ACCELERATED PUBLICATION
Cell Adhesion Regulates the Interaction between Nck and p21-activated Kinase*

Alan K. HoweDagger

Department of Pharmacology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7365

Received for publication, November 9, 2000, and in revised form, March 6, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The p21-activated kinases (PAKs) are important mediators of cytoskeletal reorganization, cell motility and transcriptional events regulated by the Rho family GTPases Rac and Cdc42. PAK activation by serum components is strongly dependent on cell adhesion to the extracellular matrix (ECM). PAK binds directly to the Nck adapter protein, an interaction thought to play an important role in regulation and localization of PAK activity. This report demonstrates that the interaction of PAK with Nck is regulated dynamically by cell adhesion. PAK-Nck binding is rapidly lost after cell detachment and rapidly restored after re-adhesion to the ECM protein fibronectin, suggesting a rapidly reversible mode of regulation. Furthermore, the loss of Nck binding correlates with changes in the phosphorylation state of PAK in nonadherent cells, as evidenced by electrophoretic mobility shift and phosphorylation within a sequence known to mediate interaction with Nck. The ability of cell adhesion to regulate PAK phosphorylation and interaction with Nck may contribute to the anchorage-dependence of PAK activation as well as to the localization of activated PAK within a cell.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The p21-activated kinase (PAK)1 family comprises the best characterized effectors for the Rho family GTPases Cdc42 and Rac (1). As such, PAK activity has been implicated in Cdc42- and Rac-mediated regulation of gene expression and modulation of the actin cytoskeleton and cell motility (2, 3). PAK is also involved in the regulation of signaling cascades involving the MAPK (4-6) and Jun N-terminal kinases (JNK) (5, 7, 8). PAK activity is stimulated by a variety of soluble factors (e.g. insulin, thrombin, PDGF (1, 2, 9)) as well as by cellular adhesion to ECM (10, 11). Recently, it has been reported that activation of PAK by soluble factors is highly dependent on cell adhesion (12, 13), and this may contribute to the anchorage-dependence of MAPK activation (13, 14). One of these reports offers strong evidence that the lack of PAK activation in nonadherent cells is due to an uncoupling of PAK from activated Rac at the plasma membrane (12). Specifically, the deficiency in PAK activation in nonadherent cells is not due to a dramatic reduction in Rac activation but correlates with a marked decrease in the recruitment of activated Rac to the cell membrane. This is particularly interesting given observations that forced localization of PAK to the plasma membrane strongly activates PAK (15-17). One hypothesis to explain this activation is that membrane localization of PAK is likely to increase the efficiency of its interaction with other membrane-associated regulatory elements, such as activated Rac (18) and sphingolipids (17, 18). Taken together, these data strongly suggest that membrane localization is a crucial aspect of PAK activation in vivo.

The recruitment of PAK to the cell membrane may be mediated by its direct interaction with the adapter protein Nck (19-22). The three SH3 domains mediate interaction with a large number of binding partners (23), while the SH2 domain binds to phosphotyrosine-containing proteins such as activated receptor tyrosine kinases (RTKs; e.g. PDGFR (20, 24, 25)) and the Crk-associated substrate p130CAS (26). Nck-PAK interaction is mediated through the second SH3 domain of Nck, which binds to a proline-rich region in the N terminus of PAK (19-21). In fibroblasts, this interaction occurs even in the absence of extracellular stimuli (19-21). This suggests that in unstimulated cells, PAK and Nck exist in a constitutive complex that can be rapidly relocated to membrane-associated, phosphotyrosine-rich sites generated by growth factor stimulation or adhesion to ECM. The importance of this interaction in regulating PAK function is underscored by the observations that expression of a membrane-targeted Nck SH3 domain leads to constitutive PAK activation (15) and that a PAK mutant unable to bind Nck is severely impaired in its ability to promote cytoskeletal organization (27).

Given the tight regulation of PAK activation by cell adhesion to ECM and the importance of Nck binding in regulating PAK activity, I investigated whether adhesion might affect PAK-Nck interaction. Herein, I show that the ability of PAK to bind Nck is highly dependent on cell adhesion and correlates with an anchorage-dependent change in PAK phosphorylation. These data have significant implications for the regulation and localization of PAK activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Antibodies-- Polyclonal antibodies against the N- or C terminus of PAK1 were from Santa Cruz Biotechnology. Antibodies against Nck included a monoclonal from Transduction Laboratories and a polyclonal from Santa Cruz Biotechnology. Polyclonal antibody against the PDGFbeta receptor was a generous gift from K. DeMali and A. Kazlauskas and was used as described (28). Phosphotyrosine was detected using monoclonal antibody 4G10 from Upstate Biotechnology.

Cell Culture-- NIH3T3 cells were cultured as described previously (13). Briefly, cells were serum-starved overnight and then either harvested or cultured in suspension. For suspension culture, cells were trypsinized, collected by centrifugation, washed once with DMEM, 2% BSA, 2 mg/ml trysin inhibitor and once with DMEM, 2% BSA, and then resuspended and rotated in the same at 37 °C for the indicated times. Where indicated, cells were replated, after 1 h in suspension, onto FN-coated tissue culture plates (13) for the indicated times.

Cell Lysis, Immunoprecipitation, and Western Blotting-- Cells were washed twice in ice-cold phosphate-buffered saline and then scraped or resuspended in lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM each EDTA and EGTA, 1% Triton X-100 containing protease and phosphatase inhibitors (Sigma)). Lysates were transferred to microcentrifuge tubes, vortexed vigorously for 30 s, incubated on ice for 10 min, and centrifuged at 4 °C for 10 min at ~15,000 × g. Precleared lysates were either frozen at -80 °C or further clarified by incubation with protein G-Sepharose beads (PharMingen) for 1 h at 4 °C and then used for immunoprecipitation. The lysates were incubated with antibodies for 1-2 h at 4 °C and then with protein G-coupled Sepharose beads (PharMingen) for 1 h. In most cases, complexes were washed three times with lysis buffer. For polyclonal Nck immunoprecipitations, complexes were washed once with lysis buffer and three times with phosphate-buffered saline containing 1% Tween 20. Washed complexes were boiled in 1× Laemmli sample buffer for 5 min. Samples were run on standard Bio-Rad mini-gels (10% for Nck Western blots, 7.5% for PAK and PDGFR Western blots) or on higher resolution gels contained 8% acrylamide and 0.5% bisacrylamide and were cast in 15 × 15-cm cassettes (Hoefer Scientific). Separated proteins were transferred to nitrocellulose or polyvinylidene difluoride membranes, which were then blocked and blotted using standard techniques.

Phosphatase Treatment-- PAK immunoprecipitates were washed three times with lysis buffer, twice with 0.05 M Tris, pH 7.0, 0.1 M NaCl, and once with buffer A (0.05 M Tris-HCl, pH 7.9, 0.1 M NaCl, 0.01 M magnesium chloride). Immunoconjugates were resuspended in 50 µl of buffer A alone or buffer A containing 0.5 units of calf intenstinal alkaline phosphatase (New England Biolabs) and incubated at 30 °C for 30 min. Immunoconjugates were then washed twice in buffer A and either resuspended in 1× Laemmli sample buffer and boiled for 5 min or resuspended in 50 µl of 0.02 M MES, pH 6.0, containing 0.75 units of potato acid phosphatase (Calbiochem) and incubated for 15 min at 30 °C. These latter samples were then washed in buffer A and boiled in Laemmli sample buffer as described above.

Phosphopeptide Analysis-- Cells were serum-starved overnight then labeled for 4 h with 1.0 mCi/ml 32P-labeled orthophosphate in phosphate-free DMEM containing 2% BSA. For the suspension culture, the labeling medium was removed, and the cells were rapidly washed, trypsinized, collected, and resuspended in the original labeling medium and then incubated at 37 °C with rotation for 1 h. Cells were harvested, and PAK was immunoprecipitated as described above. Radiolabeled PAK was purified on SDS-PAGE gels, and individual bands were excised and subjected to in-gel trypsinization as described elsewhere (13). Phosphopeptides were analyzed by two-dimensional separation on thin layer cellulose plates (13) or by HPLC (AP Biotechnology) on a reverse-phase C18 column (Vydac) developed with a linear gradient of aqueous acetonitrile. For some experiments, an excess of a synthetic peptide with the sequence NTSTMIGAGSK (single-letter code), corresponding to the tryptic peptide containing Ser21 of PAK1, was added to the mix of radioactive peptides before HPLC separation. Fractions were then analyzed by scintillation counting and spectrophotometry at 215 nm. The peptide was synthesized and purified by the University of North Carolina Peptide Synthesis Facility.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Recruitment of PAK to Activated RTK Complexes Requires Cell Adhesion-- Nck can recruit PAK to activated RTKs (e.g. PDGFR) upon growth factor stimulation, which may play a role in regulating PAK activity (20). Given the strong anchorage dependence of PAK activation by growth factors (12, 13), the effect of cell adhesion on recruitment of PAK to activated PDGFR was investigated (Fig. 1). As previously reported (29), PDGF-stimulated tyrosine phosphorylation of the PDGFR occurred efficiently in both adherent and nonadherent NIH3T3 cells. Growth factor stimulation of adherent cells induced the formation of a complex containing tyrosine-phosphorylated PDGFR, Nck, and PAK. In nonadherent cells, Nck was still recruited to the activated PDGFR, whereas PAK was completely absent from this complex; this suggested that although the SH2-dependent recruitment of Nck to activated receptor is anchorage-independent, the SH3-mediated interaction of Nck with PAK might be regulated by cell adhesion.


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Fig. 1.   Recruitment of PAK to activated PDGF receptor is anchorage-dependent. PDGFR was immunoprecipitated from adherent cells (Adh) or cells cultured in suspension for 1 h (Susp) before or after stimulation with 20 ng/ml PDGF-BB for 5 min as indicated. Immunocomplexes were separated by SDS-PAGE and analyzed by Western blotting with antibodies against the PDGFR (to confirm equal loading), Nck, and PAK. For analysis of PDGFR tyrosine phosphorylation, anti-PDGFR blots were stripped and reprobed with anti-phospotyrosine (P-Tyr) antibody.

The Interaction between PAK and Nck Is Anchorage-dependent-- To directly investigate the effects of cell adhesion on PAK-Nck interaction, anti-Nck Western blots were performed on PAK immunoprecipitates from stably adherent cells and cells that had been incubated in suspension (Fig. 2). Even in the absence of serum and growth factors, PAK immunoprecipitates from stably adherent cells contained readily detectable levels of Nck, consistent with previous reports (19, 20). However, little or no Nck was detected in PAK immunoprecipitates from nonadherent cells (Fig. 2). The lack of PAK-Nck association was not caused by a decrease in the amount of Nck protein in nonadherent cells, as shown by Nck Western blots of adherent and nonadherent cell extracts (data not shown and Fig. 4). Interestingly, the interaction between Nck and PAK was also ablated by treatment of adherent cells with cytochalasin D (Fig. 2). It is important to point out that although the concentration of cytochalasin D used in these experiments allowed cells to remain loosely attached, it completely disrupted the actin cytoskeleton and promoted cell rounding and complete tyrosine dephosphorylation of FAK (data not shown). This indicates that in NIH3T3 cells the growth factor-independent interaction between Nck and PAK requires a fully functional cellular interaction with the ECM.


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Fig. 2.   Cell adhesion regulates PAK-Nck interaction. PAK was immunoprecipitated from serum-starved, stably adherent cells (Adh), cells cultured in suspension (Susp), or adherent cells treated with 2.5 µM cytochalasin D for 30 min (CD). Immunocomplexes were separated by SDS-PAGE and analyzed by Western blotting with antibodies against PAK and Nck. 10 µg of whole cell extract (wce) from stably adherent cells was run alongside the immunoprecipitates to more easily distinguish bands of interest (indicated by arrowheads) from background Ig bands.

PAK-Nck Interaction Is Dynamically Regulated by Cell Adhesion-- To further investigate this anchorage-dependent interaction, the kinetics of PAK-Nck dissociation after cellular detachment were determined (Fig. 3). The amount of Nck co-precipitating with PAK dropped dramatically (~8-fold by densitometry) within 10 min after detachment and was essentially undetectable at 30 and 60 min. Interestingly, the loss of PAK-Nck interaction after detachment was rapidly reversed by replating cells onto the ECM protein fibronectin (FN). The amount of co-precipitating Nck in cells replated on FN for 15 min was ~90% of the level seen in stably adherent cells, whereas the level at 30 min after replating was indistinguishable from that of adherent cells (Fig. 3).


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Fig. 3.   PAK-Nck interaction is regulated reversibly and dynamically by cell adhesion. PAK was immunoprecipitated from serum-starved, stably adherent cells (Adh) and cells cultured in suspension (Susp) or replated onto FN-coated plates (FN) for the indicated periods of time (in minutes). Immunocomplexes were separated by SDS-PAGE and analyzed by Western blotting with antibodies against PAK and Nck. 10 µg of whole cell extract (wce) from stably adherent cells was run alongside the immunoprecipitates to distinguish more easily the bands of interest (indicated by arrowheads) from background Ig bands.

Nck Binding Correlates with PAK Phosphorylation-- The rapid kinetics of PAK-Nck dissociation and re-association suggest that the complex might be regulated by a rapidly reversible post-translational modification, such as phosphorylation. Both PAK and Nck are known phosphoproteins (1, 23). However, the role of phosphorylation in regulating Nck function has yet to be determined, and while many of the details of PAK phosphorylation in vitro have been elucidated, the complexity of its regulation in vivo is not completely understood, and its regulation by cell adhesion has never been investigated.

Importantly, a recent report demonstrates that the association of Nck and PAK can be regulated by phosphorylation (22). In this report, strong biochemical data indicated that phosphorylation of Ser21, a putative autophosphorylation site at the C-terminal end of the Nck-binding region of PAK1, dramatically reduced the interaction between PAK and Nck (22). Reduced Nck binding was also observed in cells in which PAK was robustly activated and hyperphosphorylated either by mutation or expression of a constitutively active mutant of Cdc42 (22), suggesting a negative feedback loop in which PAK activation down-regulates interaction with Nck.

Previous work has correlated PAK phosphorylation with an electrophoretic mobility shift on SDS-PAGE gels; specifically, increasing phosphorylation of PAK correlates with slower migration (22, 30). This correlation was utilized to investigate whether the loss of Nck binding in nonadherent cells coincided with a change in the phosphorylation state of PAK. Previous experiments in the current report were performed using standard mini-gels, and no shift in PAK mobility was readily apparent. Therefore, to better visualize potential PAK mobility shifts, subsequent experiments utilized larger, higher cross-linking gels (see "Experimental Procedures"). Using this method, PAK isolated from adherent, serum-starved cells runs as a discrete doublet, whereas in nonadherent cells, only the faster migrating form is present (Fig. 4A). A slower migrating form of PAK reappears within an hour of replating cells on FN (Fig. 4A). As expected, this mobility shift is due to a difference in phosphorylation, as the slower migrating species present in PAK immunoprecipitates from adherent cells are almost completely converted to the faster migrating form by phosphatase treatment in vitro (Fig. 4B). Significantly, this differential phosphorylation correlates closely with the loss of Nck binding in nonadherent cells (Fig. 4A) and suggests that Nck binds exclusively to the more highly phosphorylated, slower migrating form of PAK. This finding was confirmed in reciprocal co-immunoprecipitation experiments in which the PAK present in Nck immunoprecipitates co-migrated exclusively with the slower migrating form of PAK that is present in adherent cells but completely absent from nonadherent cells (Fig. 4C).


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Fig. 4.   Loss of PAK-Nck interaction correlates with dephosphorylation of PAK. A, PAK was immunoprecipitated from serum-starved, stably adherent cells (Adh), cells cultured in suspension (Susp), or cells replated onto FN-coated plates (FN) for 1 h. Immunocomplexes were separated in large, high cross-linker SDS-PAGE gels and analyzed by Western blotting with antibodies against PAK and Nck. Whole cell extracts from nonadherent or replated cells were run alongside the immunoprecipitates to distinguish bands of interest (indicated by arrowheads). B, PAK was immunoprecipitated from adherent cells or cells in suspension and incubated in phosphatase buffer alone or buffer containing potato acid phosphatase (PAP) and/or calf intenstinal phosphatase (CIP) as indicated. Proteins were resolved on high cross-linker SDS-PAGE gels and analyzed by Western blotting with antibodies against PAK. C, Nck was quantitatively immunoprecipitated from stably adherent cells, and the supernatant extract (Super) from this IP was immediately reimmunoprecipitated with antibodies against PAK. These immunocomplexes were run on high cross-linker gels alongside PAK IPs from stably adherent cells (Adh) or cells in suspension (Susp) and analyzed by anti-PAK Western blotting. The position of a 64,000-Da molecular mass marker (64) is indicated. D, 32P-labeled tryptic peptides of PAK isolated from adherent cells (white bars) or cells in suspension for 1 h (shaded bars) were doped with an excess of an unlabeled, synthetic peptide comprising amino acids 19-29 of PAK1 (see "Experimental Procedures") and separated by HPLC. Portions of the indicated fractions were analyzed by liquid scintillation counting (bars) to determine the phosphate content (in counts/minute (CPM)) or by spectrophotometry at 215 nm (dashed line; A215) to assess the presence of the synthetic peptide.

The loss of PAK-Nck interaction in nonadherent cells and its correlation with PAK dephosphorylation indicate that Nck binding is compromised under conditions in which PAK activity is very low, which is somewhat surprising in light of the recent report suggesting high levels of PAK activity are responsible for disrupting PAK·Nck complexes (22). However, that report is in stark contrast to an earlier report in which a constitutively active double-point mutant of PAK showed enhanced binding to Nck (27). The recovery of PAK·Nck complexes from cells stimulated with growth factors and other agonists also supports the notion that Nck remains associated with activated PAK under physiological conditions (15, 16, 19-21, 31, 32). Therefore, there are some apparent contradictions regarding the role of PAK activity or autophosphorylation in regulating Nck binding in vivo. However, there is still strong in vitro evidence indicating that PAK-Nck interaction can indeed be regulated by phosphorylation near the proline-rich Nck-binding region (22). How might these observations be reconciled with the current data?

One possible explanation is that Ser21 appears to be a relatively poor autophosphorylation site (30) and may be modified in this way only when PAK is activated to supraphysiological levels (e.g. by an excess of constitutively active Cdc42 or Rac). It is therefore possible that phosphorylation of this site is indeed responsible for regulating Nck binding but that in vivo this modification is catalyzed by a kinase other than PAK. Results thus far suggest that such a kinase may be activated upon cellular detachment. To directly investigate the possibility that cellular detachment might affect phosphorylation of this region, phosphopeptide mapping was performed on radiolabeled PAK immunoprecipitated from 32P-labeled adherent cells and cells in suspension. Separation of tryptic phosphopeptides by two-dimensional mapping or by HPLC revealed several adhesion-dependent changes in PAK phosphorylation (Fig. 4D and data not shown). To identify specific changes in phosphorylation in and around Ser21, an excess of purified, synthetic peptide corresponding to amino acids 19-29 of PAK1 (the tryptic fragment containing Ser21) was added to the phosphopeptide mixtures to serve as a marker during HPLC. Although PAK from adherent cells contained several phosphopeptides not present in the nonadherent sample (Fig. 4D, fractions 20, 29, and 30), PAK from nonadherent cells contained a phosphopeptide that co-eluted with the synthetic peptide (Fig. 4D, fraction 26). These data suggest that cellular detachment induces phosphorylation of PAK within a region known to regulate interaction with Nck.

Regulation of PAK phosphorylation in vivo is complex and not fully understood. The shift in electrophoretic mobility observed in Fig. 4A suggests a net dephosphorylation of PAK in nonadherent cells. However, Fig. 4D demonstrates that under these conditions PAK retains some phosphates and even acquires new modifications that are induced specifically, and perhaps uniquely, by the state of nonadherence. Considerable effort has established that PAK is a substrate for autophosphorylation (1, 30) as well as phosphorylation by other kinases (e.g. PDK1 (34), PKA (13)). Clearly, further analysis is required to address fully the regulation of PAK phosphorylation by cell adhesion. Among the most important issues to be addressed are the complete profile of PAK phosphorylation in adherent versus nonadherent cells and the kinases or phosphatases involved in this regulation.

The importance of Nck-mediated PAK localization to the membrane is likely to be 2-fold. First, relocation to the membrane places PAK in close proximity with membrane-associated activating factors, such as activated Rac (12) and sphingolipids (17), increasing the efficiency of their interaction and thereby the efficiency of PAK activation (18). Second, membrane localization of PAK places it in an appropriate setting to exert its effects on lamellipodia extension and cell motility, events that involve modulation of the actin cytoskeleton near the cell membrane (36). Another possibility is that by recruiting PAK to sites of active growth factor and/or cell adhesion signaling, the Nck-PAK interaction may be important in translating extracellular gradients into intracellular asymmetry and thus in establishing cell polarity (3). Given the ability of PAK to regulate the actin cytoskeleton, cell motility, cytoplasmic signaling cascades, and gene expression, the regulation of PAK-Nck interaction by cell adhesion provides an intriguing new facet of regulation to this important signaling complex.

    ACKNOWLEDGEMENTS

The guidance and generosity of R. L. Juliano are gratefully acknowledged. Lee Graves (University of North Carolina at Chapel Hill) generously provided advice and equipment for phosphopeptide analyses.

    FOOTNOTES

* This work was done in the laboratory of R. L. Juliano, who is supported by National Institutes of Health Grants GM26165 and HL45100.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a postdoctoral fellowship (PF-99-009-01-CSM) from the American Cancer Society. To whom correspondence should be addressed: Tel.: 919-966-4343; Fax: 919-955-5640; E-mail: Alan_Howe@med.unc.edu.

Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.C000797200

    ABBREVIATIONS

The abbreviations used are: PAK, p21-activated kinase; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; FN, fibronectin; GTPase, guanosine triphosphatase; HPLC, high-performance liquid chromatography; IP, immunoprecipitation; MAPK, mitogen-activated protein kinase; MES, 2-(N-morpholino)ethanesulfonic acid; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PAGE, polyacrylamide gel electrophoresis; SH, Src homology.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Knaus, U. G., and Bokoch, G. M. (1998) Int. J. Biochem. Cell Biol. 30, 857-862[CrossRef][Medline] [Order article via Infotrieve]
2. Bagrodia, S., and Cerione, R. A. (1999) Trends Cell Biol 9, 350-355[CrossRef][Medline] [Order article via Infotrieve]
3. Daniels, R. H., and Bokoch, G. M. (1999) Trends Biochem. Sci 24, 350-355[CrossRef][Medline] [Order article via Infotrieve]
4. Frost, J. A., Steen, H., Shapiro, P., Lewis, T., Ahn, N., Shaw, P. E., and Cobb, M. H. (1997) EMBO J. 16, 6426-6438[Abstract/Free Full Text]
5. Frost, J. A., Xu, S., Hutchison, M. R., Marcus, S., and Cobb, M. H. (1996) Mol. Cell. Biol. 16, 3707-3713[Abstract]
6. King, A. J., Sun, H., Diaz, B., Barnard, D., Miao, W., Bagrodia, S., and Marshall, M. S. (1998) Nature 396, 180-183[CrossRef][Medline] [Order article via Infotrieve]
7. Bagrodia, S., Derijard, B., Davis, R. J., and Cerione, R. A. (1995) J. Biol. Chem. 270, 27995-27998[Abstract/Free Full Text]
8. Zhang, S., Han, J., Sells, M. A., Chernoff, J., Knaus, U. G., Ulevitch, R. J., and Bokoch, G. M. (1995) J. Biol. Chem. 270, 23934-23936[Abstract/Free Full Text]
9. Avruch, J. (1998) Mol. Cell. Biochem. 182, 31-48[CrossRef][Medline] [Order article via Infotrieve]
10. Price, L. S., Leng, J., Schwartz, M. A., and Bokoch, G. M. (1998) Mol. Biol. Cell 9, 1863-1871[Abstract/Free Full Text]
11. Kiosses, W. B., Daniels, R. H., Otey, C., Bokoch, G. M., and Schwartz, M. A. (1999) J. Cell Biol. 147, 831-844[Abstract/Free Full Text]
12. del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., and Schwartz, M. A. (2000) EMBO J. 19, 2008-2014[Abstract/Free Full Text]
13. Howe, A. K., and Juliano, R. L. (2000) Nat. Cell Biol. 2, 593-601[CrossRef][Medline] [Order article via Infotrieve]
14. Howe, A., Aplin, A. E., Alahari, S. K., and Juliano, R. L. (1998) Curr. Opin. Cell Biol. 10, 220-231[CrossRef][Medline] [Order article via Infotrieve]
15. Lu, W., Katz, S., Gupta, R., and Mayer, B. J. (1997) Curr. Biol. 7, 85-94[Medline] [Order article via Infotrieve]
16. Lu, W., and Mayer, B. J. (1999) Oncogene 18, 797-806[CrossRef][Medline] [Order article via Infotrieve]
17. Bokoch, G. M., Reilly, A. M., Daniels, R. H., King, C. C., Olivera, A., Spiegel, S., and Knaus, U. G. (1998) J. Biol. Chem. 273, 8137-8144[Abstract/Free Full Text]
18. Symons, M. (2000) Curr. Biol. 10, R535-537[CrossRef][Medline] [Order article via Infotrieve]
19. Bokoch, G. M., Wang, Y., Bohl, B. P., Sells, M. A., Quilliam, L. A., and Knaus, U. G. (1996) J. Biol. Chem. 271, 25746-25749[Abstract/Free Full Text]
20. Galisteo, M. L., Chernoff, J., Su, Y. C., Skolnik, E. Y., and Schlessinger, J. (1996) J. Biol. Chem. 271, 20997-21000[Abstract/Free Full Text]
21. Bagrodia, S., Taylor, S. J., Creasy, C. L., Chernoff, J., and Cerione, R. A. (1995) J. Biol. Chem. 270, 22731-22737[Abstract/Free Full Text]
22. Zhao, Z. S., Manser, E., and Lim, L. (2000) Mol. Cell. Biol. 20, 3906-3917[Abstract/Free Full Text]
23. McCarty, J. H. (1998) Bioessays 20, 913-921[CrossRef][Medline] [Order article via Infotrieve]
24. Tang, J., Feng, G. S., and Li, W. (1997) Oncogene 15, 1823-1832[CrossRef][Medline] [Order article via Infotrieve]
25. Chen, M., She, H., Kim, A., Woodley, D. T., and Li, W. (2000) Mol. Cell. Biol. 20, 7867-7880[Abstract/Free Full Text]
26. Schlaepfer, D. D., Broome, M. A., and Hunter, T. (1997) Mol. Cell. Biol. 17, 1702-1713[Abstract]
27. Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M., and Chernoff, J. (1997) Curr. Biol. 7, 202-210[Medline] [Order article via Infotrieve]
28. DeMali, K. A., Balciunaite, E., and Kazlauskas, A. (1999) J. Biol. Chem. 274, 19551-19558[Abstract/Free Full Text]
29. Lin, T. H., Chen, Q., Howe, A., and Juliano, R. L. (1997) J. Biol. Chem. 272, 8849-8852[Abstract/Free Full Text]
30. Manser, E., Huang, H. Y., Loo, T. H., Chen, X. Q., Dong, J. M., Leung, T., and Lim, L. (1997) Mol. Cell. Biol. 17, 1129-1143[Abstract]
31. Yablonski, D., Kane, L. P., Qian, D., and Weiss, A. (1998) EMBO J. 17, 5647-5657[Abstract/Free Full Text]
32. Voisin, L., Larose, L., and Meloche, S. (1999) Biochem. J. 341, 217-223[CrossRef][Medline] [Order article via Infotrieve]
33. King, C. C., Zenke, F. T., Dawson, P. E., Dutil, E. M., Newton, A. C., Hemmings, B. A., and Bokoch, G. M. (2000) J. Biol. Chem
34. Borisy, G. G., and Svitkina, T. M. (2000) Curr. Opin. Cell Biol. 12, 104-112[CrossRef][Medline] [Order article via Infotrieve]


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