Clinical & Experimental Metastasis 20: 357–364, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Protein phosphatase-2A regulates protein tyrosine phosphatase activity in Lewis lung carcinoma tumor variants Jodi L. Jackson1 & M. Rita I. Young2−4 Departments of 1 Cell Biology, Neurobiology and Anatomy, 2 Pathology and 3 Otolaryngology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois, USA; 4 Research Services, Hines VA Hospital, Hines, Illinois, USA Received 30 October 2002; accepted in revised form 9 December 2002

Key words: metastasis, paxillin, phosphorylation, protein phosphatase-2A, protein tyrosine phosphatase, Shp-2

Abstract Cellular adherence and motility are processes that are controlled by focal adhesion assembly and disassembly. Consequently, the dynamics of focal adhesions regulate tumor cell metastasis and are influenced by the tyrosine phosphorylation state of paxillin. Metastatic LLC cells are more migratory and have reduced paxillin tyrosine phosphorylation as compared to nonmetastatic LLC cells. In nonmetastatic Lewis lung carcinoma (LLC) tumor cells, inhibition of the serine/threonine protein phosphatase-2A (PP-2A) activity results in increased motility that is associated with a reduction in the phosphotyrosine content of paxillin. Studies to determine if PP-2A can regulate protein tyrosine phosphatase activity showed that blocking PP-2A activity of nonmetastatic LLC-C8 tumor cells with okadaic acid reduces protein tyrosine phosphatase activity. Among the tyrosine phosphatases whose activity was inhibited upon PP-2A inhibition is Shp-2. In contrast, protein levels of Shp-2 are unaffected by PP-2A inhibition. While these results do not fully identify how inhibition of PP-2A results in tyrosine dephosphorylation of paxillin, they do demonstrate that PP-2A can link serine/threonine and tyrosine signaling pathways by regulating protein tyrosine phosphatases.

Introduction Cellular adherence and motility are intricate processes that regulate tumor cell metastasis. Tumor cells that have a greater capacity to metastasize in vivo are less adherent and more motile and invasive in vitro [1–6]. Numerous signaling pathways can regulate cellular adherence and motility, including the tyrosine kinases c-src and fyn [7–9] and the serine/threonine kinases protein kinase C (PKC) and protein kinase A (PKA) [10–17]. In addition to kinases, protein phosphatases are important contributors to this regulation. For example, in LLC tumor variants PP-2A negatively regulates metastasis by maintaining adherence and restricting tumor cell motility [18, 19]. Cellular adherence and motility are regulated by focal adhesion structures that link the actin cytoskeleton to the extracellular matrix [20-21]. Altering cellular adhesion, in order to facilitate motility, requires assembly and disassembly of focal adhesions to coordinate attachment and detachment of the cell from the surrounding tumor tissue. Focal adhesion dynamics are regulated by the focal adhesion protein paxillin. Paxillin binds a variety of structural and signaling molecules localized within focal adhesions and plays a vital role in coordinating focal adhesion assembly by acting as a scaffolding protein to mediate interactions between Correspondence to: M. Rita I. Young, PhD, Research Services (151-Z2), Hines VA Hospital, Hines, IL 60141, USA. Tel: +1-708-202-5749; Fax: +1-708-202-2684; E-mail: [email protected]

these molecules [22–23]. Indeed, paxillin plays a critical role in regulating adhesion and motility. Deleting the gene that encodes paxillin results in embryonic lethality by day 9.5 and defects in cell spreading and motility [24]. The ability of paxillin to regulate focal adhesion dynamics, and hence cellular adherence and motility, is regulated by tyrosine phosphorylation. For example, focal adhesion formation and cellular adhesion are accompanied by tyrosine phosphorylation of paxillin [25–29], whereas breakdown of focal adhesions and loss of cellular adherence are accompanied by tyrosine dephosphorylation of paxillin [30–34]. Blocking the tyrosine phosphorylation of paxillin results in failure to form organized stress fibers and focal adhesions and defective cellular adhesion and motility [25–26, 35]. The current dogma regarding paxillin phosphorylation is that it is maintained by signaling pathways catalyzed by protein tyrosine kinases and protein tyrosine phosphatases. However, paxillin phosphorylation can also be regulated by serine/threonine phosphorylation. In LLC tumor variants, a reduction in the phosphotyrosine content of paxillin is observed when inhibiting the enzymatic activity of PP-2A [18]. Although our previous studies demonstrated a reduction in the tyrosine phosphorylation of paxillin in nonmetastatic LLC cells whose PP-2A was inhibited with okadaic acid, they did not address the mechanistic question of how a serine/threonine phosphatase can regulate this tyrosine phosphorylation. Therefore, the aim of this study was to elucidate

358 the regulatory mechanisms that mediate cross-talk between serine/threonine and tyrosine signaling pathways. In cultured cells, increased protein tyrosine phosphatase activity has been shown to coincide with reduced adherence, a phenomenon observed in PP-2A-inhibited cells [33, 36]. Based on this information, we had hypothesized that the observed cross-talk between these two signaling pathways causing the reduction in the phosphotyrosine content of paxillin in PP2A-inhibited cells was likely mediated by increased protein tyrosine phosphatase activity. In contrast, we show here that blocking PP-2A causes a reduction in protein tyrosine phosphatase activity. While these studies are contradictory to our original expectation, they still reveal that PP-2A can regulate protein tyrosine phosphatase activity and thus facilitate interaction between two prominent cellular signal transduction pathways.

Materials and methods Cell culture The cloned nonmetastatic LLC-C8 tumor line was derived from a subcutaneous LLC tumor and a cloned metastatic LLC-LN7 tumor was derived from a metastatic lung nodule [37]. The LLC tumor variants were cultured in RPMI-1640 (Sigma) supplemented with 10% fetal bovine serum (HyClone) and maintained in a 5% CO2 atmosphere at 37 ◦ C. Cells were subcultured 48 h prior to treatment with media, DMSO (Sigma) diluent control (0.1%) or 100 nm okadaic acid (Biomol). While okadaic acid inhibits the purified PP2A enzyme and PP-2A in cell lysates at 0.1–1 nM, and then becomes inhibitory to extracellular PP-1 at concentrations greater than 10 nM, significantly higher levels are needed to inhibit PP-2A and PP-1 in intact cells. Prior extensive enzymatic analyses [38, 39] showed that at the concentration of okadaic acid that we used to treat intact tumor cells (100 nM), PP-2A is inhibited but not PP-1. Over 10-fold the concentration of okadaic acid (> 1 µM) is required for cell treatment before PP-1 inhibition is initiated. Migration assay In vitro migration of LLC cells through polycarbonate filters was measured by filling the bottom chambers of 96-well migration plates (Neuro/Probe, Cabin John, Maryland) with medium and overlaying plates with a filter of 7 µm pore size. Cells were placed onto the filters at a density of 5 × 104 cells/20 µl. In experiments that utilized okadaic acid to block PP-2A, the inhibitor was added with the cells in the upper compartment. Chambers were incubated overnight and then the cells that remained on the upper surface of the filters were removed. To the cleaned upper surface of the filter, 0.25% EDTA was added for 10 min to detach any cells that may have transversed but not detached from the filters. The 96-well migration chamber was centrifuged to pellet cells into the lower compartment. The proportion of cells in the lower compartment was then estimated with a tetrazoliumbased colorimetric assay (MTS; Promega). Control wells

J.L. Jackson & M.R.I. Young with known numbers of endothelial cells were used to calculate the percentage of cells that had migrated through the filter. Protein tyrosine phosphatase enzyme analysis Nonmetastatic LLC-C8 tumor cells were collected by scraping and washing twice in ice-cold lysis buffer (20 mM TrisCl, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 57 mM PMSF and 0.1 µg/ml leupeptin). Next, cells were lysed by sonicating for 2 min on ice and the cell lysates were centrifuged at 14,000 rpm for 30 min. The lysates were cleaned of any contaminating phosphate by spinning at 1,600 rpm through Sephadex G-25 spin columns for 5 min at 4 ◦ C. Protein samples (10 µg) were incubated with reaction buffer (60 mM sodium acetate, pH 5.2) and two protein tyrosine phosphatase specific synthetic phosphopeptides [END(pY)INASL (peptide 1) and DADE(pY)LIPQQG (peptide 2)] at 30 ◦ C for 30 min. Two peptides were used in order to measure a broad range of protein tyrosine phosphatases. Peptide 1 corresponds to a highly conserved region of the T-cell phosphatase (TC.PTP) and has been shown to be dephosphorylated by PTP-1B, CD45, PTPα and TC.PTP [40]. Peptide 2 corresponds to an autophosphorylation site of the epidermal growth factor receptor (EGFR). The EGFR has been shown to be dephosphorylated by PTP1, cdc25, low Mr PTP and Shp-1 [41–44]. The enzymatic reactions were terminated by the addition of a Molybdate dye/additive solution and incubation for 15 min at room temperature prior to reading at 630 nm. Phosphate release (pmoles) was determined by comparing sample absorbances to a standard curve generated by phosphate standards of known concentration (Promega). Immunoprecipitation LLC tumor variant cells were collected and washed twice in ice-cold Tris (20 mM Tris-Cl, pH 7.5). Next, cells were lysed by incubating in RIPA buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.2, 2 mM EDTA, 50 mM NaF, 0.2 U/ml aprotinin and 57 mM PMSF) for 30 min on ice, and the cell lysates were centrifuged at 14,000 rpm for 1 h. Lysates were precleared overnight by gently shaking at 4 ◦ C with protein G-agarose (25 µl, Sigma). Precleared lysates (1 mg/ml) were incubated with anti-paxillin (6 µg, Transduction Laboratories) or anti-Shp-2 (6 µg, Transduction Laboratories) antibodies and protein G-agarose (25 µl, Sigma) at 4 ◦ C for 90 min each. The beads were washed sequentially with dilution buffer (0.01 M Tris-Cl pH 8.0, 0.14 M NaCl, 0.025% NaN3 and 0.1% Triton X-100), TSA solution (0.01 M TrisCl pH 8.0, 0.14 M NaCl and 0.025% NaN3 ) and 0.05 M Tris-Cl, pH 6.8. Protein tyrosine phosphatase activity was measured as described above by incubating immunoprecipitates and agarose alone, to determine baseline activity, with reaction buffer (60 mM sodium acetate, pH 5.2) and a protein tyrosine phosphatase specific synthetic phosphopeptide [DADE(pY)LIPQQG] at 30 ◦ C for 30 min. Alternatively,

PP-2A regulates protein tyrosine phosphatase

Figure 1. Okadaic acid stimulates motility of the nonmetastatic LLC-C8 cells. LLC variants were seeded onto a 7 µM filter and allowed to migrate through the filter into the lower migration compartment during overnight incubation. The number of cells that migrated into the lower compartment was estimated colorimetrically and compared to a known number of seeded cells. Shown are calculated percentages of migrating cells + SEM from triplicate chambers from each of four experiments.

immunoprecipitated paxillin was subjected to Western blot analysis as described below. Western blot analysis Cell lysates of immunoprecipitates which were prepared as described above were mixed with SDS loading buffer and boiled at 100 ◦ C for 5 min. Protein samples were separated by SDS-polyacrylamide gel electrophoresis (8% gel), transferred to a nitrocellulose membrane (Bio-Rad), blocked and probed with anti-Shp-2 IgG (1:5,000, Santa Cruz Biotechnology) and horseradish-peroxidase conjugated anti-rabbit IgG (1:5,000, Upstate Biotechnology), or anti-PTP-1B IgG (1:100, Santa Cruz Biotechnology) and horseradish-peroxidase conjugated anti-goat IgG (1:2,500, Santa Cruz Biotechnology). Immunoprecipitated paxillin was probed with horseradish-peroxidase conjugated antiphosphotyrosine IgG (1:2,500, Transduction Laboratories). Membranes were then reprobed with anti-paxillin IgG (1:5,000, Transduction Laboratories) and horseradishperoxidase conjugated anti-mouse IgG (1:16,000, Amersham). Protein expression was visualized by chemiluminescence (Amersham) and quantitated by densitometric analysis of the autoradiogram (Scion Image). Analysis of data ANOVA was used to determine the significance of the differences in protein tyrosine phosphatase activity between control and experimental groups. All analyses were performed in at least triplicate with different cell preparations.

359 were conducted to determine how this stimulated motility compared to the motility of metastatic LLC variants. Shown in Figure 1 is the increased level of motility exhibited by metastatic LLC cells compared to nonmetastatic LLC cells, and that treatment of nonmetastatic LLC cells with concentrations of okadaic acid that selectively inhibit PP-2A [38, 39] stimulated their motility to a level similar to seen for metastatic LLC cells. Okadaic acid had no effect on the motility of metastatic LLC, whose PP-2A is inherently diminished compared to nonmetastatic LLC cells [19]. Since increased motility is associated with increased dynamics of the focal adhesion complexes, which is regulated by the level of tyrosine phosphorylation of the focal adhesion protein paxillin [25–34], the tyrosine phosphorylation state of paxillin from metastatic and nonmetastatic LLC variants was compared to that of nonmetastatic variants whose PP-2A was inhibited with okadaic acid. While paxillin of nonmetastatic LLC cells showed prominent paxillin tyrosine phosphorylation, paxillin of both metastatic LLC as well as nonmetastatic LLC whose PP-2A activity was inhibited with okadaic acid showed a loss in paxillin tyrosine content (Figure 2). This prompted studies described below to identify if the tyrosine phosphatase content of paxillin was modulated by inhibition of the serine/threonine phosphatase PP-2A. Protein tyrosine phosphatase activity is reduced in PP-2- inhibited cells Studies by others had shown a reduction in the phosphotyrosine content of paxillin and concomitant loss of cellular adherence when increasing protein tyrosine phosphatase activity [33, 36]. Based on this information, we hypothesized that the reduced paxillin tyrosine content in LLC cells with diminished PP-2A activity could be due to increased protein tyrosine phosphatase activity. To test this hypothesis, nonmetastatic LLC-C8 cells were treated with media, DMSO diluent control or 100 nM okadaic acid. After 24 h, protein tyrosine phosphatase activity was determined by measuring phosphate release from two synthetic phosphopeptides that are substrates for a variety of protein tyrosine phosphatases. Surprisingly, reduced protein tyrosine phosphatase activity was observed in the okadaic acid-treated LLC-C8 cells when compared to the media and DMSO diluent control cells (Figures 3 and 4). In three separate experiments, an average 57% ± 4% (peptide 1) and 57% ± 5% (peptide 2) reduction was observed when inhibiting PP-2A activity. These data demonstrate that PP-2A can negatively regulate protein tyrosine phosphatase activity and that an overall increase in protein tyrosine phosphatase activity was not a likely explanation for the loss of paxillin tyrosine phosphorylation in PP-2A-inhibited LLC cells.

Results

Shp-2 activity is reduced in PP-2A-inhibited cells

Increased motility and reduced paxillin tyrosine phosphorylation in cells with diminished PP-2A activity

Morphological transitions and motility defects have been noted in several cell types when disrupting or upregulating the function of PTP-1B and Shp-2. Interestingly, these protein tyrosine phosphatases have also been implicated in regulating the tyrosine phosphorylation of paxillin [33, 45–

Since PP-2A inhibition had previously been shown to stimulate motility of nonmetastatic LLC tumor cells [18], studies

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Figure 2. Reduced phosphotyrosine content of paxillin in metastatic LLC cells and in nonmetastatic LLC whose PP-2A is inhibited with okadaic acid. Paxillin was immunoprecipitated from metastatic LLC-LN7 cells or from nonmetastatic LLC-C8 cells that were treated for 24 h with DMSO diluent control or 100 nM okadaic acid. The immunoprecipitates were analyzed by sequentially probing with anti-phosphotyrosine and anti-paxillin antibodies. Phosphotyrosine and paxillin expression was quantitated by densitometry and results from three separate experiments ± SD are shown in the lower panel.

Figure 3. Treatment of nonmetastatic LLC-C8 tumor cells with 100 nM okadaic acid reduces protein tyrosine phosphatase activity. Lysates from nonmetastatic tumor cells treated for 24 h with media, DMSO diluent control or 100 nM okadaic acid were allowed to react with a protein tyrosine phosphatase specific phosphopeptide (peptide 1 – highly conserved region of the T-cell phosphatase). Phosphate release was determined colorimetrically. Representative data from one individual experiment are shown as phosphate released ± SD. Similar enzymatic activity was observed in three independent experiments performed in duplicate. Statistical analyses were performed using ANOVA. ∗ P < 0.05.

52]. While these studies provided the rationale to determine whether the observed reduction in the phosphotyrosine content of paxillin in PP-2A-inhibited cells was due to increased Shp-2 and/or PTP-1B activity, we first needed to verify their presence in the nonmetastatic LLC-C8 tumor variants. After 24 h in the absence or presence of okadaic acid, the nonmetastatic LLC-C8 cells were lysed and the resultant lysates were analyzed by Western blotting with anti-PTP-1B and anti-Shp-2 antibodies. While PTP-1B expression was not detectable (data not shown), equal amounts of Shp-2 were observed in the media, DMSO and okadaic acid-treated tumor cells (Figure 5). Satisfied that Shp-2 was expressed in the nonmetastatic LLC-C8 cells, enzymatic analysis was performed to determine the effects of PP-2A inhibition on Shp-2 activity. After 24 h treatment with media, DMSO diluent control or 100 nM okadaic acid, Shp-2 immunoprecipitates were analyzed for protein tyrosine phosphatase activity. Figure 6 shows that Shp-2 activity was reduced in the okadaic acid-treated nonmetastatic LLC-C8 cells compared to the LLC-C8 controls. On average, this reduction was 68% ± 13% when compared to the media and DMSOtreated LLC-C8 tumor cells. Taken together, these data demonstrate that PP-2A can downregulate Shp-2 activity, and that this regulation does not occur by affecting protein expression. Protein tyrosine phosphatase activity is not associated with paxillin in LLC-C8 cells

Figure 4. Treatment of nonmetastatic LLC-C8 tumor cells with 100 nM okadaic acid reduces protein tyrosine phosphatase activity. Lysates from nonmetastatic tumor cells treated for 24 h with media, DMSO diluent control or 100 nM okadaic acid were allowed to react with a protein tyrosine phosphatase specific phosphopeptide (peptide 2 – autophosphorylation site of the epidermal growth factor receptor). Phosphate release was determined colorimetrically. Representative data from one individual experiment are shown as phosphate released ± SD. Similar enzymatic activity was observed in three independent experiments performed in duplicate. Statistical analyses were performed using ANOVA. ∗ P < 0.05.

Although total cellular protein tyrosine phosphatase and Shp-2 activities were reduced in the okadaic acid-treated LLC-C8 cells, we investigated whether the reduction in the phosphotyrosine content of paxillin in these cells was due to increased protein tyrosine phosphatase activity associated with paxillin. To assess this possibility, paxillin was immunoprecipitated from LLC-C8 cells that were treated with

PP-2A regulates protein tyrosine phosphatase

Figure 5. Shp-2 is expressed in nonmetastatic LLC-C8 tumor cells. Cell lysates collected from nonmetastatic tumor cells treated for 24 h with media, DMSO diluent control or 100 nM okadaic acid were separated under denaturing conditions and analyzed by Western blot analysis with an anti-Shp-2 antibody. Shp-2 expression was visualized using chemiluminescence and quantitated by densitometric analysis of the autoradiogram. Data shown are representative of five independent experiments.

Figure 6. Shp-2 tyrosine phosphatase activity is reduced in okadaic acid-treated nonmetastatic LLC-C8 tumor cells. Shp-2 immunoprecipitates from nonmetastatic tumor cells treated for 24 h with media, DMSO diluent control or 100 nM okadaic acid were allowed to react with a protein tyrosine phosphatase specific phosphopeptide. Phosphate release was determined colorimetrically. Representative data from one individual experiment are shown as phosphate released ± SD. Similar enzymatic activity was observed in three separate experiments performed in duplicate. Statistical analyses were performed using ANOVA. ∗ P < 0.05

media, DMSO diluent control or 100 nM okadaic acid for 24 h and analyzed for protein tyrosine phosphatase activity. As shown in Figure 7, no significant enzymatic activity above baseline levels was detected in the media, DMSO diluent control or okadaic acid-treated cells. These results suggest that the reduced tyrosine phosphorylation of paxillin observed in PP-2A-inhibited cells cannot be explained by increased protein tyrosine phosphatase activity associated with paxillin.

Discussion Metastatic tumor cells are characterized as being more motile and less adherent than nonmetastatic tumor cells [1– 6, 10–12]. Our studies with LLC tumor variants have shown that blocking PP-2A in nonmetastatic LLC-C8 tumor cells

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Figure 7. Lack of detectable protein tyrosine phosphatase activity associated with paxillin in okadaic acid-treated nonmetastatic LLC-C8 tumor cells. Paxillin immunoprecipitates from nonmetastatic tumor cells treated for 24 h with media, DMSO diluent control or 100 nM okadaic acid were allowed to react with a protein tyrosine phosphatase specific phosphopeptide (peptide 1). Phosphate release was determined colorimetrically. Representative data from one individual experiment are shown as phosphate released ± SD. Similar enzymatic activity was observed in three separate experiments performed in duplicate. Statistical analyses were performed using ANOVA.

results in reduced adherence and increased motility [18], indicating that PP-2A may negatively regulate metastasis by maintaining adherence and restricting tumor cell motility. In addition to adherence and motility, these studies showed that PP-2A regulates the tyrosine phosphorylation of paxillin, as the phosphotyrosine content of paxillin is reduced in PP-2A-inhibited cells. Therefore, studies were conducted to determine the mechanisms that mediate this cross-talk between serine/threonine and tyrosine signaling pathways. In so doing, we evaluated whether the diminished tyrosine content of paxillin from cells whose PP-2A was inhibited with okadaic acid could be secondary to nonspecific inhibitory effects of okadaic acid on other serine/threonine phosphatases. This was, however, considered unlikely since the concentration of okadaic acid that was used to inhibit PP2A intracellularly has previously shown not to be sufficient to inhibit other serine/threonine phosphatases such as PP1, which requires over 10-fold the concentration of okadaic acid as was used in the present study [38, 39]. Because increased protein tyrosine phosphatase activity is associated with adhesive deficits [33, 36], we originally hypothesized that protein tyrosine phosphatase activity would be upregulated in PP-2A-inhibited cells. Contrary to this speculation, our results show that treating the nonmetastatic LLC-C8 tumor cells with okadaic acid caused a reduction in total cellular protein tyrosine phosphatase activity. We also measured the enzymatic activity of Shp2, believing it might be increased in PP-2A-inhibited cells. This possibility was based on studies demonstrating adherence and motility defects when modulating the activity of this tyrosine phosphatase [47, 49]. Like total cellular protein tyrosine phosphatase activity, treating the nonmetastatic tumor cells with okadaic acid also caused a reduction in the activity of Shp-2. Lastly, we sought to determine whether the reduction in the tyrosine phosphorylation of paxillin in PP-2A-inhibited cells could be explained by an increase in protein tyrosine phosphatase activity associated with paxillin. However, protein tyrosine phosphatase activity was

362 undetectable in paxillin immunoprecipitates from LLC-C8 tumor cells treated with media, DMSO or okadaic acid. Based on genomic sequencing, the estimated number of eukaryotic protein tyrosine phosphatase genes has grown from 60 to 500. To date, approximately 75 protein tyrosine phosphatases have been identified, making this family of enzymes one of the largest in the eukaryotic kingdom [53, 54]. While the results of this study contradict our original expectation that tyrosine phosphatase activity would be increased in PP-2A-inhibited cells, there remains the possibility that the enzymatic activity of a small group of protein tyrosine phosphatases capable of regulating paxillin phosphorylation may be increased in PP-2A-inhibited cells. The mechanism by which PP-2A regulates the activity of protein tyrosine phosphatases, such as Shp-2, remains unclear. Regulation does not appear to occur by modulating protein expression, as the level of Shp-2 was not altered in LLC-C8 cells treated with okadaic acid when compared to media or DMSO treated cells. Alterations in the activities of protein tyrosine phosphatases have previously been described as a consequence of serine phosphorylation [55, 56]. Although not assessed in the present study, we speculate that the PP-2A-mediated regulation of protein tyrosine phosphatase activity may involve modulation of the level of serine phosphorylation. If this speculation is accurate, we could hypothesize that the reduced protein tyrosine phosphatase activity observed in PP-2A-inhibited cells results from increased serine phosphorylation due to the inability of PP-2A to dephosphorylate these tyrosine phosphatases. In fact, our earlier studies had shown that the phosphoserine content of several proteins becomes increased after PP-2A is inhibited in nonmetastatic tumor cells with okadaic acid treatment [18], although the identity of most of these proteins is not known. Although indirect in regulation, the reduced tyrosine phosphorylation of paxillin in PP-2A-inhibited cells can alternatively be explained by the observed reduction in protein tyrosine phosphatase activity. Paxillin phosphorylation is mediated by focal adhesion kinase (FAK) and src [57– 59]. Src activity is positively regulated by a single tyrosine residue (Y527) that when phosphorylated stabilizes src in an inactive conformation [60–62]. By dephosphorylating this tyrosine residue, protein tyrosine phosphatases stimulate src activity. Several have been implicated in catalyzing this dephosphorylation including Shp-2 [46, 63–68]. We are currently examining if the reduced level of paxillin tyrosine phosphorylation that is seen in PP-2A-inhibited cells is due to the loss of Shp-2 dephosphorylation of src’s Y527 residue, which would promote the inactive form of src unable to tyrosine phosphorylate paxillin.

Acknowledgements This work was supported by the Medical Research Service of the Department of Veterans Affairs, and by Grants CA45080, CA77208, CA79768, CA78262 and CA85266 from the National Institutes of Health (M.R.I.Y.).

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