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Address correspondence to Richard L. Klemke, Dept. of Immunology, SP231, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-7750. Fax: (858) 784-7785. email: klemke{at}scripps.edu
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Abstract |
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Key Words: cell migration; apoptosis; signal transduction; focal adhesions; Abl tyrosine kinase
Abbreviations used in this paper: Lasp-1, Lim, actin, and SH3 domain; MudPIT, multidimensional protein identification technology; SH3, Src homology domain 3; siRNA, small interfering RNA; TSA, trichostatin A.
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Introduction |
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To help unravel the spatiotemporal organization of signaling cascades involved in cell polarization, including mechanisms of protein translocation, activation, post-translational modifications, and formation of complex multi-protein scaffolds, we developed a biochemical method to selectively isolate the pseudopodium and cell body of cells polarized toward a chemoattractant gradient using a microporous filter system (Cho and Klemke, 2002; Brahmbhatt and Klemke, 2003). Pseudopodia extension in this system, like traditional pseudopodia formation on two-dimensional surfaces, requires Cdc42 and Rac activity and shows normal actin cytoskeletal organization and focal adhesions. Cells extend pseudopodia projections through small openings in the vasculature and ECM in vivo as a necessary process of immune cell intravasation as well as pathological processes associated with cancer cell metastasis (Wyckoff et al., 2000). Therefore, this model recapitulates physiological events associated with cell migration and is ideal for unraveling the spatial and temporal signaling mechanisms responsible for focal adhesion changes leading to cell polarity and directional movement.
In this report, we used this technique along with a new protein sequencing method called multidimensional protein identification technology (MudPIT; Washburn et al., 2001) for rapid and large-scale proteome analysis of purified pseudopodia. By using multidimensional liquid chromatography, tandem mass spectrometry, and database searching with SEQUEST algorithm, it is possible to identify large numbers of proteins (>1,000) directly from a complex protein lysate (Washburn et al., 2001). Here, we isolated pseudopodia and cell body proteins for comparison by MudPIT to reveal proteins uniquely present in these different cellular compartments. The novel cytoskeletal-associated Lim, actin, and SH3 protein (Lasp-1; GenBank/EMBL/DDBJ accession no. X82456) was identified as a component of the pseudopodium and further characterized for functional significance. Lasp-1 was initially identified from a breast cancerderived metastatic lymph node cDNA library and is overexpressed in 812% of breast cancer (Tomasetto et al., 1995a, b). It is a ubiquitously expressed actin-binding protein with a unique domain configuration (Tomasetto et al., 1995a; Schreiber et al., 1998) that includes a LIM domain (Lin11, Isl-1, and Mec-3) in the NH2-terminal region followed by two actin-binding repeats (R1, R2), and an Src homology 3 (SH3) domain in the COOH-terminal region. There are also two tyrosine phosphorylation sites (Y52 and Y152) corresponding to SH2-binding consensus motifs (YXXP; Tomasetto et al., 1995a), suggesting that Lasp-1 is tyrosine phosphorylated and may facilitate binding of SH2 effector proteins and their downstream signals. However, the biological function and regulation of this molecule has not yet been identified. Our findings demonstrate that Lasp-1 is a dynamic, spatially regulated protein necessary for cell migration. Furthermore, we show that the cytoskeletal regulatory protein c-Abl tyrosine kinase (Woodring et al., 2003) directly phosphorylates Lasp-1, which regulates its localization to focal adhesions in apoptotic (but not migratory) cells.
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Results |
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Lasp-1 is necessary for cell migration (but not adhesion) to the ECM
Although the function of Lasp-1 is not known, the above findings suggest that it is important for cell adhesion and migration on the ECM. To directly examine this possibility, we depleted cells of Lasp-1 using small interfering RNA (siRNA) technology (Elbashir et al., 2001). Cells exposed to anti-Lasp-1 siRNA showed an 8090% reduction in Lasp-1 protein compared with cells mock treated with control siRNA directed to GL2 luciferase. Total protein staining and Western blotting for either Lasp-1 or actin revealed that only Lasp-1 was reduced and not other cellular proteins (Fig. 4 A). Interestingly, although cells depleted of Lasp-1 showed little difference in adhesion and spreading, they did show a significantly impaired ability to migrate on the ECM (Fig. 4, A and B). These findings indicate that Lasp-1 protein is necessary for cell migration, but not cell adhesion and spreading. Similar findings were obtained with NIH 3T3 fibroblast cells depleted of Lasp-1 protein (unpublished data). Interestingly, Lasp-1 expression has been reported to be increased in metastatic breast cancers, suggesting that protein amplification may contribute to the migratory properties of these cells (Tomasetto et al., 1995b). To investigate this, we ectopically expressed Lasp-1 in cells and monitored their ability to migrate in response to growth factors. Surprisingly, Lasp-1 amplification inhibited basal and growth factorstimulated cell migration (Fig. 4 C) without affecting attachment to the ECM (unpublished data). Similar findings were obtained with HEK 293 (Fig. 4 D) and MCF-7 cells (unpublished data). Importantly, expression of Lasp
C failed to inhibit cell migration or adhesion, indicating that the COOH-terminal region of Lasp-1 is required for this process. Expression of the SH3 domain alone strongly inhibited cell migration, which supports this notion (Fig. 4 C). It is not yet clear why both depletion and amplification of Lasp-1 inhibits cell migration. However, it is possible that global amplification of Lasp-1 throughout the cell body disrupts signaling polarity and the normal restricted localization of this protein, as has been shown for Cdc42 (Allen et al., 1998). In this regard, disruption of signaling polarity by constitutive relocalization of Lasp-1 to the plasma membrane caused dramatic membrane blebbing and detachment from the ECM leading to cell death, whereas targeting of Lasp
C to the membrane failed to induce this response (Fig. 4 E). In any case, these findings demonstrate that Lasp-1 plays an important spatial role in cell migration, and that this process is regulated by the COOH-terminal region of Lasp-1 containing the SH3 domain.
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Interestingly, Abl also induces cell apoptosis in response to DNA-damaging agents (cisplatin; Gong et al., 1999) and oxidative stress (H2O2; Sun et al., 2000). Although the mechanisms are not yet understood, the death process involves deregulation of the actin cytoskeleton and focal adhesions during the execution phase of death (Huot et al., 1998; Houle et al., 2003). Indeed, exposure of cells to either H2O2 or cisplatin caused strong Lasp-1 tyrosine phosphorylation that required endogenous Abl kinase activity (Fig. 6). Moreover, H2O2-induced Lasp-1 phosphorylation was significantly impaired in embryonic mouse fibroblast cells isolated from abl/arg/ animals, compared with these cells stably reconstituted with Abl (Fig. 6 B). However, at later times (>45 min) a small level of Lasp-1 phosphorylation is detected, suggesting that another kinase(s) may phosphorylate Lasp-1. Similar findings were obtained with cells treated with pervanadate, which strongly (>17-fold) activates Abl (unpublished data; Woodring et al., 2003). These findings demonstrate the tyrosine phosphorylation of Lasp-1 by endogenous Abl activation in response to apoptotic agents.
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Discussion |
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We chose to characterize Lasp-1 because its biological function is not known. In this report, we provide several lines of evidence that demonstrate Lasp-1 is a dynamic protein that transits to focal adhesions and is necessary for proper cell migration. First, depletion of Lasp-1 protein from cells strongly inhibits cell migration in response to ECM proteins. The inhibitory effect is specific to migration, as cell attachment and spreading appear normal in cells without Lasp-1 protein. Second, exogenous amplification of Lasp-1 also inhibits cell migration, but not adhesion and spreading. We believe that exogenous expression of Lasp-1 disrupts its normal signaling polarity. Lasp-1 is strongly polarized in migrating cells, where it localizes to the leading edge of the pseudopodium and to nascent focal adhesions in this structure, but not the central body of the cell. That constitutive relocalization of Lasp-1 to the plasma membrane strongly induced membrane blebbing and detachment from the ECM supports this idea. Third, stimulation of nonmigratory cells with growth factors or ECM proteins that induce cell migration cause the rapid relocalization of Lasp-1 from the peripheral membrane to a subset of focal adhesions in the spreading edge of the pseudopodium. Because cells depleted of Lasp-1 still attach to the ECM and form focal adhesions, it would appear that Lasp-1 plays a supportive role in focal adhesion dynamics during cell migration rather than in the actual formation of these structures. That Lasp-1 localizes to focal adhesions at the tips of retracting tails of migrating cells also supports this notion, as these adhesive sites must turnover for proper cell body translocation and tail release (Palecek et al., 1996). Together, these findings demonstrate that Lasp-1 is a dynamic focal adhesion protein necessary for cell migration.
Although the cellular factors responsible for translocation of Lasp-1 to focal adhesions are not yet known, it appears that the COOH-terminal region of Lasp-1 is critical for this response. Our biochemical and time-lapse analyses (unpublished data) of Lasp-1 indicate that translocation occurs rapidly within 12 min of growth factor stimulation and is independent of tyrosine phosphorylation. Indeed, repeated attempts to demonstrate changes in tyrosine phosphorylation of Lasp-1, in response to ECM proteins or growth factors, failed to show this response, even though these factors activate several tyrosine kinases including src and Abl (Lewis et al., 1996; Plattner et al., 1999). Furthermore, we did not detect a mobility shift of Lasp-1 in SDS-PAGE in stimulated cells or purified pseudopodia, which is characteristic of phosphorylation by PKA (Chew et al., 1998) as well as tyrosine phosphorylation (Fig. 6, A and B). This suggests that phosphorylation does not regulate focal adhesion targeting of Lasp-1 during cell spreading or pseudopodial extension. However, in some cases PKA may regulate Lasp-1 in focal adhesions as well as its association with actin in gastric parietal cells where it localizes to the actin-rich canalicular membrane (Chew et al., 2002; Butt et al., 2003). It is also possible that low levels of Abl and PKA activity regulate Lasp-1 dynamics in the microenvironment of the migrating cell, but this activity was below biochemical detection in our analyses.
Our findings indicate that the COOH-terminal portion of Lasp-1 is for targeting to focal adhesions. The SH3 domain of Lasp-1 may play a pivotal role in focal complex targeting, as these structures can direct proteins like p130CAS (Crk-associated substrate) to focal adhesions (Harte et al., 2000). However, a GFP fusion of the Lasp-1 SH3 domain failed to translocate by itself to focal complexes (unpublished data). It is possible that the GFP tag interfered with the normal targeting function of this domain. Additional truncations in the COOH terminus as well as point mutations that disrupt SH3 domain function will be necessary to pinpoint the region of Lasp-1 responsible for translocation to focal complexes and for identification of binding proteins that mediate this process. In any case, the Lim domain does not play a critical role in focal adhesion targeting, as truncation of this domain did not prevent Lasp-1 localization to these structures (unpublished data).
It is intriguing that Abl tyrosine kinase is activated by growth promoting as well as by apoptotic stimuli (Lewis et al., 1996; Gong et al., 1999; Plattner et al., 1999; Sun et al., 2000). This suggests that Abl activity is a complex process tightly regulated by temporal and spatial mechanisms that couple to specific effector molecules. Our findings indicate that growth factor/motility factors like serum, PDGF-BB, EGF, and ECM proteins do not mediate coupling of Abl to Lasp-1. Rather, Abl activation in response to apoptotic stimuli appears to selectively phosphorylate Lasp-1 on tyrosine 171, preventing its translocation into focal complexes. This event is specific to focal complex targeting, as Abl-induced phosphorylation of Lasp-1 does not inhibit its localization to actin-rich structures including membrane ruffles and actin cables. Thus, Abl-dependent tyrosine phosphorylation of Y171 is a distinct signaling event that specifically blocks Lasp-1 transiting to focal complexes in apoptotic cells. Although the mechanism of Lasp-1 translocation is not known, these findings have important implications, as Abl-mediated death has been reported to involve nuclear and mitochondrial events involving p53/p73 and cytochrome c release, respectively (Agami et al., 1999; Yuan et al., 1999; Kumar et al., 2001; Goldberg et al., 2002). Our findings suggest that Abl activation also targets cytoplasmic and focal complex substrates in response to these apoptotic agents. An early step in the apoptotic process involves remodeling of focal adhesions and detachment from the ECM (Mills et al., 1999). Interestingly, an intermediate adhesive strength is optimal for transmitting survival signals from focal adhesions, suggesting that this process is tightly coupled to focal adhesion dynamics and cell motility processes (Murphy-Ullrich, 2001; Truong et al., 2003). Prevention of Lasp-1 localization to focal complexes by Abl may disrupt critical survival signals and may contribute to the execution phase of the apoptotic response. Recent evidence indicates that cytoplasmic Abl inactivates the focal adhesion protein c-CrkII, which induces apoptosis of carcinoma cells (Kain et al., 2003). Interestingly, Abl coordinately regulates both the migration and apoptotic machineries of cells through c-CrkII phosphorylation (Kain and Klemke, 2001; Kain et al., 2003). However, we found that Abl only couples to Lasp-1 under conditions that induce cell apoptosis and not motility. The ability of Abl to differentially target cytoplasmic and focal adhesion substrates like Lasp-1 and c-CrkII under migratory or apoptotic conditions may be related to the duration of kinase activation. Apoptotic agents tend to induce strong and persistent Abl activity (>1 h), whereas survival stimuli like integrin and growth factor receptor activation typically promote only a transient response (<30 min; Lewis et al., 1996; Gong et al., 1999; Plattner et al., 1999; Sun et al., 2000). Interestingly, recent evidence indicates that integrins and cell adhesion to the ECM is necessary for Abl-mediated death in response to DNA-damaging agents like cisplatin (Lewis et al., 2002; Truong et al., 2003). This suggests that at least part of the Abl death signal requires integrins and focal adhesion structures. Although the kinetics of Abl activity was not addressed in these reports, it seems reasonable that part of the suicide program involves not only localization of Abl to the nucleus where it regulates p53/73 (Lewis et al., 2002; Truong et al., 2003), but also its localization to the cytoplasm where it inactivates specific focal adhesion proteins like Lasp-1 and c-CrkII. In this way, Abl activation would have widespread impact on the apoptotic machineries that operate at multiple compartments within the cell. This widespread insult is likely important in the cell's decision to commit suicide or to attempt a repair and rescue program. In any case, the current evidence indicates that Abl-mediated death involves a cooperative effort with integrins, cytoskeleton, focal adhesions, and mitochondrial and nuclear proteins. The challenge now is to understand how Abl coordinately regulates these diverse processes and whether these events contribute to cancer development and progression.
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Materials and methods |
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Details of MudPIT and data analysis have been described elsewhere (Washburn et al., 2001; Tabb et al., 2002). Tandem mass spectroscopy spectra were searched against a combined database of human, mouse, and rat proteins using SEQUEST, and the combined database was constructed from the NCBI protein database (August 20, 2002). The results were further analyzed by DTASelect and Contrast software. Peptides with nontryptic cleavage sites were not included. All the proteins identified with a minimum of a single peptide were used to make protein lists for the pseudopod and for the cell body. A total of 980 proteins was identified by SEQUEST search. For the proteins with only single peptide match, relatively high Xcorr values (+1, 1.8; +2, 2.5; +3, 3.5; Eng et al., 1994) and probability-based scoring system of peptide identification (Pep Probe) was used to filter peptide matches, and then every tandem mass spectroscopy spectra was manually evaluated for final verification purposes. The Xcorr values represent a cross-correlation function used to provide measurement of similarity between the mass-to-charge ratios for the fragment ions predicated from amino acid sequences obtained from the database and the fragment ions actually observed in the tandem mass spectrum (Eng et al., 1994). Unique proteins in each sample and proteins showing significant changes (>50%) in the number of peptides identified were combined to make a list of enriched proteins in the pseudopodium and in the cell body. Proteins identified via a single peptide were included only if identified in a single compartment. Only those proteins positively identified in each of the five independent experiments via MudPIT were included in the list.
Immunofluorescence analysis of Lasp-1 dynamics
NIH 3T3 cells were transfected with the appropriate GFP constructs on glass coverslips as described above. Cells were washed 2x with PBS, fixed with 4% PFA in PBS, permeabilized with 0.1% Triton X-100 in PBS for 1 min, and then blocked with 0.5% BSA for 1 h. Anti-vinculin antibodies or TRITC-conjugated phalloidin (Sigma-Aldrich) was diluted in blocking solution and incubated with the fixed cells for 60 min. Anti-vinculintreated cells were then incubated with Alexa Fluor® 568conjugated goat antimouse IgG antibodies and were washed with PBS and mounted on coverslips using a ProLong® anti-fade kit (Molecular Probes, Inc.). Immunofluorescence microscopy was performed using a microscope (model IX70; Olympus) and data acquisition by a liquid cooled CCD camera (500 KHz, 12 bit, 2MP, KAF1400GI, 1317 x 1035; model CH350L, Photometrics). Image data were deconvolved with DeltaVision softWoRx version 2.5 software (Applied Precision). To examine the role of Abl activation in Lasp-1 localization to focal adhesions, 25 µM STI 571 was added to serum-starved cells for 16 h. Cells were then stimulated for the indicated times with either 1 mM H2O2, 25 µM cisplatin, 2 mM sodium orthovanadate, 20 ng PDGF-BB, or 10% FBS, which was added for 15 min in addition to other chemicals. Vinculin-positive focal adhesions and the F-actin cytoskeleton were examined as described above.
siRNA silencing
21-nucleotide double-stranded RNAs (Dharmacon Research) were synthesized by targeting human Lasp-1 (5'-AACUACAAGGGCUACGAGAAG-3'; corresponds to the coding region 127147 relative to the first nucleotide of the start codon). Luciferase GL2 duplex (Dharmacon Research) was used as a negative control. The siRNAs were transfected into Cos-7 cells using OligofectamineTM (Invitrogen) according to the manufacturer's instructions. Specific depletion of Lasp-1 was confirmed 48 h after transfection by Western blotting using anti-Lasp-1 and anti-actin antibodies.
Online supplemental material
A complete list of all pseudopodia and cell bodyassociated proteins is shown in Tables S1S4 and Fig. S1. Materials and methods for cell lines, plasmids, constructs, antibodies, and cell-based assays, kinase assays, immunoprecipitation, and Western blotting are shown. Fig. S1 is a comparative schematic of the number of proteins identified in the each of the subcompartments of the cell body and pseudopodial fractions. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200311045/DC1.
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Acknowledgments |
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Y.H. Lin is supported by California Breast Cancer Research Program grant 7FB-0117. R.L. Klemke is funded by National Institute of Health grant CA097022. This is manuscript no. 16012-IMM from The Scripps Research Institute.
Submitted: 7 November 2003
Accepted: 5 April 2004
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References |
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Agami, R., G. Blandino, M. Oren, and Y. Shaul. 1999. Interaction of c-Abl and p73 and their collaboration to induce apoptosis. Nature. 399:809813.[CrossRef][Medline]
Aizawa, H., Y. Fukui, and I. Yahara. 1997. Live dynamics of Dictyostelium cofilin suggests a role in remodeling actin latticework into bundles. J. Cell Sci. 110:23332344.
Allen, W.E., D. Zicha, A.J. Ridley, and G.E. Jones. 1998. A role for Cdc42 in macrophage chemotaxis. J. Cell Biol. 141:11471157.
Bailly, M., L. Yan, G.M. Whitesides, J.S. Condeelis, and J.E. Segall. 1998. Regulation of protrusion shape and adhesion to the substratum during chemotactic responses of mammalian carcinoma cells. Exp. Cell Res. 241:285299.[CrossRef][Medline]
Bailly, M., I. Ichetovkin, W. Grant, N. Zebda, L.M. Machesky, J.E. Segall, and J. Condeelis. 2001. The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension. Curr. Biol. 11:620625.[CrossRef][Medline]
Barila, D., R. Mangano, S. Gonfloni, J. Kretzschmar, M. Moro, D. Bohmann, and F. Superti. 2000. A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK. EMBO J. 19:273281.
Brahmbhatt, A.A., and R.L. Klemke. 2003. ERK and RhoA differentially regulate pseudopodia growth and retraction during chemotaxis. J. Biol. Chem. 278:1301613025.
Buchdunger, E., J. Zimmermann, H. Mett, T. Meyer, M. Muller, B.J. Druker, and N.B. Lydon. 1996. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res. 56:100104.[Abstract]
Butt, E., S. Gambaryan, N. Gottfert, A. Galler, K. Marcus, and H.E. Meyer. 2003. Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146. J. Biol. Chem. 278:1560115607.
Chew, C.S., J.A. Parente, C. Zhou, E. Baranco, and X. Chen. 1998. Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell. Am. J. Physiol. 275:C56C67.[Medline]
Chew, C.S., X. Chen, J.A. Parente, S. Tarrer, C. Okamoto, and H.Y. Qin. 2002. Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo. J. Cell Sci. 115:47874799.[CrossRef][Medline]
Cho, S.Y., and R.L. Klemke. 2002. Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold. J. Cell Biol. 156:725736.
Conrad, P.A., M.A. Nederlof, I.M. Herman, and D.L. Taylor. 1989. Correlated distribution of actin, myosin, and microtubules at the leading edge of migrating Swiss 3T3 fibroblasts. Cell Motil. Cytoskeleton. 14:527543.[Medline]
Cooper, J.A. 2002. Actin dynamics: tropomyosin provides stability. Curr. Biol. 12:R523R525.[CrossRef][Medline]
Daley, G.Q., E. Van, and D. Baltimore. 1990. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 247:824830.[Medline]
Elbashir, S.M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494498.[CrossRef][Medline]
Eng, J.K., A.L. McCormack, and J.R. Yates III. 1994. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5:976989.[CrossRef]
Funamoto, S., R. Meili, S. Lee, L. Parry, and R.A. Firtel. 2002. Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell. 109:611623.[Medline]
Goldberg, Z., S. Vogt, M. Berger, Y. Zwang, R. Perets, E. Van, M. Oren, Y. Taya, and Y. Haupt. 2002. Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation. EMBO J. 21:37153727.
Gong, J.G., A. Costanzo, H.Q. Yang, G. Melino, W.G. Kaelin, M. Levrero, and J.Y. Wang. 1999. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature. 399:806809.[CrossRef][Medline]
Grosveld, G., T. Verwoerd, T. van Agthoven, A. de Klein, K.L. Ramachandran, N. Heisterkamp, K. Stam, and J. Groffen. 1986. The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript. Mol. Cell. Biol. 6:607616.[Medline]
Harte, M.T., M. Macklem, C.L. Weidow, J.T. Parsons, and A.H. Bouton. 2000. Identification of two focal adhesion targeting sequences in the adapter molecule p130(Cas). Biochim. Biophys. Acta. 1499:3448.[CrossRef][Medline]
Helfman, D.M., E.T. Levy, C. Berthier, M. Shtutman, D. Riveline, I. Grosheva, Z. Lachish, M. Elbaum, and A.D. Bershadsky. 1999. Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions. Mol. Biol. Cell. 10:30973112.
Houle, F., S. Rousseau, N. Morrice, M. Luc, S. Mongrain, C.E. Turner, S. Tanaka, P. Moreau, and J. Huot. 2003. Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: impact on membrane blebbing. Mol. Biol. Cell. 14:14181432.[CrossRef][Medline]
Huot, J., F. Houle, S. Rousseau, R.G. Deschesnes, G.M. Shah, and J. Landry. 1998. SAPK2/p38-dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis. J. Cell Biol. 143:13611373.
Kain, K.H., and R.L. Klemke. 2001. Inhibition of cell migration by Abl family tyrosine kinases through uncoupling of Crk-CAS complexes. J. Biol. Chem. 276:1618516192.
Kain, K.H., S. Gooch, and R.L. Klemke. 2003. Cytoplasmic c-Abl provides a molecular rheostat controlling carcinoma cell survival and invasion. Oncogene. 22:60716080.[CrossRef][Medline]
Kumar, S., A. Bharti, N.C. Mishra, D. Raina, S. Kharbanda, S. Saxena, and D. Kufe. 2001. Targeting of the c-Abl tyrosine kinase to mitochondria in the necrotic cell death response to oxidative stress. J. Biol. Chem. 276:1728117285.
Lauffenburger, D.A., and A.F. Horwitz. 1996. Cell migration: a physically integrated molecular process. Cell. 84:359369.[Medline]
Laukaitis, C.M., D.J. Webb, K. Donais, and A.F. Horwitz. 2001. Differential dynamics of 5 integrin, paxillin, and
-actinin during formation and disassembly of adhesions in migrating cells. J. Cell Biol. 153:14271440.
Lewis, J.M., R. Baskaran, S. Taagepera, M.A. Schwartz, and J.Y. Wang. 1996. Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport. Proc. Natl. Acad. Sci. USA. 93:1517415179.
Lewis, J.M., T. Truong, and M.A. Schwartz. 2002. Integrins regulate the apoptotic response to DNA damage through modulation of p53. Proc. Natl. Acad. Sci. USA. 99:36273632.
Liu, F., D.E. Hill, and J. Chernoff. 1996. Direct binding of the proline-rich region of protein tyrosine phosphatase 1B to the Src homology 3 domain of p130(Cas). J. Biol. Chem. 271:3129031295.
Mills, J.C., N.L. Stone, and R.N. Pittman. 1999. Extranuclear apoptosis. The role of the cytoplasm in the execution phase. J. Cell Biol. 146:703708.
Murphy-Ullrich, J.E. 2001. The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? J. Clin. Invest. 107:785790.
Palecek, S.P., C.E. Schmidt, D.A. Lauffenburger, and A.F. Horwitz. 1996. Integrin dynamics on the tail region of migrating fibroblasts. J. Cell Sci. 109:941952.
Parent, C.A., and P.N. Devreotes. 1999. A cell's sense of direction. Science. 284:765770.
Plattner, R., L. Kadlec, K.A. DeMali, A. Kazlauskas, and A.M. Pendergast. 1999. c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF. Genes Dev. 13:24002411.
Ruefli, A., M. Ausserlechner, D. Bernard, V. Sutton, K. Tainton, R. Kofler, M. Smith, and R. Johnstone. 2001. The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell death pathway characterized by cleavage of Bid and production of reactive oxygen species. Proc. Natl. Acad. Sci. USA. 98:1083310838.
Schreiber, V., L. Moog, C.H. Regnier, M.P. Chenard, H. Boeuf, J.L. Vonesch, C. Tomasetto, and M.C. Rio. 1998. Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions. Mol. Med. 4:675687.[Medline]
Smith, J.M., S. Katz, and B.J. Mayer. 1999. Activation of the Abl tyrosine kinase in vivo by Src homology 3 domains from the Src homology 2/Src homology 3 adaptor Nck. J. Biol. Chem. 274:2795627962.
Sun, X., P. Majumder, H. Shioya, F. Wu, S. Kumar, R. Weichselbaum, S. Kharbanda, and D. Kufe. 2000. Activation of the cytoplasmic c-Abl tyrosine kinase by reactive oxygen species. J. Biol. Chem. 275:1723717240.
Tabb, D.L., W.H. McDonald, and J.R. Yates. 2002. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1:2126.[Medline]
Tomasetto, C., L. Moog, C.H. Regnier, V. Schreiber, P. Basset, and M.C. Rio. 1995a. Lasp-1 (MLN 50) defines a new LIM protein subfamily characterized by the association of LIM and SH3 domains. FEBS Lett. 373:245249.[CrossRef][Medline]
Tomasetto, C., C. Regnier, L. Moog, M.G. Mattei, M.P. Chenard, R. Lidereau, P. Basset, and M.C. Rio. 1995b. Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17. Genomics. 28:367376.[CrossRef][Medline]
Truong, T., G. Sun, M. Doorly, J.Y. Wang, and M.A. Schwartz. 2003. Modulation of DNA damage-induced apoptosis by cell adhesion is independently mediated by p53 and c-Abl. Proc. Natl. Acad. Sci. USA. 100:1028110286.
Wang, B., E.A. Golemis, and G.D. Kruh. 1997. ArgBP2, a multiple Src homology 3 domain-containing, Arg/Abl-interacting protein, is phosphorylated in v-Abl-transformed cells and localized in stress fibers and cardiocyte Z-disks. J. Biol. Chem. 272:1754217550.
Washburn, M.P., D. Wolters, and J.R. Yates. 2001. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19:242247.[CrossRef][Medline]
Woodring, P.J., T. Hunter, and J.Y. Wang. 2003. Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases. J. Cell Sci. 116:26132626.
Wyckoff, J.B., J.G. Jones, J.S. Condeelis, and J.E. Segall. 2000. A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer Res. 60:25042511.
Yuan, Z.M., H. Shioya, T. Ishiko, X. Sun, J. Gu, Y.Y. Huang, H. Lu, S. Kharbanda, R. Weichselbaum, and D. Kufe. 1999. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature. 399:814817.[CrossRef][Medline]