Tumor Cell Pseudopodial Protrusions

The pseudopodial protrusions of Moloney sarcoma virus (MSV)-Madin-Darby canine kidney (MDCK)-invasive (INV) variant cells were purified on 1-μm pore polycarbonate filters that selectively allow passage of the pseudopodial domains but not the cell body. The purified pseudopodial fraction contains phosphotyrosinated proteins, including Met and FAK, and various signaling proteins, including Raf1, MEK1, ERK2, PKBα (Akt1), GSK3α, GSK3β, Rb, and Stat3. Pseudopodial proteins identified by liquid chromatography tandem mass spectrometry included actin and actin-regulatory proteins (ERM, calpain, filamin, myosin, Sra-1, and IQGAP1), tubulin, vimentin, adhesion proteins (vinculin, talin, and β1 integrin), glycolytic enzymes, proteins associated with protein translation, RNA translocation, and ubiquitin-mediated protein degradation, as well as protein chaperones (HSP90 and HSC70) and signaling proteins (RhoGDI and ROCK). Inhibitors of MEK1 (U0126) and HSP90 (geldanamycin) significantly reduced MSV-MDCK-INV cell motility and pseudopod expression, and geldanamycin treatment inhibited Met phosphorylation and induced the expression of actin stress fibers. ROCK inhibition did not inhibit cell motility but transformed the pseudopodial protrusions of MSV-MDCK-INV cells into extended lamellipodia. Dominant negative Rho disrupted pseudopod expression and, in serum-starved cells, l-α-lysophosphatidic acid (oleoyl) activation of Rho induced pseudopodial protrusions or, in the presence of the ROCK inhibitor, extended lamellipodia. RNA was localized to the actin-rich pseudopodial domains of MSV-MDCK-INV cells, but the extent of colocalization with dense actin ruffles was reduced in the extended lamellipodia formed upon ROCK inhibition. Rho/ROCK activation in epithelial tumor cells therefore regulates RNA translocation to a pseudopodial domain that contains proteins involved in signaling, cytoskeleton remodeling, cell adhesion, glycolysis, and protein translation and degradation. The pseudopodial protrusions of Moloney sarcoma virus (MSV)-Madin-Darby canine kidney (MDCK)-invasive (INV) variant cells were purified on 1-μm pore polycarbonate filters that selectively allow passage of the pseudopodial domains but not the cell body. The purified pseudopodial fraction contains phosphotyrosinated proteins, including Met and FAK, and various signaling proteins, including Raf1, MEK1, ERK2, PKBα (Akt1), GSK3α, GSK3β, Rb, and Stat3. Pseudopodial proteins identified by liquid chromatography tandem mass spectrometry included actin and actin-regulatory proteins (ERM, calpain, filamin, myosin, Sra-1, and IQGAP1), tubulin, vimentin, adhesion proteins (vinculin, talin, and β1 integrin), glycolytic enzymes, proteins associated with protein translation, RNA translocation, and ubiquitin-mediated protein degradation, as well as protein chaperones (HSP90 and HSC70) and signaling proteins (RhoGDI and ROCK). Inhibitors of MEK1 (U0126) and HSP90 (geldanamycin) significantly reduced MSV-MDCK-INV cell motility and pseudopod expression, and geldanamycin treatment inhibited Met phosphorylation and induced the expression of actin stress fibers. ROCK inhibition did not inhibit cell motility but transformed the pseudopodial protrusions of MSV-MDCK-INV cells into extended lamellipodia. Dominant negative Rho disrupted pseudopod expression and, in serum-starved cells, l-α-lysophosphatidic acid (oleoyl) activation of Rho induced pseudopodial protrusions or, in the presence of the ROCK inhibitor, extended lamellipodia. RNA was localized to the actin-rich pseudopodial domains of MSV-MDCK-INV cells, but the extent of colocalization with dense actin ruffles was reduced in the extended lamellipodia formed upon ROCK inhibition. Rho/ROCK activation in epithelial tumor cells therefore regulates RNA translocation to a pseudopodial domain that contains proteins involved in signaling, cytoskeleton remodeling, cell adhesion, glycolysis, and protein translation and degradation. The repeated, directional extension and stabilization by substrate adhesive contacts of pseudopodia constitute the basic mechanism by which cells move over a substrate (1Lauffenberger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3291) Google Scholar). Cell motility is therefore associated with the polarized formation of a distinct plasma membrane domain, the pseudopod, whose stabilization determines the directionality of cell movement (2Nabi I.R. J. Cell Sci. 1999; 112: 1803-1811Crossref PubMed Google Scholar). Receptor stimulation of Rho GTPases and phosphatidylinositol 4,5-bisphosphate (PIP2) activates WASp/Scar proteins at the leading edge recruiting Arp2/3 and actin monomers to induce actin filament branching and membrane protrusion (3Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3303) Google Scholar). Complex interactions between these signaling modules, and others, are implicated in the protrusive and adhesive modes of cell migration. MSV-MDCK-INV 1The abbreviations used are: MSV, Moloney sarcoma virus; HGF, hepatocyte growth factor; HGFR, hepatocyte growth factor receptor; p-, phospho-; p-Tyr, phosphotyrosine; MAPK, mitogen-activated protein kinase; ROCK, Rho kinase; p97(VCP), valosin-containing protein; EF1α, elongation factor 1α; NPC, nuclear pore complex proteins; ERM, ezrin/radixin/moesin; LPA, l-α-lysophosphatidic acid, oleoyl; MDCK, Madin-Darby canine kidney cells; PPF, purified pseudopodial fraction; GFP, green fluorescent protein; EGFP, enhanced GFP; MOPS, 4-morpholinepropanesulfonic acid; MS, mass spectrometry; ER, endoplasmic reticulum; DN, dominant negative; ERK, extracellular signal-regulated kinase; INV, invasive. cells, selected from Moloney sarcoma virus (MSV)-transformed MDCK cells for their ability to invade across Matrigel-coated filters, exhibit multiple protrusive, actin-rich, blebbed pseudopodial domains whose formation is dependent on autocrine Met activation (4Le P.U. Nguyen T.N. Drolet-Savoie P. Leclerc N. Nabi I.R. Cancer Res. 1998; 58: 1631-1635PubMed Google Scholar, 5Vadnais J. Nault G. Daher Z. Amraei M. Dodier Y. Nabi I.R. Noel J. J. Biol. Chem. 2002; 277: 48342-48350Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). Using polycarbonate filters of defined 1-μm pore size that selectively allow passage of the pseudopodial domains but not the cell body, we established a method to purify MSV-MDCK-INV pseudopodia (6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). The pseudopodia of MSV-MDCK-INV cells are strongly labeled for β-actin by immunofluorescence, and the purified pseudopodial fraction (PPF) is enriched for β-actin and depleted of mitochondrial proteins relative to the total cell lysate (4Le P.U. Nguyen T.N. Drolet-Savoie P. Leclerc N. Nabi I.R. Cancer Res. 1998; 58: 1631-1635PubMed Google Scholar, 6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). Glycolysis represents a critical energy supply for tumor cell motility (7Beckner M.E. Stracke M.L. Liotta L.A. Schiffmanm E. J. Natl. Cancer Inst. 1990; 82: 1836-1840Crossref PubMed Scopus (116) Google Scholar), and identification of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase by Edman degradation in purified pseudopodia of MSV-MDCK-INV cells led to the demonstration that glycolysis regulates the formation and protrusion of tumor cell pseudopodia (6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). Using a scaled up pseudopod purification approach, we show here that the pseudopodial domain of MSV-MDCK-INV cells contains phosphorylated Met and FAK as well as a number of downstream signaling proteins, including Raf1, MEK1, ERK2, PKBα (Akt1), GSK3α, GSK3β, Rb, and Stat3. A subsequent proteomic analysis identified other regulators of Met activity, including RhoGDI, ROCK2, and HSP90, as well as major classes of proteins that included cytoskeleton-associated proteins, such as actin, tubulin, vimentin, and actin- and tubulin-associated proteins, glycolytic enzymes and protein chaperones, as well as proteins associated with RNA translocation, protein translation, and ubiquitin-mediated protein degradation. Pseudopodial protrusion was inhibited by the MEK1 inhibitor U0126 and by inhibition of HSP90 activity with geldanamycin, which was also shown to inhibit constitutive Met activation. ROCK inhibition with Y27632 did not affect cell motility but transformed the protrusive pseudopodia into extended lamellipodia. Inhibition of ROCK was associated with reduced RNA targeting to the lamellipodia showing that Rho/ROCK activation recruits RNA to the pseudopodial domain of MSV-MDCK-INV cells for local translation. Antibodies and Reagents—Anti-β-actin, anti-BiP/GRP78, and antitalin were purchased from Sigma;, anti-FAK and anti-phosphotyrosine (p-Tyr; PY-99), anti-phospho-RB1 (S780), anti-HSC70, and anti-lamin A/C were from Santa Cruz Biotechnology; anti-phospho-Met (Y1230, Y1234, and Y1235), anti-phospho-FAK Y397, and anti-phospho-FAK Y577 were from BIOSOURCE International; anti-p97(VCP) was from Abcam and were from was from was from pore complex proteins were from and were from and was from was purchased from and was from polycarbonate filters pore were purchased from and 1-μm pore filters were from geldanamycin, and to were purchased from acid, was purchased from and and U0126 were from Cell and cells were in with and and in a as (4Le P.U. Nguyen T.N. Drolet-Savoie P. Leclerc N. Nabi I.R. Cancer Res. 1998; 58: 1631-1635PubMed Google Scholar). MSV-MDCK-INV cells were with by and cells were selected with MSV-MDCK-INV cells were on 1-μm pore filters at a of for MSV-MDCK-INV pseudopodia were purified a to that (6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). MSV-MDCK-INV cells were on 1-μm pore filters between in a and the was with to cell to the of the a the was with and and the and of the with a The pseudopodia cell were from the in and and and The was for on for at and the was and the protein was used for the were in and at as the of cells for on on 1-μm pore was as (6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). were with and with cells and or, for and A/C with RNA was with and, cells were with at for and filters with the of the up were with the of a with the of The extent of of labeled cells with pseudopodial domains was were to of the cell and actin-rich pseudopodial which were to the to for the actin-rich pseudopodial domain and the of the cells from a of were were for to of the and cells to were and in as of cell and the cell and pseudopodial of MSV-MDCK-INV cells was the protein of protein was by and and with or, for with to with the and to The labeled were by and to was on and were by on to the in were by liquid spectrometry of liquid chromatography with and were on with The was into the of the mass The used for was of and the total was mass were the the A protein was as a with were with a were on their tandem mass spectrometry of Cell pseudopodia purified by the cells on polycarbonate filters of 1-μm pore that the passage of MSV-MDCK-INV pseudopodia but not the cell (6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). in the and cell on the of the protrusions to the of the The pseudopodial protrusions that the are labeled for on pseudopodia purification used (6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar), and we scaled up the purification filters and MSV-MDCK-INV cells exhibit total expression that in the pseudopodial domain J. Nault G. Daher Z. Amraei M. Dodier Y. Nabi I.R. Noel J. J. Biol. Chem. 2002; 277: 48342-48350Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), and we the of phosphorylation in the pseudopodial fraction by with the in the expression between the pseudopodial fraction and the cell lysate were The major in at and with to phosphorylated Y1234, and and to FAK that with major phosphorylated at and immunofluorescence, and with actin and in the pseudopodial domain of Met and FAK are therefore in MSV-MDCK-INV the for expression of the phosphorylated of various signaling proteins a of with the in MSV-MDCK-INV pseudopodia the phosphorylated of multiple signaling proteins with localized autocrine Met including Rb, and Met signaling are therefore the pseudopodia of MSV-MDCK-INV The also identified phosphorylated Raf1, and as enriched in the pseudopodial fraction The subsequent proteomic analysis of the pseudopodial fraction by liquid chromatography tandem mass spectrometry analysis identified proteins from and other proteins were up in the and proteins of were from the proteins were into major including cytoskeleton-associated proteins adhesion proteins glycolytic enzymes protein chaperones proteins proteins S. M. M. E. A. Cancer Res. 2000; Google Scholar), proteins signaling proteins as well as proteins associated with membrane and proteins not into these for the of the domain that the is from the of actin, proteins, tubulin, and glycolytic The proteomic up the proteins, and a of from the number of identified that to a the size of a protein on the number of of number that the pseudopodial fraction is enriched for proteins, glycolytic enzymes, protein chaperones, and translation associated proteins on the number of the proteins in the pseudopodial fraction in to actin the proteins and and tubulin, vimentin, various glycolytic enzymes, the chaperones HSP90, and elongation and the and the signaling proteins and of the pseudopod on the proteomic analysis and of MSV-MDCK-INV pseudopodia in the number of proteins and total number of identified protein are as the presence of a number of the major proteins by of the and cell EF1α, HSP90, FAK, and are in the pseudopodial fraction at to that of β-actin with and in the pseudopodial domain relative to for and A/C were in the PPF, and that pseudopodial expression of these proteins was relative to the cell and to that of mitochondrial shown to from MSV-MDCK-INV pseudopodia (6Nguyen T.N. Wang H.J. Zalzal S. Nanci A. Nabi I.R. Exp. Cell Res. 2000; 258: 171-183Crossref PubMed Scopus (49) Google Scholar). used to protein to the pseudopodial domain relative to the of the cell the proteins a to distinct of the proteins to the pseudopodial domain were Pseudopodial expression was by in the pseudopodial domain relative to the of the that protein to pseudopodia is not a and to of proteins, we cells with that a reduced in the pseudopodial domain relative to the of the cell A and in a number of proteins (vinculin, talin, EF1α, and in the pseudopodia relative to GFP, a and and were depleted and The of pseudopodial to the of the cell relative to the the that the pseudopodial domain that the is not to the actin-rich of the pseudopodial protrusions of MSV-MDCK-INV actin was enriched in the pseudopodial domain by but significantly enriched on by The number of proteins in the proteomic analysis represents a of the of the proteins were identified and and mitochondrial proteins The major protein was which is on the cell and identified as a for tumor J. 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Full Text Full Text PDF PubMed Scopus Google Scholar). and their in the proteomic analysis therefore not their pseudopodial but their relative expression not a in Pseudopodial in MSV-MDCK-INV the Met regulators involved in MSV-MDCK-INV cell motility and pseudopod we the cells with of and (U0126) identified in the and of ROCK and HSP90 (geldanamycin) identified in the proteomic of the cells with U0126 and geldanamycin inhibited MSV-MDCK-INV cell motility and also disrupted MSV-MDCK-INV cell in enhanced cell cells actin stress as in cells constitutive activation was disrupted J. Nault G. Daher Z. Amraei M. Dodier Y. Nabi I.R. Noel J. J. Biol. Chem. 2002; 277: 48342-48350Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). HSP90 is to associated with Met signaling in G. J. E. E. Cancer Res. 2002; Google Scholar, S. M. M. E. A. 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