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Specific Requirement for the p85-p110α Phosphatidylinositol 3-Kinase during Epidermal Growth Factor-stimulated Actin Nucleation in Breast Cancer Cells*

Open AccessPublished:February 11, 2000DOI:https://doi.org/10.1074/jbc.275.6.3741
      We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells withepidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nm wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110α and p110β, and do not contain p110δ. Injection of specific inhibitory antibodies to p110α induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110β antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110α antibodies. In summary, the p110α isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.
      PI 3-kinase
      phosphatidylinositol 3-kinase
      EGF
      epidermal growth factor
      FITC
      fluorescein isothiocyanate
      GEF
      GTP exchange factor
      PI 3-kinases1 are important signaling intermediates in a variety of regulated cellular processes (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). They are classified based on their regulation and substrate specificity (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). Class I enzymes produce PI[3]P, PI[3,4]P2, and PI[3,4,5]P3, whereas class II and III enzymes produce PI[3]P and PI[3,4]P2, or only PI[3]P, respectively. Class Ia enzymes exhibit the greatest diversity of the known PI 3-kinases, with multiple isoforms of both the regulatory (p85) and catalytic (p110) subunits (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). Differential phosphorylation of p85α and p85 and differential activation of p85α- and p85β-associated PI 3-kinase have been reported (
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ,
      • Baltensperger K.
      • Kozma L.M.
      • Jaspers S.R.
      • Czech M.P.
      ). Knockouts of p85α further suggest that the p85α and p85β are not redundant (
      • Suzuki H.
      • Terauchi Y.
      • Fujiwara M.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      • Koyasu S.
      ,
      • Fruman D.A.
      • Snapper S.B.
      • Yballe C.M.
      • Davidson L., Yu, J.Y.
      • Alt F.W.
      • Cantley L.C.
      ). Distinct class Ia catalytic subunit isoforms also have different functions. Both p110α and p110β play a role in mitogenesis, although p110α is required for responses to a broader range of growth factors (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Roche S.
      • Downward J.
      • Raynal P.
      • Courtneidge S.A.
      ). Recently, distinct signaling properties for p110 isoforms have been demonstrated in macrophages (
      • Vanhaesebroeck B.
      • Jones G.E.
      • Allen W.E.
      • Zicha D.
      • Hooshmand-Rad R.
      • Sawyer C.
      • Wells C.
      • Waterfield M.D.
      • Ridley A.J.
      ).
      We examined the specific functions of p110α and p110β in MTLn3 cells, a metastatic variant of the 13762NF rat mammary adenocarcinoma. MTLn3 cells undergo chemotaxis in an EGF gradient (
      • Neri A.
      • Welch D.
      • Kawaguchi T.
      • Nicolson G.L.
      ,
      • Segall J.E.
      • Tyerch S.
      • Boselli L.
      • Masseling S.
      • Helft J.
      • Chan A.
      • Jones J.
      • Condeelis J.
      ). This response involves the actin-dependent extension of a lamellipod in the direction of increasing EGF concentrations, with a zone of newly polymerized F-actin at the leading edge (

      Chan, A. Y., Raft, S., Bailly, M., Wykoff, J. B., Segall, J. E., and Conddelis, J. S. (1998) J. Cell Sci. 199–211

      ). Using isoform-specific inhibitory antibodies against p110α and p110β, we now show that EGF-stimulated lamellipod extension requires p110α but not p110β. Significantly, anti-p110α antibodies blocked the formation of new barbed ends during an in situ actin nucleation assay. These studies provide direct evidence that p110α is required for the regulation of actin nucleation by EGF.

      DISCUSSION

      We have defined differential roles for class Ia PI 3-kinase isoforms during EGF-stimulated rearrangements of the actin cytoskeleton in breast cancer cells. Whereas both p110α and p110β are required for maximal mitogenic responses, we have demonstrated a specific requirement for p110α during EGF-stimulated lamellipod extension. Moreover, we have shown that inhibition of p110α blocks the formation of new barbed ends in EGF-stimulated cells, providing the first direct evidence that a specific isoform of PI 3-kinase is responsible for the regulation of actin nucleation. Our data provide a mechanistic basis for previous studies showing that growth factor-induced ruffling is blocked by PI 3-kinase inhibitors (
      • Wennström S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      • Jackson T.
      • Claesson-Welsh L.
      • Stephens L.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ) and directly implicate p110α in the acute regulation of the actin cytoskeleton in breast cancer cells
      There is not at present a clear explanation for the distinct intracellular functions of different class Ia PI 3-kinases. Thein vitro lipid and protein kinase activities of the class Ia isoforms are similar. Furthermore, p110α and p110β form heterodimers with the same p85 regulatory subunits (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). Both p110α (
      • Yu J.
      • Zhang Y.
      • McIlroy J.
      • Rordorf-Nikolic T.
      • Orr G.A.
      • Backer J.M.
      ) and p110β (data not shown) are labile as monomers at 37 °C, but are stabilized by association with p85 regulatory. It is therefore likely that, in intact cells, both isoforms function exclusively as dimers with p85 because dissociation would lead to rapid inactivation and degradation. However, p110α and p110β are only 42% identical and could form additional direct interactions with cellular effectors or targeting proteins (
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). Thus, p110β binds directly to activated Rab5 in vitro and is found in clathrin-coated vesicles, whereas p110α is not (
      • Christoforidis S.
      • Miaczynska M.
      • Ashman K.
      • Wilm M.
      • Zhao L.
      • Yip S.-C.
      • Waterfield M.D.
      • Backer J.M.
      • Zerial M.
      ). Similarly, a differential association of p110α with cytoskeletal components in MTLn3 cells could explain its role in EGF-stimulated lamellipod extension.
      Injection of anti-p110α antibodies causes a rounding up of MTLn3 cells similar to that seen in cells treated with cytochalasin D (data not shown). Barbed end number is also reduced in anti-p110α-injected cells, demonstrating an inhibition of actin nucleation. Thus, our data directly implicate the p85-p110α PI 3-kinase in the formation of new barbed ends at the leading edge of the cell. Although the mechanism involved is not yet clear, activation of Rho-family GTPases appears to be central in growth factor-mediated actin reorganization (
      • Allen W.E.
      • Jones G.E.
      • Pollard J.W.
      • Ridley A.J.
      ,
      • Rohatgi R.
      • Ma L.
      • Miki H.
      • Lopez M.
      • Kirchhausen T.
      • Takenawa T.
      • Kirschner M.W.
      ). PI 3-kinase has been linked to Rac activation in platelet-derived growth factor-stimulated cells (
      • Hawkins P.T.
      • Eguinoa A.
      • Qiu R.-G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennström S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • Stephens L.
      ), where the products of PI 3-kinase presumably signal to Rac through a protein analogous to Vav, a PI 3-kinase-dependent GTP exchange factor (GEF) in hematopoietic cells (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ). Direct regulation of CDC42 by PI 3-kinase products has not been demonstrated. However, the faciogenital dysplasia protein (FGD-1) and its homologues are GEFs for CDC42 and contain both FYVE and PH domains that may bind 3-phosphoinositides (
      • Olson M.F.
      • Pasteris N.G.
      • Gorski J.L.
      • Hall A.
      ). Activated Rac and CDC42 may affect the cytoskeleton through the effector kinase PAK-1, which phosphorylates and activates LIM kinase (
      • Edwards D.C.
      • Sanders L.C.
      • Bokoch G.M.
      • Gill G.N.
      ). LIM kinase in turn phosphorylates and inhibits the actin severing protein cofilin (
      • Yang N.
      • Higuchi O.
      • Ohashi K.
      • Nagata K.
      • Wada A.
      • Kangawa K.
      • Nishida E.
      • Mizuno K.
      ), leading to a decrease in the turnover of actin filaments. Cofilin may also contribute to generation of new barbed ends through severing actin filaments (
      • Bailly M.
      • Macaluso F.
      • Cammer M.
      • Chan A.
      • Segall J.E.
      • Condeelis J.S.
      ). Alternatively, activated CDC42 has been shown to form a complex with N-WASP and Arp2/3, which increase the actin nucleating activity of Arp2/3 (
      • Rohatgi R.
      • Ma L.
      • Miki H.
      • Lopez M.
      • Kirchhausen T.
      • Takenawa T.
      • Kirschner M.W.
      ). It is not yet clear if PI 3-kinases signal preferentially to one or the other of these pathways.
      It is interesting to compare our studies with the recently published work of Vanhaesebroeck et al. (
      • Vanhaesebroeck B.
      • Jones G.E.
      • Allen W.E.
      • Zicha D.
      • Hooshmand-Rad R.
      • Sawyer C.
      • Wells C.
      • Waterfield M.D.
      • Ridley A.J.
      ) who show that membrane ruffling and motility in macrophages is blocked by inhibitory antibodies to p110δ, and to a lesser extent p110β, but not by inhibitory antibodies to p110α. A unique role for p110δ in macrophages is not surprising because this isoform is restricted to hematopoietic cells and p110δ cannot be detected in MTLn3 cells by Western blot analysis (data not shown). However, the difference in the responses of MTLn3 cells versus macrophages to inhibitory anti-p110α and anti-p110β antibodies is unexpected. The difference is not because of variability in the antibody preparations because our anti-p110α antibodies do not inhibit CSF-1-stimulated cytoskeletal rearrangements in macrophages (data not shown), and the anti-p110β antibodies produced by Vanhaesebroeck and co-workers do not block EGF-stimulated lamellipod extension in MTLn3 cells (data not shown). The differential utilization of p110 isoforms in the two systems may reflect distinct signaling mechanisms used by the different receptors (EGF versus CSF-1). In this regard, it will be interesting to compare the coupling of EGF and CSF-1 receptors to class Ia PI 3-kinases in the same cell.
      Alternatively, the differential utilization of p110 isoforms could reflect the specific cell backgrounds in the two experimental systems. The finding of a specific requirement for p110α during EGF-stimulated motile responses in breast cancer cells suggest that p110α may be an important target for the development of anti-metastatic pharmaceuticals in the treatment of breast cancer.

      Acknowledgments

      We thank Dr. Bart Vanhaesebroeck (Ludwig Institute for Cancer Research, London), for anti-p110β antibodies and for sharing unpublished data, and Dr. Michael Waterfield (Ludwig Institute for Cancer Research, London), for the p110α and p110β constructs. We thank Jeffrey Wykoff, Michael Cammer, and the Analytical Imaging Facility at AECOM for assistance with image acquisition.

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