Phosphorylation of tyrosine 285 of PAK1 facilitates βPIX/GIT1 binding and adhesion turnover

Abstract

The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to β-PAK1-interacting guanine-nucleotide exchange factor (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced βPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.—Hammer, A., Oladimeji, P., De La Casas, L. E., Diakonova, M. Phosphorylation of tyrosine 285 of PAK1 facilitates bPIX/GIT1 binding and adhesion turnover.

Cell migration is critical for many vital biologic functions, including embryogenesis, tissue remodeling and growth, the inflammatory immune response, atherosclerosis, wound repair, tumor formation, and metastasis. Directed cell motility is complex and requires the highly coordinated assembly and disassembly of adhesion complexes (1, 2). The first structures observed at the sites of adhesion are nascent adhesions that are small and highly transient. They may either disassemble or mature into focal complexes. Focal complexes are adhesions in the early stage of maturation. They are larger than nascent adhesions and may either disassemble (turnover) or maturate and elongate into focal adhesions. The fast turnover of nascent adhesions and focal complexes facilitates cell migration, whereas large and temporally more stable focal adhesions tend to inhibit cell migration (3, 4). The mechanisms that regulate adhesion assembly, maturation, and turnover are not well understood and have become a critical area of emerging interest. Over 180 proteins are found in adhesions, many of which exhibit multiple protein-protein interactions (5). The principal cell surface proteins responsible for regulating the binding of cells to the extracellular matrix are the integrins. Clustering of integrins leads to activation and autophosphorylation of focal adhesion kinase (FAK) on tyrosine 397 and subsequent phosphorylation of paxillin primarily on tyrosines 31 and 118 by FAK in conjunction with Src (reviewed elsewhere) (6, 7). This tyrosyl phosphorylation indirectly enhances the binding of paxillin to FAK and has been implicated in adhesion turnover (8). Cytoskeletal proteins such as talin, α-actinin, vinculin, and zyxin are recruited sequentially to adhesions and regulate integrin avidity by clustering integrin and actin filaments (reviewed elsewhere) (5, 9–11).

Numerous studies have implicated PAK1 in regulation of adhesion complexes. PAK1 is targeted to adhesion complexes via the PAK1-interacting guanine-nucleotide exchange factor PIX (12). A proline-rich motif of PAK1 (residues 182–203) binds directly to the SH3 domain of PIX, and this interaction is negatively regulated by autophosphorylation of PAK1 Ser199/Ser204 (12, 13). Interaction with α/βPIX contributes to PAK1 activity (14, 15). PAK1 phosphorylates Ser340 on βPIX although the physiologic significance of this phosphorylation is still not clear (16). The PIX proteins associate with GIT1, a GTPase activating protein (GAP) for Arf, that targets adhesion complexes by binding to paxillin (17) and FAK (18). PIX and GIT1 can homodimerize and form large aggregates in the cell (19, 20). This oligomerization is essential for localization to sites of adhesion because mutations that disrupt either GIT-PIX association or PIX homodimerization result in diffuse cytoplasmic localization of both proteins (21, 22). PAK1 is an important component of this complex and formation of the 4-molecule PAK1/βPIX/GIT1/paxillin signaling module transiently targets PAK1 to the sites of adhesion (18, 23–25).

Recent studies implicated PAK1 in regulation of adhesion turnover. Overexpression of constitutively active PAK1 increases adhesion assembly and disassembly rates (4), and overexpression of the autoinhibitory domain of PAK1 significantly impairs adhesion turnover and decreases mature focal adhesion numbers (26). PAK1 promotes focal adhesion disassembly (13, 27), an effect that is blocked by the PAK1 inhibitory domain (13) or by overexpression of kinase-dead PAK1 K299R (28, 29). A molecular mechanism of PAK1-dependent regulation of adhesion dynamics has been proposed (4). According to this mechanism, PAK1 phosphorylates paxillin on Ser273 within the paxillin LD4 motif, which up-regulates adhesion turnover by increasing paxillin-GIT1 binding and Rac activation via guanine nucleotide–exchange factor activity of PIX in a positive-feedback loop. This phosphorylation also mediates recruitment of PAK1/βPIX/GIT1 complexes to the leading edge (4). It is important to note that GIT1 and GIT2 binds directly to the paxillin LD4 motif (17, 18, 25, 30), and recent studies have shown that phosphorylation of Ser273 within the LD4 motif of paxillin negatively affects GIT1 binding in vitro (31, 32). Although Ser273 phosphorylation of paxillin has been demonstrated for PAK4 as well (33), participation of PAK1 in this process is still questionable (32).

Another possible mechanism of PAK1-dependent regulation of cell adhesion involves tyrosine kinase Etk/Bmx, a member of the Tec family of nonreceptor cytoplasmic kinases (see elsewhere for review) (34). Src activates Etk by directly phosphorylating Etk at Tyr 566 (35). Furthermore, Etk is a substrate of FAK that is activated through extracellular matrix/integrin-dependent pathway (36). Etk phosphorylates PAK1 on tyrosines and activates PAK1, and Tyr kinase inhibitor AG 879 blocks the specific interaction between Etk and PAK1 in cells (37, 38). It still remains to be determined whether Etk-dependent phosphorylation of PAK1 is activated by Src/FAK and required for adhesion regulation.

We have previously implicated PAK1 as a novel substrate of the JAK2 tyrosine kinase in vivo and in vitro. PAK1 tyrosines 153, 201, and 285 were identified as JAK2 tyrosyl phosphorylation sites by mass spectrometry and 2-dimensional peptide mapping (39). Phosphorylation of these 3 tyrosines of PAK1 impacts filamin A-dependent changes of the actin cytoskeleton and cell motility (40). These 3 PAK1 tyrosines are required for maximal invasion of breast cancer cells and are implicated in enhancing expression of matrix metalloproteinase (MMP)-1 and MMP-3 in 3-dimensional collagen IV (41). We have also implicated the hormone/cytokine PRL in PAK1-dependent regulation of cell motility and invasion (40, 41). PRL binding to its receptor activates JAK2 (and subsequent tyrosyl phosphorylation of PAK1), promotes PRL receptor phosphorylation and phosphorylation of signal transducers and activators of transcription, MAPKs, protein kinase C, and phosphatidylinositol 3-kinase (reviewed elsewhere) (42, 43). There is now clear evidence that PRL is involved in breast cancer (see elsewhere for review) (44–47), where it increases motility of breast cancer cells (40, 48, 49). These data, combined with animal studies reporting either increased metastases with PRL administration (50) or prevention of neoplasia progression into invasive carcinoma in PRL receptor-deficient mice (51), suggest that PRL is involved in the development of metastasis and tumor progression, although the exact mechanism remains to be clarified.

In this study, we extend our findings implicating tyrosyl phosphorylated PAK1 in PRL-stimulated motility and adhesion of breast cancer cells. We have used human breast cancer T47D cell lines that stably overexpress PAK1 WT or PAK1 Y3F mutant in which the 3 JAK2 phosphorylation sites [Tyr(s) 153, 201, and 285] were mutated to phenylalanine. We have demonstrated that these tyrosines regulate cell adhesion, contribute to maximal PAK1 kinase activity, and increase ability to bind βPIX and GIT1. We have generated phosphospecific Abs to single tyrosines 153, 201, and 285 and demonstrated that Tyr285 is a site of JAK2 phosphorylation in response to PRL. We show that a PAK1 Y285F mutant abolishes PRL-dependent PAK1/βPIX/GIT1 binding and inhibits PRL-dependent adhesion turnover. This observation is significant because immunohistochemical analysis reveals that PAK1 tyrosyl phosphorylation on Tyr285 is higher in breast carcinomas than in normal breast tissue. Thus, our data emphasize a role for JAK2/PAK1 interplay in human breast cancer. Collectively, we demonstrate a novel phosphotyrosyl-PAK1-dependent mechanism that regulates adhesion dynamics through a paxillin-GIT1/βPIX/PAK1 complex.

MATERIALS AND METHODS

Plasmids

Construction of PAK1 WT in the pLNCX2 retroviral vector containing the internal ribosomal entry site 2-enhanced green fluorescence protein (IRES2-EGFP) element was described previously (52). Tyrosines 153, 201, and 285 in PAK1 WT were mutated to phenylalanines using the QuikChange site-directed mutagenesis kit (PAK1 Y285F and PAK1 Y3F; Stratagene, Santa Clara, CA, USA) as described previously (39). The final plasmids PAK1 WT, PAK1 Y285F, and PAK1 Y3F with N-terminal myc tags were expressed from retroviral constructs that include IRES elements that allow the transcription of a single bicistronic mRNA of myc-PAK1-IRES2-EGFP, and so produce myc-PAK1 together with EGFP as a reporter for expression of PAK1. RFP-vinculin was a generous gift from Dr. Garcia-Mata (University of Toledo, Toledo, OH, USA). GST-tagged PAK1 binding domain (GST-PBD) was described previously (53). cDNAs encoding JAK2 WT, kinase-inactive JAK2 K882E (K-E), and constitutive active JAK2 V617F (all in pCDNA3 vector) were a gift of Dr. Martin Myers (University of Michigan, Ann Arbor, MI, USA); see elsewhere for description (54, 55).

Abs and reagents

Rabbit polyclonal phosphospecific Abs against pY201-PAK1 and pY285-PAK1were generated and affinity purified by Proteintech Group, Incorporated (Chicago, IL, USA) using phosphorylated peptides HTKSVpYTRSVIC and CASGTVpYTAMDV, respectively, as antigens. Rabbit polyclonal phosphospecific Ab against pY153-PAK1 was generated and affinity purified by AbMax Biotechnology Company, Limited (Beijing, China) using phosphorylated peptide KSAEDpYNSSNAC as antigen. Primary Abs used in this study were polyclonal αPAK (N-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal αPY (clone 4G10; EMD Millipore, Billerica, MA, USA); rabbit monoclonal αJAK2 (Cell Signaling, Beverly, MA, USA); polyclonal αpPAK1(Thr423)/PAK2(Thr402) (Cell Signaling); polyclonal αPAK1 (#2606, Cell Signaling); monoclonal αRac1 (EMD Millipore); monoclonal αactin (pan AB-5, clone ACTN05; Thermo Fisher Scientific, Waltham, MA, USA); monoclonal αHA (Roche Applied Science, Indianapolis, IN, USA); monoclonal αγ-tubulin (Sigma-Aldrich, St. Louis, MO, USA); monoclonal αGFP (Thermo Fisher Scientific); polyclonal αPIXβ (Cell Signaling); monoclonal αGIT1 (BD Transduction Laboratories, BD Biosciences, Franklin Lakes, NJ, USA); monoclonal αFAK (EMD Millipore); polyclonal αpY397-FAK (Abcam, Cambridge, MA, USA); polyclonal αpY31-paxillin, polyclonal αpY118-paxillin, and polyclonal αpSer273-paxillin (all 3 from Invitrogen Incorporated, Carlsbad, CA, USA); monoclonal αpaxillin (BD Biosciences); monoclonal αHA (Covance, Princeton, NJ). Ascites containing monoclonal αmyc Abs, produced by the Michigan Diabetes Research and Training Center Hybridoma Core (Ann Arbor, MI, USA) was used for immunoprecipitation and monoclonal αmyc Ab (9E10; Santa Cruz Biotechnology) was used for immunoblotting. Phalloidin-AlexaFluor 594, goat-αmouse-AlexaFluor 480 Ab (Invitrogen Incorporated), and goat-αrabbit-AlexaFluor 594 Ab (all 3 are from Invitrogen Incorporated) were used for immunofluorescence. Human PRL was purchased from the National Hormone and Peptide Program (Parlow, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA).

Cells

T47D cells stably overexpressing GFP, myc-tagged PAK1 WT, and myc-tagged PAK1 Y3F were described previously (40) (see also Fig. 12A). T47D clones were maintained in RPMI 1640 medium (Corning Cellgro, Corning, Incorporated, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and insulin (Sigma-Aldrich). MCF-7 clones were maintained in DMEM (Corning Cellgro) supplemented with 10% FBS (Sigma-Aldrich).

Figure 12.

Phosphorylation of tyrosine 285 of PAK1 participates in regulation of adhesion turnover. A) Characterization of T47D cell line stably expressing GFP with myc-tagged PAK1 Y285F. T47D cells stably expressing GFP (lane 1), myc-PAK1 WT (lane 2), myc-PAK1 Y285F (lane 3), and myc-PAK1 Y3F (lane 4) were lysed, and proteins were resolved by SDS-PAGE. Overexpressed proteins were visualized by immunoblotting with αmyc Ab (upper panel). The same PVDF membrane was stripped and reblotted with αPAK1 Ab to visualize endogenous (lane 1) and overexpressed PAK1 (lanes 2–4) (middle panel). Immunoblotting with αactin Ab was used as a loading control (bottom panel). B) T47D PAK1 Y285F clone was plated on collagen IV and treated with PRL for indicated times. PAK1 was IPed and examined for phosphotyrosine 285-PAK1, co-IPed βPIX, and GIT1. C) Equal amount of T47D cells stably overexpressing GFP, PAK1 WT, or PAK1 Y285F were allowed to attach to collagen IV-coated wells for 30 min in the absence or presence PRL (200 ng/ml). Adherent cells were stained and quantified at optical density 570 nm after extraction; n = 3 for each experimental condition. Bars represent mean ± se. *P < 0.05 compared with the same cells untreated with PRL. D) Example of RFP-vinculin fluorescence time-lapse images of T47D clones expressing GFP, PAK1 WT, or PAK1 Y285F, plated on collagen IV and treated with PRL for indicated times. White boxes in the whole images (left) indicate the localization of the magnified regions shown in the right panels. Arrowheads and arrows indicate assembling and disassembling AC, respectively. Scale bar, 10 μm. E) Rate constant for the assembly and disassembly of RFP-vinculin in adhesion complexes. The rate of assembly/disassembly of ACs was slower in PAK1 Y285F clone than those in PAK1 WT clone. Data are mean ± se, measure from 8–15 individual adhesions in 5–6 cells for each condition from independent experiments. *P < 0.05, compared with GFP cells.

Cell adhesion assay

A 96-well flat bottom microwell plate was coated with collagen I (1 μg/ml; BD Biosciences) or collagen IV (1 μg/ml; Sigma-Aldrich) The T47D cell clones were serum-deprived in deprivation medium (RPMI 1640 medium with 1% bovine serum albumin) for 72 h, detached with 0.05% trypsin-EDTA (Thermo Fisher Scientific), washed with defined trypsin inhibitor (DTI, Life Technologies) to quench the trypsin activity, then pelleted and added to each well (10⁵ cells per well). Cells were allowed to adhere in adhesion buffer (56) for 30 min at 37°C and then fixed with 96% ethanol for 10 min. The adherent cells were stained with 0.1% crystal violet for 30 min, lysed with 0.2% Triton-X-100, and absorbance at 570 nm was read in SpectraMax M5 (MDS Analytical Technologies, Sunnyvale, CA). Data from 5 wells were polled and plotted. Each experiment was repeated at least 3 times.

Gene silencing

For small interference siRNA (siRNA) transfection, PAK1 siRNA or negative control nontargeting siRNA (Cell Signaling Incorporated) were transiently transfected using the Lipofectamine RNAiMAX reagent (Invitrogen Incorporated) according to the manufacturer’s instructions. The final concentration of the siRNA was 100 nM. In siRNA rescue experiments, 24 h after PAK1 siRNA transfection, cells were transiently transfected with cDNA encoding PAK1 WT using a modification of the polyethylenimine method (57). PAK1 knock-down and re-expression were confirmed by Western blot analysis with αPAK1 at the day of adhesion assay.

In vitro kinase assay

To assess PAK1 WT and PAK1 Y3F in vitro kinase activity, myc-PAK1 were immunoprecipitated (IPed) with αmyc (T47D clones) or αHA (MCF-7 clones) Ab from cells treated with or without PRL (200 ng/ml, 20 min) and subjected to an in vitro kinase assay in the presence of 10 μCi [γ-³²P]ATP (MP Biomedical, Santa Ana, CA, USA), and 5 μg of histone H4 (substrate of PAK1; New England Biolabs, Ipswich, MA). Relative levels of incorporation of ³²P into histone H4, an indicator of phosphorylation, were assessed by autoradiography and estimated by a phosphoimager screen. The same membrane was blotted with αPAK1 to assess the amount of PAK1 for each condition. Nitrocellulose patterns were scanned and the amount of PAK1 was quantified using Multi-Analyst (Bio-Rad Laboratories, Hercules, CA, USA) software. Relative PAK1 kinase activity was then normalized by the amount of IPed PAK1 for each lane.

Detection of activated Rac

Activated Rac1 was detected using the PBD method (58). T47D clones were deprived for 72 h and treated with or without 200 ng/ml PRL for the indicated times. Cells were lysed and solubilized proteins incubated with GST-PBD and glutathione-agarose beads (Sigma-Aldrich). Bound proteins were immunoblotted with αRac1.

Immunoprecipitation and immunoblotting

Cells were serum deprived for 72 h and treated with or without PRL with the indicated concentrations for the indicated times. myc-PAK1 was IPed from the cell lysate using αmyc and protein A-agarose. Proteins were resolved by SDS-PAGE followed by immunoblotting with the indicated Abs. All immunoblots presented in the same figure derive from the same gel.

Immunofluorescence analysis

T47D clones were plated on the collagen IV-covered coverslips, serum deprived for 72 h, and treated with or without 200 ng/ml PRL for the indicated time. The coverslips were fixed for 15 min in 4% paraformaldehyde in intracellular buffer (53). Cells were permeabilized with methanol for 15 min at −20°C, blocked with 2% human serum and incubated with αpaxillin followed by goat-αmouse-AlexaFluor 594 (Invitrogen) to localize adhesion complexes. For colocalization of pY285-PAK1 and paxillin, the coverslips were incubated with rabbit αpY285-PAK1 followed by goat-αrabbit-AlexaFluor 594 and monoclonal αpaxillin followed by goat-αmouse-AlexaFluor 488 (Invitrogen). Staining by secondary Ab reagent alone was negligible (not shown). Immunofluorescence images of fixed cells were acquired on an inverted microscope (Olympus IX81) using a PlanApo N 60x/1.42 oil objective lens (Olympus, Tokyo, Japan).

Fluorescence intensity quantifications and adhesion complexes (AC) measurements were performed in MetaMorph (Molecular Devices, Sunnyvale, CA, USA) software. All images were background subtracted before intensity measurement. To quantify the distribution of peripheral adhesion complexes, all the detectable ACs within 5 μm of the cell border were measured. Only cells not contacted with neighboring cells were analyzed. To quantify the frequency of pY285-PAK1 levels in ACs, paxillin immunofluorescence images were thresholded to include only ACs, and fluorescence intensity of pY285-PAK1 was measured in each thresholded region. All experiments were repeated at least 3 times with n ≥ 30 cells for each condition.

Adhesion turnover quantification

T47D clones were transfected with RFP-vinculin using the polyethylenimine method (57), plated on the collagen IV-covered glass-bottomed dishes, and serum deprived for 24 h. RFP-vinculin images were acquired at 30 s intervals for 25 min on a Leica SP8 TCS confocal scanning microscope using a 63×/1.4 NA HC PL APO oil CS2 objective lens (Leica Microsystems, Buffalo Grove, IL, USA). During live-cell imaging, cells were maintained at 37°C with 5% CO₂. PRL (200 ng/ml) was added to cells after first 5 min of imaging. Quantification of AC was performed as described previously (2). Rate constant measurements for each cell type were obtained from 8 to 15 adhesions for 5 to 6 cells.

Immunohistochemistry

Normal mammary tissue collected at the Department of Pathology, University of Toledo Hospital and commercial breast cancer tissue microarray (BR1002a; US Biomax, Rockville, MD, USA) were studied. Immunohistochemistry using paraffin-embedded sections was done as described elsewhere (59). Briefly, formalin-fixed, paraffin-embedded sections were boiled for 15 min in 0.01 M sodium citrate buffer (pH 6.0) to expose antigenic epitopes. Sections were blocked with 2.5% normal horse serum for 30 min and then incubated 2 h with αpY285-PAK1 or with αPAK (N-20; Santa-Cruz Biotechnology). The sections were then counterstained with Mayer’s hematoxylin (Thermo Fisher Scientific). Staining by secondary Ab alone was negligible (not shown). To determine labeling intensity, the cytoplasm of 100 total cells in 5 randomly chosen microscopic fields (at ×40) in divergent region of each sample were counted by an investigator blind to the experimental status. The brown stain intensity was quantified by using the MetaMorph (Molecular Devices) software.

Statistical analysis

Data from at least 3 separate experiments per each condition were pooled and analyzed using 1-way ANOVA plus Tukey’s honest significant difference test. Differences were considered to be statistically significant at P < 0.05. Results are expressed as the mean ± se. When individual experiments were analyzed, the results were indistinguishable from those obtained from the pooled data.

RESULTS

PAK1 tyrosyl phosphorylation induces motile cell phenotype upon adhesion and inhibits cell adhesion

We sought to determine whether tyrosyl phosphorylation of PAK1 participates in regulation of cell spreading upon adhesion. For that, we assessed the effect of PAK1 WT and PAK1 Y3F (phosphotyrosyl-deficient mutant) on spreading of T47D cells to collagen IV in the presence of PRL (Fig. 1). T47D clones stably overexpressing either GFP, PAK1 WT, or PAK1 Y3F were described previously (40) (see also Fig. 12A). At 60 min, the spread area of T47D PAK1 WT and T47D GFP clones was significantly less in the presence of PRL than in the absence of PRL while spreading of T47D Y3F cells was not affected by PRL (Fig. 1B). Next, we used a standard shape factor (defined by 4πA/P², where A is the area and P the perimeter) for quantitative analysis of cell shape (60). This shape factor varies from 0 to 1, for elongated or circular shapes, respectively. WT and GFP cells had decreased shape factor by 60 min of PRL treatment, demonstrating an elongated phenotype characteristic of motile cells. Compared with WT cells, Y3F cells had an increased shape factor, indicative of a more regular, round shape regardless of PRL presence (Fig. 1A and C, right black bars).

Figure 1.

PAK1 tyrosyl phosphorylation affects cell spreading upon adhesion. A) Equal amount of T47D cells stably overexpressing GFP, PAK1 WT, or PAK1 Y3F were allowed to attach to collagen IV (1 μg/ml) and PRL (200 ng/ml) or vehicle were added during the adhesion. The cells were fixed at indicated times and stained with phalloidin-AlexaFluor 594. Scale bar, 10 μm. B) The images in (A) were analyzed to quantify the spread area of GFP, WT, and Y3F clones. C) The graph depicts morphology. A shape factor (4π area/perimeter²) was determined in GFP, WT, and Y3F clones imaged in (A). D) Equal amount of T47D cells stably overexpressing GFP, PAK1 WT, or PAK1 Y3F were allowed to attach to collagen IV-coated wells for 30 min in the absence or presence of PRL (200 ng/ml). Adherent cells were stained and quantified at optical density 570 nm after extraction. In (B) and (C), n = 150 cells in 3 independent experiments for each experimental conditions; in (D), n = 3 for each experimental condition; in (B–D), bars represent mean ± se. *P < 0.05 compared with the same cells untreated with PRL.

PAK1 facilitates integrin-mediated cell adhesion (61–63). To assess the impact of tyrosyl-phosphorylated PAK1 (pTyr-PAK1) on cell adhesion, cell adhesion to collagen IV was measured in the presence or absence of PRL. In concordance with previously published data demonstrating abolishment of focal adhesions and reduction of cell attachment in cells overexpressing either activated PAK1 (27, 64) or PAK4 (56, 65), overexpression of PAK1 WT decreased cell adhesion on collagen IV as compared with GFP- and PAK1 Y3F-transfected cells (white bars in Fig. 1D), suggesting that PAK1 tyrosyl phosphorylation participates in integrin-dependent adhesion. PRL addition during adhesion led to a further decrease in the number of attached WT and GFP cells, and the number of attached PAK1 Y3F cells were not affected (black bars in Fig. 1D), suggesting that tyrosyl phosphorylation of the 3 tyrosines of PAK1 participates not only in integrin-dependent but also in PRL-dependent cell adhesion.

To directly establish the significance of PAK1 signaling on cell adhesion, we examined the effect of PAK1 knockdown by PAK1-specific siRNA in T47D and MCF-7 cells. In agreement with previously published data that PAK1 silencing enhanced cell adhesion and/or spreading and led to increased size and number of mature focal adhesion (26, 66–68), we found that PAK1-depleted cells exhibited enhanced collagen-dependent cell attachment (Fig. 2A, B). Re-expression of PAK1 WT decreased collagen-dependent adhesion to control levels, and re-expression of PAK1 Y3F led to a partial rescue of adhesive properties in both cell lines (Fig. 2A, B).

Figure 2.

PAK1 participates in collagen-mediated cell adhesion. T47D cells (A) or MCF-7 cells (B) were transfected with control (ctrl) or PAK1 siRNA. In siRNA rescue experiments, 24 h after PAK1 siRNA transfection, cells were transiently transfected with PAK1 WT or PAK1 Y3F. The cells were then plated on collagen I or collagen IV and assessed for adhesion assay. Adherent cells were stained and quantified at optical density 570 nm after extraction. Silencing efficiency was judged by immunoblotting with αPAK. The expression levels of γ-tubulin were used as an internal loading control. Upper plots represent densitometric analysis of the bands obtained for PAK1 expression; n = 5 for each experimental condition. Bars represent mean ± se. *P < 0.05 compared with control.

PAK1 tyrosyl phosphorylation contributes to PAK1 activity

PAK1 kinase activity is regulated by integrin-dependent adhesion and by soluble factors in the media (61–63, 69, 70). We sought to examine if tyrosyl phosphorylation of PAK1 could affect its kinase activity in an integrin-dependent manner. We assessed the kinase activity of PAK1 in the cells kept in suspension, plated on plastic or collagen IV in the presence or absence of PRL (Fig. 3). PAK1 activity in suspended T47D clones was undetectable in the presence or absence of PRL (Fig. 3A, left blot). As shown previously (40, 41), the basal kinase activities of PAK1 WT and Y3F mutant in the cells plated on the plastic were similar because PAK1 Y3F is functionally kinase active (middle blot and 2 left white bars in plot Fig. 3A). PRL increased PAK1 WT kinase activity by 2.5-fold, and PAK1 Y3F was activated by PRL to a lesser extent (2 left black bars in plot Fig. 3A). Plating cells on collagen IV stimulated activity of WT beyond that on plastic alone, but this stimulation was not observed in Y3F PAK1 (right blot and 2 right white bars in plot Fig. 3A), suggesting that PAK1 tyrosyl phosphorylation participates in integrin-dependent PAK1 activation that will be discussed in the Discussion section. Furthermore, PRL activates PAK1 to the maximal extent in WT cells plated on collagen IV (third black bars in plot Fig. 3A). Similar results were obtained with MCF-7 clones (Fig. 3B). These data show that both integrin-dependent adhesion and PRL-dependent PAK1 tyrosyl phosphorylation contribute to PAK1 activity.

Figure 3.

Tyrosyl phosphorylation of PAK1 increases PAK1 kinase activity in a PRL- and an integrin-dependent manner. PAK1 WT or PAK1 Y3F clones of T47D (A) or MCF-7 (B) cells were kept in suspension or plated on plastic or collagen IV, then treated with or without PRL. PAK1 was IPed and subjected to an in vitro kinase assay with H4 histone as a substrate and probed with αPAK1. Relative PAK1 kinase activity was then normalized by the amount of IPed PAK1 for each lane and plotted; n = 3. All blots are representative of at least 3 experiments. Bars represent mean ± se. *P < 0.05 compared with the same cells untreated with PRL.

PAK1 tyrosyl phosphorylation participates in paxillin Ser273 phosphorylation

To investigate regulation of pTyr-PAK1-dependent changes in cell morphology, we first tested Rac1 because PRL activates Rac1 (49, 71, 72) and Rac 1 plays a critical role in PAK1-dependent cell functions (4, 61, 63). We set out to determine whether PRL treatment altered Rac1 activation differently in PAK1 WT and PAK1 Y3F clones. Examination of the time course of Rac1 activation showed that Rac1 activity was increased at 7.5 min (the earliest time point measured) and peaked at 15 min in all cell clones studied (Fig. 4). PAK1 WT and PAK1 Y3F clones demonstrated more sustained Rac1 activation up to 30 min as compared with control GFP cells in which Rac1 activity fell to the near-baseline level by 30 min. These data show that PRL equally activates Rac1 in PAK1 WT and PAK1 Y3F clones. Next, treatment of T47D cells with increasing concentrations of PRL led to increased JAK2 tyrosyl phosphorylation, as expected (Fig. 5A) and to increased phosphorylation of FAK at tyrosine 397, which is critical for FAK-mediated signaling (6) (Fig. 5B). Because FAK/Src phosphorylates paxillin on tyrosine 31 and tyrosine 118, we assessed the effect of PRL on phosphorylation of these 2 tyrosines. Although both sites were phosphorylated upon plating of cells on collagen IV, PRL augmented phosphorylation of Tyr118-paxillin but not Tyr31-paxillin in a dose-dependent manner (Fig. 5C). PRL induced autophosphorylation of endogenous PAK1 on threonine 423 and endogenous PAK2 on threonine 402 (phosphorylated Thr423 and Thr402 are markers of PAK1 and PAK2 activation, respectively) and tyrosyl phosphorylation of PAK1 but not PAK2 (Fig. 5D). Collectively, in T47D cells PRL stimulated activation of JAK2 and FAK and subsequent activation of downstream targets paxillin (pTyr118) and PAK1 (tyrosyl phosphorylation and pT423).

Figure 4.

Prolactin activates Rac1 in GFP, PAK1 WT, and PAK1 Y3F cells. T47D cells stably overexpressing GFP, PAK1 WT, or PAK1 Y3F were serum deprived for 72 h and treated with 200 ng/ml PRL for the time indicated. Cells were lysed, active Rac was pulled down with GST-PBD, and proteins were resolved by SDS-PAGE and analyzed by immunoblotting with αRac1. The graphs represent densitometric analysis of the band obtained for activated Rac1 normalized with total Rac1 for each lane. Each experiment was repeated 3 times.

Figure 5.

Prolactin activates focal adhesion proteins. T47D cells were treated with the indicated concentrations of PRL for 15 min. A) JAK2 was IPed from cell lysates and probed for tyrosyl phosphorylation with αpY (clone 4G10). The graph represents the densitometric analysis of the band obtained for phosphorylated JAK2 (pY-JAK2) normalized with total JAK2 for each lane for 1 representative experiment. B–D) Whole cell lysates (WCL) were immunoblotted for the indicated Ab. αphospho-PAK1 (T423)/PAK2 (T402)-specific PAK Ab recognizes endogenous PAK1 phosphorylated at Thr423 and endogenous PAK2 phosphorylated at Thr402 (2 arrows in D). Anti-total PAK1 Ab recognizes predominantly PAK1 form. The graphs represent the densitometric analysis of the bands obtained for phosphorylated proteins normalized with total proteins for each lane. Fold induction compared with the untreated control was plotted for 3 independent experiments. All blots are representative of at least 3 experiments. Each figure represents the same blot reblotted with indicated Ab.

We tested whether tyrosyl phosphorylation of 3 tyrosines of PAK1 altered phosphorylation of Ser273 of paxillin because it might be mediated by PAK1 and regulate adhesion turnover (4, 73) (Fig. 6). PRL treatment induced transient Ser273 phosphorylation of paxillin at 7.5 min and did not affect Ser273 phosphorylation of Y3F clones. We were unable to detect Ser273 of paxillin in GFP cells because this PRL-dependent effect was probably below the detection level in these cells. These data suggest that tyrosyl phosphorylated PAK1 participates in regulation of phosphorylation of paxillin on Ser273 at an early time point of PRL treatment.

Figure 6.

Tyrosyl phosphorylation of PAK1 affects phosphorylation of paxillin on Ser273. T47D GFP, PAK1 WT, or PAK1 Y3F clones were plated on collagen IV and treated with PRL for indicated times. Whole cell lysates were immunoblotted for the indicated Ab. The graphs represent the densitometric analysis of the bands obtained for phosphorylated paxillin (pSer273-Pxl) normalized with total paxillin (Pxl) for each lane. Fold induction compared with the untreated control was plotted for 3 independent experiments. All blots are representative of at least 3 experiments. Each figure represents the same blot reblotted with indicated Ab.

Tyrosyl phosphorylation of PAK1 facilitates βPIX and GIT1 binding

PAK1 associates with the Cdc42/Rac-specific guanine-nucleotide exchange factor βPIX, which itself is a partner of the Arf-GAP GIT1, a protein that binds to paxillin. This 4-member signaling complex (PAK/βPIX/GIT1/paxillin) has a well-established role in regulating focal adhesion turnover (23). To provide insight into whether tyrosyl phosphorylation of PAK1 increases the ability of PAK1 to bind βPIX and GIT1, we IPed PAK1 from the lysates of PAK1 WT and PAK1 Y3F cells treated with PRL over a time course and assessed these immunoprecipitates for endogenous βPIX. The quantification of PAK1 and βPIX bands in the immunoprecipitates showed that PRL increased association of βPIX with PAK1 WT about 8.5-fold (Fig. 7A). The amount of endogenous βPIX bound to PAK1 Y3F was left unchanged during PRL treatment. Next, we also assessed GIT1 associated with PAK1 WT and PAK1 Y3F upon PRL treatment. Figure 7B demonstrated that 3-fold more GIT1 was associated with tyrosyl phosphorylated PAK1 WT than with PAK1 Y3F. These data demonstrate that tyrosyl phosphorylation of PAK1 facilitates its binding activity toward βPIX and GIT1.

Figure 7.

PAK1 tyrosyl phosphorylation increases the formation of the PAK1/βPIX/GIT1 complex. T47D PAK1 WT or PAK1 Y3F clones were plated on collagen IV and treated with PRL. PAK1 was IPed and examined for co-IPed βPIX (A) and GIT1 (B). The graphs represent the densitometric analysis of the bands obtained for co-IPed βPIX (A) or co-IPed GIT1 (B) normalized with amount of IPed PAK1 for each lane. Fold induction compared with the untreated control was plotted for 3 independent experiments. All blots are representative of at least 3 experiments. Each figure represents the same blot reblotted with indicated Ab.

PAK1 tyrosyl phosphorylation affects adhesion complex distribution

Because PAK1 regulates adhesion turnover (4, 26, 27), we next assessed how pTyr-PAK1 could affect distribution of ACs (the abbreviation AC is used here to refer to all integrin-extracellular matrix adhesions). To this end, paxillin was localized to the AC in T47D clones treated with or without PRL. Quantification of the paxillin-positive ACs revealed that PRL treatment significantly increased the number of ACs in GFP and PAK1 WT clones whereas the number of ACs in PAK1 Y3F cells was unchanged (Fig. 8A, B). We next categorized the ACs into small (<1.0 μm²), medium (from ≥1.0 to ≤2.0 μm²), and large (>2.0 μm²) complexes. Treatment of PAK1 WT cells with PRL resulted in a shift in the AC population toward smaller structures (54.9% without PRL and 76.1% with PRL), whereas the percentage of small AC did not change substantially in the control and PAK1 Y3F cells in response to PRL (Fig. 8C). This finding indicates that tyrosyl phosphorylation of PAK1 alters distribution of small adhesion complexes.

Figure 8.

Tyrosyl phosphorylation of PAK1 affects distribution of adhesion complexes. A) Immunofluorescence of paxillin in GFP, PAK1 WT, and PAK1 Y3F cell lines without and with PRL (200 ng/ml, 15 min). Scale bar, 10 μm. B) Quantification of AC numbers at the cellular periphery. C) Quantification of AC sizes at the cellular periphery. The AC sizes are categorized into small (<1.0 μm²), medium (1.0–2.0 μm²), and large (>2.0 μm²) adhesions. (B and C) Experiments were repeated 3 times with n ≥ 30 cells for each condition, and all detectable peripheral ACs were quantified in each cell (corresponding to a minimum of 30 ACs per cell). Bars represent mean ± se. *P < 0.05.

PAK1 phosphorylated on tyrosine 285 localizes to small paxillin-containing adhesion complexes

To determine which tyrosine on PAK1 is phosphorylated in response to PRL, we raised polyclonal Abs that specifically recognize single phosphorylated tyrosines pY153-PAK1, pY201-PAK1, or pY285-PAK1 in 293T and T47D cells (Fig. 9A, C, and D). Ab specificity was confirmed by phosphopeptide competition (Fig. 9B). We assessed PAK1 tyrosyl phosphorylation in T47D PAK1 WT and T47D Y3F PAK1 clones treated with PRL by using the αphospho-Tyr-PAK1-specific as well as α-phosphotyrosine (4G10, αPY) Abs (Fig. 10). PRL induced Y285 (Fig. 10A) but not Y153 or Y201 phosphorylation in a time-dependent manner in T47D PAK1 WT cells, suggesting that JAK2 phosphorylates tyrosine 285 of PAK1 in response to PRL. αPY Abs detected weak tyrosyl phosphorylation of the PAK1 Y3F mutant, suggesting that phosphotyrosines in the PAK1 Y3F molecule are present but not responding to PRL (Fig. 10B).

Figure 9.

Characterization of pY153-PAK1, pY201-PAK1, and pY281-PAK1 Abs. A) WT or mutated PAK1 (PAK1 Y3F, PAK1 Y153F, PAK1 Y201F, and PAK1 Y285F) were IPed from lysates of 293T cells overexpressing WT or mutated PAK1 with WT or kinase-inactive K882E (K-E) JAK2 and immunoblotted with the indicated Ab. The pY153-PAK1, pY201-PAK1, and pY281-PAK1 Abs specifically detect tyrosyl phosphorylated WT PAK1 but not corresponding PAK1 mutants. B) Phosphopeptide competition. WT PAK1 was IPed from 293T cells overexpressing WT JAK2 and WT PAK1 and immunoblotted with either phosphospecific Ab dilutions preincubated with 200-fold molar excess of no peptide (left), corresponding phosphopeptide (middle), or corresponding non-phosphopeptide (Np, right). No signals were detected in the presence of the competitive phosphopeptides (middle), whereas the signals were retained in the presence of the non-phosphopeptides (right), confirming the Abs specificity. C) WT or mutated PAK1 were IPed from lysates of 293T cells overexpressing WT or mutated PAK1 with constitutively active JAK2 V617F and immunoblotted with the indicated Abs. D) WT or mutated PAK1 Y3F were IPed from lysates of T47D cells overexpressing PAK1 WT or PAK1 Y3F with JAK2 V617F and immunoblotted with the indicated Abs. All blots are representative of at least 3 experiments. Each figure represents the same blot reblotted with indicated Ab.

Figure 10.

JAK2 phosphorylates tyrosine 285 of PAK1 in response to PRL. A) T47D PAK1 WT or PAK1 Y3F clones were plated on collagen IV and treated with PRL. PAK1 was IPed and the phosphorylation statuses of tyrosines 153, 201, and 285 were examined using αphosphospecific Abs (indicated). B) The cells were treated as in (A). IPed PAK1 WT and PAK1 Y3F were assessed for overall tyrosine phosphorylation with αphosphotyrosine Ab [αPY (4G10)]. The graphs represent the densitometric analysis of the bands obtained for phosphorylated PAK1 normalized with total PAK1 for each lane. Fold induction compared with the untreated control was plotted for 3 independent experiments. All blots are representative of at least 3 experiments. Each figure represents the same blot reblotted with indicated Abs.

To determine the localization of pY285-PAK1, T47D PAK1 WT and PAK1 Y3F cells were serum-deprived and treated with PRL for 0, 7.5, and 15 min, and then fixed. PAK1 phosphorylated on tyrosine 285 was detected by immunostaining with αpY285-PAK1 in WT but not in Y3F clones (Fig. 11A). There was no αpY285-PAK1 signal observed in ACs of WT cells without PRL, although pY285-PAK1 was colocalized with paxillin in AC after 7.5 and 15 min of PRL treatment (arrows in Fig. 11A). To further investigate the ability of PRL to cause PAK1 tyrosyl phosphorylation in AC, we quantified pY285-PAK1 average fluorescence intensity in paxillin-positive AC. PRL induced PAK1 phosphorylation of Tyr285 in 7.5 min, which was maintained for at least 15 min (Fig. 11B). Furthermore, 26.3% of small ACs (<1.0 μm²) had a pY285-PAK1 fluorescence intensity > 500 as compared with 8.8% of medium ACs (from ≥1.0 to ≤2.0 μm²) and 1.4% of large ACs (>2.0 μm²) at 7.5 min of PRL treatment (Fig. 11C). Together, our data demonstrate that PRL induces phosphorylation of tyrosine 285 of PAK1 and that PAK1 tyrosyl phosphorylated on tyrosine 285 localizes to small adhesion complexes.

Figure 11.

PAK1 phosphorylated on tyrosine 285 localizes to small paxillin-containing adhesion complexes. A) T47D PAK1 WT and PAK1 Y3F clones were treated with PRL (200 ng/ml) for indicated times, fixed, and stained for pY285-PAK1 (left column, red in merge image) and paxillin (second left column, green in merge image). White boxes in the merge column indicate the position of insets for higher magnification shown in the right column. pY285-PAK1 colocalized with paxillin at small peripheral adhesion complexes after PRL treatment in PAK1 WT cells (arrows). Scale bar, 10 μm. B) Quantification of pY285-PAK1 fluorescence intensity in the ACs at different times of PRL treatment (200 ng/ml). Bars represent mean ± se. *P < 0.05 compared with cells with no PRL treatment. C) Frequency histogram of pY285-PAK1 fluorescence intensity in ACs at 0, 7.5, and 15 min of PRL treatment (200 ng/ml). The data shown are representative of 1 experiment and are averaged from n ≥ 30 cells for each condition. The experiment was repeated 3 times with similar results. The arrow indicates the increased frequency of small ACs containing a high pY285-PAK1 level observed in cells treated with PRL for 7.5 min. a.u., arbitrary units.

Phosphorylated tyrosine 285 of PAK1 participates in adhesion turnover regulation

To determine whether pY285-PAK1 is involved in the regulation of adhesion assembly and/or disassembly, we established a T47D cell line that stably expressed myc-tagged PAK1 Y285F mutant in which only tyrosine 285 was mutated to phenylalanine (Fig. 12A). PAK1 Y285F mutant failed to exhibit a PRL-dependent increase in binding to endogenous βPIX and GIT1 (Fig. 12B), and re-expression of Y285F in PAK1-depleted cells only partly rescued the cells adhesion (data not shown). Furthermore, overexpression of PAK1 Y285F increased cell adhesion on collagen IV as compared with PAK1 WT-transfected cells (white bars in Fig. 12C), suggesting that phosphorylation of tyrosine 285 participates in an integrin-dependent adhesion. The presence of PRL during adhesion led to a decreased number of attached WT cells but the number of attached PAK1 Y285F cells was not affected (black bars in Fig. 12C), suggesting that tyrosyl phosphorylation of the tyrosine 285 of PAK1 participates not only in integrin-dependent but also in PRL-dependent cell adhesion.

To assess adhesion turnover, GFP, PAK1 WT, and PAK1 Y285F clones were transiently transfected with RFP-vinculin, plated on collagen IV, and subjected to time-lapse confocal fluorescence microscopy during treatment with PRL. The rates for adhesion assembly and disassembly were quantified by measuring the fluorescence intensity for RFP-vinculin as a marker protein, as described previously (2). The cells expressing PAK1 WT exhibited more than a 2-fold increase in rates of assembly and disassembly as compared with GFP cells. The rate for adhesion formation and disassembly was similar to controls in the cells expressing PAK1 Y285F (Fig. 12D, E). Similar data were obtained with T47D clones transiently transfected with mCherry-paxillin as another marker of adhesion complexes (data not shown). These results indicate that phosphorylated tyrosine 285 of PAK1 modulates the rate at which adhesion complexes assemble and disassemble, thus confirming a role of PRL-induced pTyr-PAK1 in regulating AC dynamics.

PAK1 tyrosyl phosphorylation on Y285 is highest in breast carcinoma

Phosphorylation levels of Y285-PAK1 as well as levels of endogenous PAK1 expression were evaluated by immunohistochemical staining on microarray tissue sections of invasive ductal carcinoma and adjacent normal breast tissue. The level of total PAK staining was similar in normal breast tissue and in carcinoma tissue samples (Fig. 13). In contrast, PAK1 tyrosine 285 phosphorylation was significantly higher in carcinoma than in normal breast tissue. Thus, coupled with the above in vitro studies, these in vivo data suggest that tyrosyl-285-phosphorylated PAK1 may modulate PAK1 signaling during breast cancer progression (Supplemental Fig. 1 and Supplemental Fig. 2).

Figure 13.

Phosphorylation of tyrosine 285 of PAK1 in breast carcinoma and normal mammary tissue. A) Characterization of anti-pY285-PAK1 and anti-total PAK (N-20, Santa Cruz Biotechnology) Abs for immunocytochemistry. Anti-pY285-PAK1 and anti-PAK Abs were preincubated with 200-fold molar excess of no peptide, blocking peptide for anti-total PAK (sc-882P, Santa Cruz Biotechnology), or corresponding phosphopeptide and non-phosphopeptide for anti-pY285-PAK1. Immunohistochemical labeling of breast carcinoma was performed as described in Material and Methods. Counterstaining with hematoxylin was omitted. No signals were detected in the presence of the competitive peptides, whereas the signals were retained with no peptides or with non-phosphopeptide (for anti-pY285-PAK1) confirming the Ab specificity for immunohistochemistry. Scale bar, 50 μm. B) Representative immunohistochemical staining of normal mammary tissue and breast carcinoma. Tissues were stained with anti-pY285-PAK1 Ab (brown). Nuclei are blue with hematoxylin counterstain. Scale bar, 50 μm. C) Quantification of total PAK (N-20 Ab) and pY285-PAK1 immunostaining in normal mammary tissue (white bars) and carcinoma tissue (black bars); n = 33 for normal tissue and n = 46 for carcinoma tissue; 100 total cells in 5 randomly chosen microscopic fields were evaluated for each sample. Bars represent mean ± se. *P < 0.05.

DISCUSSION

The role of PAK1 in actin-dependent cell functions is well documented (reviewed elsewhere) (74–77). PAK1 is localized in and regulates areas of the cortical actin cytoskeleton (78, 79); PAK1 kinase activity participates in directional motility (27, 80, 81) and PAK1 directly phosphorylates cytoskeletal proteins, including LIM kinase (82), myosin light chain kinase (83), paxillin (17), filamin A, p41-Arc, and merlin (84–86). The role of PAK1 in the regulation of cell adhesion is also well documented and at least 1 mechanism has been proposed (4). See elsewhere for review (74). According to this mechanism, PAK1 phosphorylates paxillin on Ser273, leading to increased paxillin-GIT1 binding and adhesion turnover (4). PAK1 has been found in adhesion complexes (26, 27, 87, 88), and localization of PAK1 to adhesion sites is mediated by direct binding of a proline-rich motif of PAK1 (residues 182–203) to SH3 domain of PIX proteins (12, 13).

We have previously implicated PRL/JAK2-dependent tyrosyl phosphorylation of PAK1 in regulation of cell motility and invasion (40, 41). Here we extend our findings and demonstrate that pTyr-PAK1 induces a motile cell phenotype and decreases cell adhesion in response to PRL. We implicated phosphorylation of tyrosine 285 of PAK1 in regulation of adhesion turnover, which may cause this phenotype. In an attempt to understand the molecular mechanism of the pTyr-PAK1 action, we focused on PAK1/βPIX/GIT1 complex because this complex has a well-established role in regulating focal adhesion turnover (23). We have demonstrated that PRL-dependent tyrosyl phosphorylation of PAK1 enhances the ability of PAK1 to complex with βPIX/GIT1. Deletion of either 3 tyrosines or tyrosine 285 alone abolishes the increase in PRL-dependent PAK1-βPIX/GIT1 binding. The proline-rich motif of PAK1 (residues 182–203) directly binds to SH3 domain of βPIX (12, 13), and phosphorylation at position Tyr285 may affect the interaction with βPIX by inducing a conformational change that makes the proline-rich motif more accessible to βPIX. α/βPIX are GDP/GTP exchange factors for Cdc42/Rac1; however, PIX proteins may stimulate PAK1 through a guanine nucleotide–exchange factor-independent mechanism and physical interaction with PIX may directly enhance PAK1 activity (14, 15). This may explain why Rac1 was equally activated in PAK1 WT and PAK1 Y3F clones in response to PRL (Fig. 4).

PRL treatment induces transient Ser273 phosphorylation of paxillin at 7.5 min in PAK1 WT clone (Fig. 6). We have also observed that PRL promotes sustained elevation of pTyr-PAK1 WT association with βPIX/GIT1, which starts at 7.5 min and lasts at least 45 min (Fig. 7). Why did we not see pSer273-paxillin for this prolonged period of time? Nayal et al. (4) demonstrated a 3-fold increase in GIT1 binding for phosphomimetic S273D-paxillin as compared with WT paxillin in cells. In contrast, direct in vitro binding of LD4 motif of paxillin to GIT1 protein was reduced when Ser273 was either phosphorylated or mutated to aspartate (S273D mutant) (31). It is possible that pTyr-PAK1 WT phosphorylates Ser273 on paxillin to start formation of GIT/PIX/pTyr-285PAK1 complexes. Indeed, we have seen increased GIT1/βPIX/pTyr-285PAK1 association at the same time point as Ser273 phosphorylation of paxillin (7.5 min). However, when established, GIT1/βPIX occupation of LD4 motif of paxillin may not allow PAK1 to recognize and further phosphorylate Ser273 within this domain that leads to decline of Ser273-paxillin phosphorylation that we have demonstrated in Fig. 6 after 7.5 min. We do not have experimental data to support this hypothesis, and it will be addressed in detail in our planned future studies.

Paxillin tyrosyl phosphorylation can be triggered by adhesion-associated integrin ligation via activation of FAK and Src kinases (89–91) or by ligation of growth factor/cytokine receptors (reviewed elsewhere) (92), including epidermal growth factor (93), growth hormone (94, 95), hepatocyte growth factor (HGF) (96), IGF-I (97–99), and platelet-derived growth factor (100, 101). PRL also stimulates paxillin tyrosyl phosphorylation (102–104), and we show for the first time that both Tyr31 and Tyr118 are phosphorylated because of plating the cells on collagen IV, and also that PRL causes additional phosphorylation of Tyr118 but not Tyr31 in a dose-dependent manner (Fig. 5C). Studies have previously demonstrated different phosphorylation of Tyr31 and Tyr118 in response to stimuli, although the physiologic role of this differential phosphorylation is unclear. HGF causes phosphorylation of Tyr31 but not Tyr118 in H69 cells (105); the antibiotic mitomycin C, which activates FAK, subsequently up-regulates phosphorylation of Tyr31 but down-regulates Tyr118 in human corneal fibroblasts (106). A critical role for Src in the differential phosphorylation on these sites has been recently proposed because Src induces higher phosphorylation of Tyr118 but not Tyr31 in FAK^−/− mouse embryo fibroblasts (107). In this regard, PRL activates FAK with subsequent Src-dependent tyrosyl phosphorylation of paxillin and overexpression of dominant negative Src mutant, or treatment cells with Src inhibitor PP1 abrogate PRL-dependent FAK activation (103). It is possible that plating cells on collagen promotes integrin-dependent phosphorylation of Tyr31 and Tyr118, whereas PRL treatment stimulates additional Tyr118 phosphorylation through Src activation.

In addition to JAK2, PAK1 can be tyrosyl phosphorylated and activated by Etk, a member of the Tec/Btk family of nonreceptor tyrosine kinase (37, 38). Etk, in turn, is phosphorylated and activated by FAK in an integrin-dependent manner (36). Although the sites of tyrosine phosphorylation of PAK1 by Etk were not mapped, it is possible that these site(s) are the same as the sites of JAK2 phosphorylation. It would explain why PAK1 Y3F and Y285F mutants had similar adherence to collagen IV as compared with the GFP-transfected cells (Figs. 1D and 12C, white bars) and failed to be activated by ligation of integrin in the cells plated on collagen IV (Fig. 3A, right white bar).

In the present study we demonstrated that phosphorylation of Tyr285 of PAK1 is significantly higher in breast carcinomas than in normal breast tissue. From our in vitro studies we know that this phosphorylation can be triggered by PRL. There is now clear evidence that PRL is involved in breast cancer (see elsewhere for review) (44–47, 108, 109), and an autocrine/paracrine loop for biologically active PRL has been demonstrated in breast cancer cells (108–111). These data include epidemiologic studies indicating that postmenopausal women with high-normal levels of PRL are at increased risk of breast cancer (44). The PRL receptor is detected in 80% of human breast cancers (112) and is overexpressed in breast cancer samples (113). PRL is a mitogen for normal and breast cancer cells (reviewed elsewhere) (42) and is a survival factor (114). PRL increases motility of breast cancer cells (40, 48, 49). In addition, PRL regulates activation of proteins participating in breast cancer cell adhesion (102, 103, 115, 116). These data, combined with animal studies reporting either increased metastases with PRL administration (50) or prevention of neoplasia progression into invasive carcinoma in PRL receptor-deficient mice (51), suggest that PRL is involved in the development of metastasis and tumor progression. However, the molecular mechanism of the PRL action is still unclear. We have previously shown that PRL facilitates breast cancer cell motility via pTyr-PAK1-dependent phosphorylation of filamin A and via enhanced secretion of MMP-1 and -3 (40, 41). The research presented here establishes an important role for pTyr285-PAK1 in the adhesive properties of breast carcinoma cells, a cell function relevant to breast cancer progression. Our in vivo data demonstrate that phosphorylation of Tyr285 of PAK1 is significantly higher in carcinoma than in normal breast tissue, implicating pTyr285-PAK1 in breast cancer progression.

We propose a model for PRL-dependent regulation of cell motility and adhesion that integrates our findings with previous studies. PRL binding to its receptor leads to phosphorylation and activation of JAK2 tyrosine kinase. Activated JAK2 phosphorylates PAK1 at Tyr285 and stimulates both PAK1 activities: kinase activity and ability of PAK1 to form the GIT1/PIX/pTyr285 PAK1 complex. This complex localizes to small adhesion complexes, the amount of which is increased by PRL treatment. In these small adhesion complexes at cell periphery, PAK1 phosphorylates serine 273 on paxillin, which results in enhanced adhesion turnover. We cannot exclude the possibility that tyrosyl phosphorylated PAK1 may create additional docking sites for proteins that bring an additional activity toward cell adhesion. Thus, our current data add insight to the mechanism of PRL-stimulated motility and adhesion of breast cancer cells.

Glossary

AC

adhesion complexes

FAK

focal adhesion kinase

GIT1

G protein-coupled receptor kinase-interacting target 1

HGF

hepatocyte growth factor

IPed

immunoprecipitated

IRS2-EGFP

internal ribosomal entry site 2-enhanced green fluorescence protein

PAK1

p21-activated serine-threonine kinase

PIX

PAK1-interacting guanine-nucleotide exchange factor

PRL

prolactin

pTyr

tyrosyl phosphorylated

RFP

red fluorescence protein

siRNA

small interference RNA