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Originally published In Press as doi:10.1074/mcp.M600101-MCP200 on April 26, 2006.
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Molecular & Cellular Proteomics 5:1359-1367, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Functional Proteomics Identifies Protein-tyrosine Phosphatase 1B as a Target of RhoA Signaling*,S

Yukihito Kabuyama{ddagger},{ddagger}{ddagger},, Stephen J. Langer||, Kirsi Polvinen{ddagger},§,**, Yoshimi Homma{ddagger}{ddagger}, Katheryn A. Resing{ddagger} and Natalie G. Ahn{ddagger},§,§§

From the Departments of {ddagger} Chemistry and Biochemistry and || Molecular, Cellular, and Developmental Biology and § Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado 80309-0215 and {ddagger}{ddagger} Department of Biomolecular Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.

Rho family members (Rho, Rac, and Cdc42) belong to the Ras superfamily of small GTPases. Members of this class of signaling molecules function as molecular switches by cycling between active, GTP-bound and inactive, GDP-bound forms (1–3). This cycle is known to be activated by various guanine nucleotide exchange factors and inactivated by GTPase-activating proteins. Once activated, Rho GTPases directly bind to effector target molecules to initiate downstream responses. A major function of Rho GTPases is to regulate polymerization of the actin cytoskeleton and related cellular processes such as cell morphogenesis, migration, invasion, and axon guidance (4–6). In addition to their well established function in actin polymerization, Rho GTPases also control a wide range of biological processes, including gene transcription, cell cycle regulation, microtubule dynamics, membrane transport, and cell transformation (7–9). This functional diversity is reflected by a large number of regulators and effector molecules. Recent reports estimate more than 70 guanine nucleotide exchange factors, 80 GTPase-activating proteins, and 60 effector proteins for Rho, Rac, and Cdc42 in the human genome (5, 10).

Key effectors that link Rho GTPases to actin include (i) p21-associated protein kinase (PAK), 1 which activates the LIM kinase-cofilin pathway for controlling actin severing and polymerization in response to Rac and Cdc42 (11); (ii) Wiskott-Aldrich syndrome protein and Wiskott-Aldrich verprolin homologous protein family members, effectors of Cdc42 and Rac, respectively, which bind the Arp2/3 complex and promote cortical actin polymerization at filopodia and lamellipodia (12); (iii) Rho kinase, which promotes actomyosin contractility by phosphorylation and inactivation of myosin light chain phosphatase (1); and (iv) Diaphanous/formin family members, effectors of Cdc42 and Rho, which drive profilin-actin polymerization coupled to ATP hydrolysis (13). However, less well understood are downstream targets that presumably regulate other cellular processes that may be dependent on the cellular context. This prompts the need for proteomic screening of Rho GTPase pathways.

Protein expression profiling using two-dimensional electrophoresis (2-DE) combined with protein identification by MS represents an important strategy to monitor molecular responses induced by the activation or inhibition of specific signaling pathways (14). Studies using this approach have led to the successful identification of novel molecular responses downstream of mitogen-activated protein kinase (MAPK) (15), transforming growth factor ß (16), endothelin1 (17), and Fas (18) signaling pathways. Alternative protein profiling strategies using ICAT and electrospray ionization tandem mass spectrometry have also identified targets of Myc signaling (19). These studies have revealed novel targets and new cellular functions of signaling pathways, clearly indicating the validity of proteomics as an important discovery-based experimental approach.

Here we used 2-DE/MS to screen for molecular targets of RhoA, Cdc42, and Rac1 signaling pathways. A total of 22 proteins were identified by this screen of which 19 were novel targets of Rho GTPases. We further demonstrate that one of these new targets, protein-tyrosine phosphatase 1B (PTP1B), is controlled by post-translational modification in a manner that correlates with RhoA-dependent inactivation of phosphatase activity. This occurs in parallel with RhoA-dependent tyrosine phosphorylation of p130Cas, a critical mediator of focal adhesion turnover, and both events diverge from the well established Rho kinase effector pathway. Thus, PTP1B represents a novel mediator of RhoA signaling that controls phosphorylation of p130Cas by a Rho kinase-independent mechanism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—
WM35 human melanoma cell lines obtained from Dr. Meenhard Herlyn (Wistar Institute) were maintained in 10% fetal bovine serum + RPMI 1640 medium. Adenoviruses expressing constitutively active mutants RhoA-V14, Rac1-V12, or Cdc42-V12 were prepared by recombination into the Ad5 genome using a ß-galactosidase shuttle vector (20). Infection was carried out at 20 plaque-forming units/cell for 48 h, yielding >95% cell expression efficiency. Controls were performed by parallel infections using empty adenovirus (Ad-CMV). In some cases cells were pretreated for 30 min with Y27632 (30 µg/ml, Calbiochem) to inhibit Rho kinase/ROCK. Retinal pigment epithelial cells immortalized by expression of human telomerase reverse transcriptase (hTERT-RPE, Clontech) were grown in 10% fetal bovine serum + Dulbecco’s modified Eagle’s medium/F-12. Transient transfection of plasmid pMT2-GST-PTP1B-wt (from Nicholas Tonks, Cold Spring Harbor Laboratories) to 90% efficiency was obtained in retinal pigment epithelial (RPE) cells using FuGENE (Stratagene) at a 6:2 ratio of FuGENE:cDNA.

2-DE—
Cells were washed two times with PBS, extracted, and processed for isoelectric focusing as described previously (21) using IPG gel strips (pI 4–7 and 6–11, 18 cm, Amersham Biosciences) and 8–18% SDS-PAGE. Analytical (150 µg of protein) or preparative (400 µg) gels were silver-stained using formaldehyde or methanol fixation, respectively (22). Gels were analyzed using Melanie III software (GeneBio), measuring protein intensities by percent volume (pixel intensities integrated over each area and divided by the sum of all intensities) and correcting each spot volume by subtracting a background volume of equal area. Statistics carried out on at least three gels from two independent experiments showed significant change in intensity by Student’s t test (p < 0.01).

Mass Spectrometry—
Proteins were excised from wet gels, destained, digested in-gel with modified porcine trypsin (100 ng/digestion, Promega), and desalted on C18 ZipTips (Millipore). Peptides were cocrystallized with 2,5-dihydroxybenzoic acid on MALDI plates and analyzed using a Pulsar QqTOF mass spectrometer (Applied Biosystems). Peptide fingerprint spectra were summed over 50 acquisitions, and masses were matched against the National Center for Biotechnology non-redundant (NCBInr) database using MS-Fit (prospector.ucsf.edu). At least two peptides in each digest were sequenced by MS/MS to confirm protein identifications using MS-Tag. Mass tolerances of precursor and fragment ions were 0.1 Da, and searches specified trypsin cleavages.

Antibodies and Western Blotting—
Cells were lysed in 50 mM Tris-HCl, pH 7.2, 10 mM dithiothreitol, and protein was determined using the DC assay (Bio-Rad). Extracts (30 µg of protein) were separated by SDS-PAGE and transferred to PVDF membranes. Primary antibodies used included anti-PTP1B (1:1000, rabbit, HS135, Santa Cruz Biotechnology), anti-FAK-Tyr(P)397 (1:1500, rabbit, BIOSOURCE), anti-paxillin-Tyr(P)118 (1:500, mouse, BD Transduction Laboratories anti-RhoA (1:1000, mouse, Santa Cruz Biotechnology), anti-phosphatidylinositol transfer protein ß (mouse, BD Transduction Laboratories, 1:1000), anti-tryptophanyl-tRNA synthetase (rabbit, 1:10,000; Ref. 23), and anti-fatty acid-binding protein 5 (FABP5) (rabbit, 1:2500; Ref. 24). Blots were probed with donkey anti-mouse or anti-rabbit secondary antibody (1:10,000) coupled to horseradish peroxidase (Jackson Immunoresearch) and visualized by enhanced chemiluminescence (Amersham Biosciences). For 2-DE-Western blotting, extracts (75 µg of protein) were separated by 2-DE and transferred to PVDF membranes.

PTP1B and F-actin Staining—
RPE cells were grown on coverslips, fixed with 4% formalin (Sigma), and permeabilized with 0.1% Triton X-100. Coverslips were incubated with rhodamine-coupled phalloidin (3.3 nM, Molecular Probes) and mounted on glass slides, and cells were visualized with a Zeiss Axioplan II fluorescence microscope. Alternatively cells were infected for 48 h with Ad-CMV or Ad-RhoA-V14 and immunostained with anti-PTP1B antibody (1:1000, FG6 monoclonal, kindly provided by Dr. Tonks) and goat anti-mouse Alexa Fluor 488 (Molecular Probes).

PTP1B Assays—
Cells were lysed in 50 mM Hepes buffer, pH 7.4, containing 0.5% Triton X-100, 10% glycerol, 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 µg/ml pepstatin A. Activity of PTP1B was measured spectrophotometrically using a protein-tyrosine phosphatase assay kit (Sigma). Briefly PTP1B was immunoprecipitated from cell lysates (200 µg of protein), and the immunoprecipitates were washed three times with phosphate-free water (Sigma) and then resuspended in 25 mM imidazole, pH 7.0, containing 2 mM EDTA, 50 mM NaCl, and 5 mM DTT. The reactions were initiated by the addition of 200 µM synthetic phosphorylated peptide substrate (corresponding to insulin receptor-Tyr(P)1146 (residues 1142–1153), Sigma). After incubation at room temperature for 10 min, the reactions were stopped by addition of malachite green/ammonium molybdate, which complexed with inorganic phosphate formed in the reaction, and was quantified by UV-visible spectrophotometry at 620 nm (Shimadzu UV1601 spectrophotometer). To inhibit PTP1B expression, cells were incubated for 48 h with a mixture of four RNAi oligonucleotide sequences (human PTP1B SmartPool, Dharmacon).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Rho GTPase Targets by Functional Proteomics—
To screen for Rho GTPase targets, we carried out 2-DE and monitored changes in protein expression in response to Rho GTPases. Protein responses were induced in WM35 human melanoma cell lines by using adenoviral delivery to express constitutively active mutants RhoA-V14, Rac1-V12, and Cdc42-V12, or empty virus (CMV). Cell lysates were prepared 48 h post-infection, and proteins were separated by 2-DE over pI ranges 4–7 and 6–11, which resolved ~3500 and ~1500 protein spots, respectively (Fig. 1). Proteins were scored when spot intensities changed by more than 1.5-fold in three replicate gels from two independent experiments (Fig. 2A). This analysis reproducibly revealed 17, nine, and six protein spot changes in response to Rac1, Cdc42, and RhoA, respectively (Fig. 2B). Interestingly most of the Rac1 targets found in this study (14 of 17) were not regulated by Cdc42, a pathway that shares many effectors with Rac1. This result suggests that several targets are controlled by Rac1 but not Cdc42 in these cells.


Figure 1
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  		FIG. 1. Detection of Rho GTPase-responsive proteins. Shown are representative silver-stained 2-DE gels of whole cell extracts from WM35 cells (150 µg of protein for pI 4–7; 100 µg for pI 6–11). WM35 cells were infected with empty adenovirus (CMV) or adenovirus expressing constitutively active Cdc42-V12 (multiplicity of infection = 20) for 48 h. Arrows show proteins increased in intensity following activation of Cdc42. Similar experiments were carried out in WM35 cells expressing constitutively active Rac1-V12 or RhoA-V14 (not shown).

 

Figure 2
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  		FIG. 2. Proteins altered in response to Rac1, Cdc42, and RhoA. A, selected regions of 2-DE gels illustrating candidate protein spots U2, U15, U20, U23, U25, and U26 (indicated by arrows). Large arrowheads represent protein forms that were differentially up- or down-regulated in response to activation of Rac1, Cdc42, or RhoA. B, Venn diagram showing protein spots identified by their responses to activation of Rac1, Cdc42, or RhoA. U15, U16, and U17 were regulated by both Rac1 and Cdc42, whereas U21, U22, and U23 were regulated by both Cdc42 and RhoA. These proteins were identified by in-gel digestion, peptide mass fingerprinting, and MS/MS sequencing as summarized in Table I.

 
Protein spots were subjected to in-gel digestion, and proteins were identified by peptide mass fingerprinting and confirmed by peptide sequencing using tandem mass spectrometry. Twenty-three protein spots were reliably identified by mass spectrometry, representing 22 distinct proteins (Table I and Supplemental Tables 1 and 2). Surprisingly only three proteins were previously reported to be regulated by Rho GTPases. These included tropomyosin, cofilin, and stathmin, which are known downstream targets of RhoA, Rac1/Cdc42, and Rac1, respectively (25–27). The remaining 19 targets include proteins involved in cytoskeleton regulation (tubulin cofactor, calponin 3, and destrin), phospholipid turnover (phosphatidylinositol transfer protein), protein adduct formation with oxidized lipids (fatty acid-binding protein 5), protein folding (BiP), and phospholipid binding/cellular apoptosis (annexin 5). Four targets represented hypothetical proteins without matches to characterized genes. Thus, proteomic profiling effectively identified novel targets and suggested new functions of Rho GTPases in previously unrecognized cellular processes.


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  		TABLE I Identification of protein targets by peptide fingerprinting and MS/MS sequencing

Obs., observed; Calc., calculated; UNK, unknown.

 
Next the effects of Rho GTPases on the expression and covalent modification of four of the novel targets (FABP5, tryptophanyl-tRNA synthetase (TrpRS), PTP1B, and phosphatidylinositol transfer protein ß (PITPß)) were analyzed. Extracts were separated by 2-DE, and proteins were visualized by Western blotting using selective antibodies (Fig. 3). In the case of FABP5, we found that the intensity of one immunoreactive protein spot decreased significantly in response to active Rac1. Antibodies against TrpRS reacted with three protein spots with similar mass but running at different pI values, suggesting the existence of various post-translationally modified forms. All forms increased to the same extent upon activation of Rac1, suggesting that Rac1 regulates TrpRS at the level of expression. Antibodies against PTP1B and PITPß each reacted with several protein spots. Expression of active Cdc42 enhanced the intensity of a form of PITPß migrating at more acidic pI, suggesting regulation of post-translational modification. Likewise activation of RhoA induced a significant shift in distribution of spots corresponding to PTP1B toward acidic pI. These results clearly demonstrated the ability of 2-DE-based approaches to detect protein modifications regulated by signaling pathway activation. Taken together, Western blotting analyses confirmed selective responses of several Rho GTPase targets identified by 2-DE, supporting the accuracy of the initial protein screen.


Figure 3
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  		FIG. 3. 2-DE-Western blotting reveals evidence for post-translational modifications of novel Rho GTPase targets. Extracts of WM35 cells stimulated with Rac1, Cdc42, and RhoA were separated by 2-DE, and proteins were transferred to membranes. Western blots were probed with specific antibodies recognizing FABP5, TrpRS, PITPß, and PTP1B. Antibodies specific for the latter three proteins reacted with protein spots with similar mass but varying pI, suggesting the existence of covalently modified forms. Arrowheads indicate those protein spots detected by their altered intensities on silver-stained gels in response to Rho GTPase stimulation.

 
RhoA Promotes Phosphorylation of p130Cas through the Inhibition of PTP1B—
Studies were carried out to examine the functional regulation of PTP1B, which was shown to be regulated post-translationally by RhoA. Although PTP1B has never been linked to RhoA signaling, a potential connection between these proteins was suggested by their common regulation of p130Cas. p130Cas is a component of focal adhesion complexes that is known to be tyrosine phosphorylated in response to various stimuli, leading to enhanced binding interactions with the proto-oncogene Crk and increased cell migration. Previous studies by Narumiya and co-workers (28) reported that RhoA promotes tyrosine phosphorylation of p130Cas, whereas a study by Chernoff and co-workers (29) showed that PTP1B directly binds and dephosphorylates p130Cas. Thus, it seemed possible that RhoA regulates tyrosine phosphorylation of p130Cas by inactivation of PTP1B.

To test this possibility, we analyzed the effect of stimulating various Rho GTPases on PTP1B activity. Endogenous PTP1B was immunoprecipitated from WM35 cells, and its activity was measured by an in vitro assay measuring release of inorganic phosphate upon dephosphorylation of a phosphopeptide corresponding to insulin receptor-Tyr(P)1146. Fig. 4A shows the specific phosphatase activity of PTP1B (top panel) normalized by the levels of PTP1B protein in the immunoprecipitates (bottom panel). The specific activity of PTP1B was reduced by ~50% in response to expression of active mutant RhoA. This represents a significant reduction, given that most PTP1B protein in cells is associated with various intracellular compartments, predicting differential regulation of spatially distinct forms. In contrast, significant effects on phosphatase activity were not observed in cells expressing active Rac1 or Cdc42, indicating that PTP1B activity is regulated selectively by RhoA.


Figure 4
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  		FIG. 4. PTP1B is inactivated in response to RhoA. A, endogenous PTP1B was immunoprecipitated from WM35 cells stimulated by Rac1, Cdc42, and RhoA, and its specific activity (top panel) was measured by in vitro protein-tyrosine phosphatase assays normalized to total protein in the immunoprecipitates (bottom panel). B, extracts at 12, 18, and 24 h after adenoviral delivery of RhoA-V14 were monitored for tyrosine phosphorylation of p130Cas using anti-phosphotyrosine and anti-p130Cas antibodies to probe Western blots of immunoprecipitated p130Cas and anti-RhoA to probe blots of whole cell lysates. C, time course of PTP1B inactivation, measured as in A, correlates with the time course of PTP1B modification (D), measured by Western blotting of 2-DE gels.

 
Next we analyzed the time course of PTP1B reactivity by 2-DE-Western blotting and correlated the onset of its covalent modification with the loss of phosphatase-specific activity. Constitutively active RhoA was expressed by 12 h after infection and remained elevated for 24 h (Fig. 4B). This correlated well with the time course of inactivation and modification of PTP1B (Fig. 4, C and D). Furthermore the time course of p130Cas phosphorylation correlated with PTP1B inactivation (Fig. 4B). Such results suggested that RhoA regulates inactivation of p130Cas via covalent modification.

To confirm the importance of PTP1B toward p130Cas, we overexpressed PTP1B and examined its effect on tyrosine phosphorylation of p130Cas under conditions of RhoA activation. These experiments were carried out using human RPE cells, which enable expression in 90% of cells following transient transfection (data not shown). Upon activation of RhoA by expression of RhoA-V14, RPE cells showed characteristic responses of enhanced cell adhesion and stress fiber formation as indicated by enhanced F-actin polymerization (Fig. 5A, top, panel 2) and elevated tyrosine phosphorylation of focal adhesion kinase (FAK-Tyr(P)397) and paxillin (Tyr(P)118) (Fig. 5B, lane 2). In parallel, RhoA activation enhanced the covalent modification of PTP1B as shown by visualization of forms shifted to acidic pI by 2-DE-Western blotting (Fig. 5A, bottom, panel 2). RhoA also enhanced the tyrosine phosphorylation of p130Cas (Fig. 5B, lane 2). Expression of dominant negative RhoA to inhibit RhoA signaling also suppressed tyrosine phosphorylation of p130Cas to levels below basal (Fig. 5C). Thus, the responses to RhoA in RPE cells were consistent with those observed in WM35 cells.


Figure 5
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  		FIG. 5. PTP1B mediates RhoA-dependent tyrosine phosphorylation of p130Cas and diverges from Rho kinase-dependent stress actin fiber formation and phosphorylation of focal adhesion proteins. Shown are RPE cells expressing RhoA-V14, PTP1B-wild type, or RhoA + PTP1B or pretreated with Y27632 prior to expression of RhoA-V14. A, top panels, F-actin visualized by immunofluorescence after cell staining with phalloidin. Bottom panels, 2-DE-Western blotting of PTP1B. F-actin as well as acidic forms of PTP1B were increased in response to RhoA, but only F-actin was suppressed by Y27632, an inhibitor of Rho kinase. PTP1B covalent modification was unaffected by Y27632, and its expression did not affect F-actin. B, Western blots showing tyrosine phosphorylation of p130Cas and focal adhesion complex proteins, FAK and paxillin. PTP1B blocks tyrosine phosphorylation of p130Cas, but not FAK or paxillin, in response to RhoA. On the other hand, Y27632 inhibits RhoA-dependent phosphorylation of FAK and paxillin but not p130Cas, indicating coregulation of PTP1B and p130Cas through a pathway separate from Rho kinase. C, Western blots showing enhancement of phospho-p130Cas by active RhoA-V14 (CA) and suppression of this signal below basal levels by dominant negative RhoA-N19 (DN). D, transfection of RPE cells, titrating GST-PTP1B at constant RhoA-V14 and titrating RhoA-V14 at constant GST-PTP1B. E, treatment of RPE cells with RhoA-V14 in the presence or absence of PTP1B-RNAi. Knockdown of PTP1B increases phospho-p130Cas levels and allows no further increase in response to RhoA-V14. F, cellular localization of PTP1B by indirect immunofluorescence of RPE cells infected with Ad-CMV or Ad-RhoA-V14. pY, phosphotyrosine.

 
We then expressed full-length PTP1B into RPE cells using a mammalian expression vector. The recombinant GST-PTP1B appeared as a higher mass form on 2-DE gels (Fig. 5A, bottom, panel 3) with no effect on actin stress fibers (Fig. 5A, top, panel 3) or tyrosine phosphorylation of p130Cas, FAK, or paxillin (Fig. 5B, lane 3). However, when transfected cells were subsequently stimulated with RhoA, PTP1B effectively blocked tyrosine phosphorylation of p130Cas (Fig. 5B, lane 4). Upon titrating PTP1B against CA-RhoA, we observed that increasing PTP1B caused further reduction in the level of phospho-p130Cas induced by CA-RhoA, whereas increasing CA-RhoA reversed the suppression of phospho-p130Cas by submaximal levels of PTP1B (Fig. 5D). Furthermore knockdown of PTP1B by RNAi led to elevated phospho-p130Cas, whereas CA-Rho transfected in the presence of PTP-1B RNAi showed little ability to further increase p130Cas phosphorylation (Fig. 5E). Together these results signify that p130Cas is regulated physiologically by PTP1B and that the mechanism by which RhoA increases p130Cas phosphorylation is dependent on PTP1B.

Importantly PTP1B expression had no effect on RhoA-dependent stress fiber formation (Fig. 5A, top, panel 4) or FAK/paxillin tyrosine phosphorylation (Fig. 5B, lane 4), indicating that the effect of PTP1B was selective for RhoA-stimulated tyrosine phosphorylation events occurring on p130Cas. This was confirmed by examining responses to inhibition of Rho kinase/ROCK, a key effector of RhoA known to be required for focal adhesion complex formation and stress fiber bundling. Pretreatment of cells with a cell-permeable inhibitor of Rho kinase (Y27632) prior to RhoA stimulation resulted in suppressed stress fiber formation (Fig. 5A, top, panel 5) and suppressed FAK/paxillin tyrosine phosphorylation (Fig. 5B, lane 5) as expected. However, Y27632 had no effect on RhoA-dependent covalent modification of PTP1B (Fig. 5A, panel 5), and neither did it affect the tyrosine phosphorylation of p130Cas (Fig. 5B, lane 5), confirming that these targets are co-regulated through a pathway distinct from Rho kinase, phosphorylation of focal adhesion proteins, and formation of stress fibers. Finally we examined the cellular localization of PTP1B by indirect immunofluorescence. The characteristic localization of PTP1B around the endoplasmic reticulum was identical in the presence or absence of active RhoA (Fig. 5G), suggesting that RhoA does not affect compartmentalization of the phosphatase. Taken together, the results support a role for PTP1B in regulating p130Cas and suggest that one mechanism by which RhoA enhances tyrosine phosphorylation of p130Cas may involve derepression of p130Cas phosphorylation by inactivation of PTP1B.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study provide two new insights into the regulation of Rho GTPase signaling pathways. First, proteomic screening revealed many previously unidentified targets of RhoA, Rac1, and Cdc42, suggesting novel connections between Rho GTPases and cellular processes not previously linked to these pathways. Second, we identified a novel role for PTP1B as a mediator of RhoA signaling and supported this finding with evidence that PTP1B functions to link RhoA with tyrosine phosphorylation of p130Cas, acting within a branch point pathway that bifurcates upstream of Rho kinase (Fig. 6).


Figure 6
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  		FIG. 6. A model for RhoA-dependent regulation of p130Cas via PTP1B. Evidence presented in this study indicates that RhoA regulates covalent modification and activity of PTP1B and that PTP1B inhibits p130Cas tyrosine phosphorylation, presumably by catalyzing phosphotyrosine dephosphorylation. Thus PTP1B functions downstream of RhoA and upstream of p130Cas. PTP1B is likely to function downstream of the RhoA effector mDia/formin, which is known to mediate tyrosine phosphorylation of p130Cas. Consistent with this, we found that PTP1B and p130Cas regulation diverge from RhoA-dependent increases in stress fiber formation and tyrosine phosphorylation of focal adhesion complex components FAK and paxillin. PTP1B may indirectly inhibit Rac and cell migration, which are known to be downstream of mDia-p130Cas. MLC, myosin light chain.

 
PTP1B has received much attention due to its proposed role in cancer, type 2 diabetes, and obesity (30). It is ubiquitously expressed in mammalian cells and present at many cellular locations. Particularly interesting is its localization at focal adhesions as well as the tips of growing neurites (31, 32), intracellular compartments known to be regulated by Rho GTPases. A recent study has suggested the possibility of functional interactions between Rho GTPases and PTP1B. Dadke and Chernoff (33) have reported that in rat 3Y1cells overexpression of PTP1B leads to activation of Rac1 and promotes the formation of membrane ruffles, whereas reduction of PTP1B expression causes significant inhibition of these events. In contrast, overexpression of PTP1B blocks inactivation of RhoA by insulin. This report suggests that PTP1B may be involved in signaling upstream of Rac or Rho. In addition, PTP1B has been found to either positively (34) or negatively (35, 36) regulate integrin signaling by direct dephosphorylation of c-Src and p130Cas (29, 34), and Arregui et al. (31) have reported that catalytically inactive mutants of PTP1B negatively regulate cell adhesion, cell spreading, and focal adhesion formation. Such reports suggest cross-regulatory interactions between Rho and PTP1B, prompting our further investigation.

Our study provides novel evidence that PTP1B also functions downstream of RhoA and that RhoA-mediated modification and inactivation of PTP1B plays a role in regulating p130Cas phosphorylation. These results are consistent with previous studies showing that phosphorylation of p130Cas is regulated positively by RhoA (28) and negatively by PTP1B (29). In addition, Tsuji et al. (28) have shown that p130Cas tyrosine phosphorylation is promoted by RhoA through the formin effector mDia, consistent with our results indicating that RhoA-PTP1B-p130Cas diverges from pathways involving RhoA-Rho kinase and stress fiber formation. Recently RhoA-mDia-p130Cas has been shown to promote Rac activation, membrane ruffling formation, and cell migration (28). Thus, we speculate that PTP1B may indirectly inhibit Rac activation and cell migration and that Rho may depress this regulation by lowering the specific activity of PTP1B (Fig. 6). A recent report showing that PTP1B negatively regulates migration of HeLa cells supports such a model (37).

Our screen represents the first application of 2-DE proteomics toward exploring molecular targets of Rho GTPase signaling pathways. Overall 26 protein spots were differentially regulated by Rho GTPases. These yielded successful identifications of 22 different proteins, including three that were previously characterized as signaling targets of Rho, Rac, or Cdc42, confirming the reliability of the screen. However, the majority of targets were novel, suggesting that the cellular functions of Rho GTPase pathways are likely to be broader than previously recognized. This result, as well as those from other studies (15, 16), illustrates the validity of proteomic approaches for discovery of novel signaling pathway functions.

Approximately 4500 protein spots could be visualized by silver staining of 2-DE gels in these experiments, suggesting that ~0.5% of cellular proteins may be responsive to Rho GTPases. However, many protein targets previously shown to be regulated by Rho GTPases (e.g. PAK and Rho kinase) were not observed. This was due to several factors. First, 2-DE-based protein profiling lacks sufficient depth of sampling to comprehensively survey all proteins represented in human proteomes. Insolubility adds to the problem of protein separation, selecting against cytoskeletal protein targets. Second, known targets of Rho GTPases are predominantly represented by effectors that bind to their GTP-bound forms, whereas binding interactions would not be observable by our profiling method. Compared with the number of direct effectors for Rho GTPases, the number of cellular proteins known to be regulated by these pathways at the level of expression or covalent modification seems relatively small. Third, not all targets of a given signaling pathway characterized in any given cell type are necessarily regulated by the same pathway in all cells. Thus, stathmin, which is phosphorylated in response to Rac and Cdc42 signaling in Hep-2 cells (27), was observed to be modulated in response to Rac, but not Cdc42, in WM35 cells.

The identification of cytoskeletal protein (tropomyosin) and their regulators (stathmin, cofilin, destrin, acidic calponin, and tubulin cofactor A) in this screen was consistent with the well known functions of Rho GTPases as regulators of the cytoskeleton. Cofilin is a known target of Rac and is regulated by LIM kinase. Phosphorylation by LIM kinase down-regulates the actin severing activity of cofilin, which is believed to function in facilitating Rac-dependent actin polymerization (26). Although a role for destrin in Rac signaling had not been described up to now, a corresponding function in Rac-dependent cell migration is likely based on its actin severing activity and sequence similarity to cofilin. Acidic calponin is an actin- and myosin-binding protein that is involved in the fixation of actin cytoskeleton to the plasma membrane (38). A recent report indicated that µ-calpain can cleave acidic calponin, which could regulate focal adhesion turnover and actin dynamics in migrating cells (39). Tropomyosin is an actin-binding protein that functions to strengthen actin stress fibers induced by Rho (25). Stathmin is a known target of Rac and is regulated by LIM kinase (27). Phosphorylation and inactivation of stathmin is believed to induce localized stabilization of microtubules at the leading edge of migrating cells. Tubulin cofactor A is a ß-tubulin-binding protein and one of the key regulators of tubulin subunit synthesis, dimer formation, and microtubule stability (40). Interestingly a recent report (41) indicated that tubulin cofactor B ({alpha}-tubulin-binding protein), a functional homologue of tubulin cofactor A, can be phosphorylated by PAK1, a kinase effector of Rac and Cdc42. This phosphorylation was shown to be important in regulating microtubule dynamics. Our result suggests that tubulin cofactor A is coordinately regulated by Cdc42 and RhoA, suggesting convergence between these pathways. Taken together, our results suggest many targets of Rho GTPases that may function to regulate cytoskeleton dynamics and related cell behavior (such as cell migration) in WM35 cells.

The other proteins regulated in response to Rho GTPase suggest other biological processes, many of which point to potentially novel aspects of pathway functions. The identification of PITPß, which has previously been shown to be involved in calcium-regulated vesicle exocytosis (42), is of interest in light of previous observations suggesting functional interactions with phosphatidylinositol-4-phosphate 5-kinase, an effector of RhoA and Rac1 (43, 44). A potential role of Rho GTPase in calcium-regulated exocytosis is also suggested by the identification of CRHSP24, which functions in Ca2+/calmodulin-regulated amylase secretion in pancreatic acinar cells (45). Other cell functions of Rho GTPases suggested by this screen include apoptosis (BTF3 and annexin), protein folding (BIP), and metabolic pathway regulation (adenylosuccinate lyase and ATP synthase). However, the precise regulations and physiological functions of these Rho targets remain to be determined.

In summary, our study illustrates the utility of proteomics for identifying novel targets of signaling pathways. Our results identified PTP1B as a new mediator of RhoA signaling that may function in physiologically important processes such as cell migration. Follow-up analyses of other novel targets should provide further insight into the regulation and function of Rho GTPase signaling pathways.


   ACKNOWLEDGMENTS
 
We are indebted to Dr. David Bernlohr (University of Minnesota) for providing FABP5 antibody, Dr. Karla Ewalt (Scripps Research Institute) for providing TrpRS antibody, Dr. Nicholas Tonks (Cold Spring Harbor Laboratories) for providing mammalian expression vector pMT2-GST-PTP1B, and Dr. Meenhard Herlyn (Wistar Institute) for providing the WM35 cell line.


  FOOTNOTES
 
Received, March 24, 2006

Published, MCP Papers in Press, April 26, 2006, DOI 10.1074/mcp.M600101-MCP200

1 The abbreviations used are: PAK, p21-associated protein kinase; 2-DE, two-dimensional electrophoresis; PTP1B, protein-tyrosine phosphatase 1B; RPE, retinal pigment epithelial; FAK, focal adhesion kinase; RNAi, RNA interference; CMV, cytomegalovirus; FABP5, fatty acid-binding protein 5; TrpRS, tryptophanyl-tRNA synthetase; PITPß, phosphatidylinositol transfer protein ß; CA, constitutively active; LIM, Lin-11/Isl-1/Mec-3; ROCK, Rho kinase. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

Present address: Dept. of Biomolecular Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan. Back

** Present address: Orion Pharma, Orionintie 1, P. O. Box 65, FI-02101 Espoo, Finland. Back

§§ To whom correspondence should be addressed. Tel.: 303-492-4799; Fax: 303-492-2439; E-mail: natalie.ahn{at}colorado.edu


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P. Mertins, H. C. Eberl, J. Renkawitz, J. V. Olsen, M. L. Tremblay, M. Mann, A. Ullrich, and H. Daub
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