunoprecipitates from PDGF-stimulated cells with wortmannin abolished the formation of PtdIns-P (Fig. 4B, lane 5), confirming the presence of PI 3-kinase in the immunoprecipitates. Negligible PI kinase activity was detected in the control immunoprecipitate with non-immune serum (Fig. 4B, lane1). These results demonstrate that Rac interacts specifically with both PtdIns-4-P 5-kinase and PI 3-kinase in vivo and that the PI 3-kinase association is regulated by PDGF.

Only a small fraction of the total PtdIns-4-P 5-kinase and PI 3-kinase in Swiss 3T3 cells is capable of binding to Rac. At saturating concentrations of GST-Rac, about 0.3% of the total PtdIns-4-P 5-kinase activity and 0.6% of total PI 3-kinase activity associate with Rac (not shown). Approximately one-third of the PtdIns-4-P 5-kinase activity and one-half of the PI 3-kinase activity that binds to exogenously added GST-Rac can be immunoprecipitated with antibodies to Rac. The association of only a small fraction of PtdIns-4-P 5-kinase with Rac suggests that either a minor isoform or a modified, more prevalent isoform of PtdIns-4-P 5-kinase binds to Rac. The same may also be true for PI 3-kinase.

Association of PI 3-Kinase with Cdc42 Immunoprecipitates

To determine whether a complex between Cdc42 and PI 3-kinase forms in vivo, Cdc42 immunoprecipitates were assayed for PI 3-kinase activity. Although we detected a small amount of PI 3-kinase in Cdc42 immunoprecipitates from PDGF-stimulated Swiss 3T3 cells (not shown), a more marked association of PI 3-kinase activity was found in Cdc42 immunoprecipitates from COS7 cells (Fig. 5, lane2). The presence of PI 3-kinase activity in Cdc42 immunoprecipitates was blocked by peptide antigen, indicating that PI 3-kinase specifically associates with Cdc42 (Fig. 5, lane3). Treatment of Cdc42 immunoprecipitates with wortmannin inhibited the synthesis of PtdIns-P (Fig. 5, lane4), confirming that the PI kinase activity is PI 3-kinase. Negligible PI kinase activity was detected in the control immunoprecipitate with non-immune serum (Fig. 5, lane1). The amount of PI 3-kinase that immunoprecipitated with Cdc42 was approximately 0.1% of the total PI 3-kinase activity in COS7 cells (data not shown). In comparison, about 0.4% of the total PI 3-kinase activity in COS cell lysate precipitated with GST-Cdc42.


Figure 5: Association of PI 3-kinase with Cdc42 immunoprecipitates. Cdc42 was immunoprecipitated from Cos cell lysates and analyzed for PI 3-kinase activity (lane2). Control immunoprecipitates were done with non-immune (NI) serum (lane1), and Cdc42 antibodies were pretreated with peptide antigen (lane3). Cdc42 immunoprecipitates were also treated with 100 nM wortmannin (Wort., lane4). PIP, phosphatidylinositol phosphate.



The finding that Cdc42 immunoprecipitates from COS7 cells contain PI 3-kinase activity in the absence of stimulation is unexpected given that the interaction between Cdc42 and PI 3-kinase is GTP-dependent. Since Swiss 3T3 cells and COS7 cells contain an equivalent amount of Cdc42 as judged by immunoblotting (not shown), these results suggest that Cdc42 is constitutively activated in COS7 cells. There are several possible explanations for this: an exchange factor might be activated, a GTPase-activating protein might be inhibited, or less Cdc42 might be bound to Rho GDP dissociation inhibitor.

We have shown that Rac specifically binds to a type I PtdIns-4-P 5-kinase and that both Rac and Cdc42 interact with PI 3-kinase. To our knowledge, this is the first demonstration of enzymatic activities associated with Rho family members in vivo. The in vivo association of Rac with PI 3-kinase was regulated by PDGF, suggesting that this interaction may be important in the PDGF-induced Rac signaling pathway leading to membrane ruffling. An obvious implication of our work is that PI 3-kinase is an effector for Rac and Cdc42. However, we do not detect an effect of Rac on PI 3-kinase activity using either immunoprecipitated or purified PI 3-kinase (data not shown). Additionally, preliminary experiments in which Rac is injected into wortmannin-treated cells suggest that PI 3-kinase is upstream of Rac.()In light of these data, it is interesting to speculate that PI 3-kinase may serve to localize the Rac/PtdIns-4-P 5-kinase complex to growth factor receptors. Local synthesis of PtdIns-4,5-P could then reorient the actin cytoskeleton by binding to actin-binding proteins such as gelsolin and profilin and promoting actin filament growth as proposed by Stossel(2) .


FOOTNOTES

*
This work was supported by a Parke-Davis American Heart Association Clinician Scientist Award, an award from the Hood Foundation (to C. L. C.), and National Institutes of Health Grant GM36624 (to L. C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Warren Alpert Bldg., Rm. 151, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3048; Fax: 617-278-3131.

The abbreviations used are: PDGF, platelet-derived growth factor; PI, phosphoinositide; PtdIns, phosphatidylinositol; GTPS, guanosine 5`-3-O-(thio)triphosphate; GST, glutathione S-transferase; DTT, dithiothreitol; GDPS, guanyl-5`-yl thiophosphate; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography.

L. Ma, M. Kirschner, K. F. Tolias, L. C. Cantley, and C. L. Carpenter, unpublished data; A. Hall, personal communication.


ACKNOWLEDGEMENTS

We thank Larry Feig for providing the Rac, Rho, and Cdc42 GST fusion proteins.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.