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First published online 13 July 2004
doi: 10.1242/jcs.01206

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Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive

Larisa Y. Romanova^1,*, Shigeru Hashimoto³, Kee-Oh Chay^1,, Mikhail V. Blagosklonny^2,, Hisataka Sabe³ and J. Frederic Mushinski¹

¹ Molecular Genetics Section, Laboratory of Genetics, National Cancer Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA
² Medicine Branch, National Cancer Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA
³ Department of Molecular Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka, 565-0874, Japan

View larger version (28K): [in a new window]   Fig. 1. Effects of IL-3 and PMA on paxillin phosphorylation. Baf3 cells were deprived of IL-3 for 6 hours or grown on 10 ng/ml IL-3, and aliquots were treated 30 minutes with 4 µM cytochalasin D, 1 µM GF109203X, with 100 nM PMA alone or with 1 µM GF109203X and 100 nM PMA. Paxillin was immunoprecipitated (IP:Paxillin) and analyzed on western blot with a mixture of anti-phosphotyrosine antibodies 4G10 and PY20 (IB:PY) or with anti-paxillin (IB:Paxillin) antibody.

View larger version (30K): [in a new window]   Fig. 2. IL-3 stimulates phosphorylation of paxillin tyrosine residues 31 and 118. (A) Baf3 cells were stably transfected with EGFP (vector) or EGFP-conjugated wild-type -paxillin or the -paxillin YF tyrosine phosphorylation mutants 31, 118, 31/118, 40/181, 31/40/118/181, 31/40/181/434/488 and 40/118/181/434/488. After six hours of IL-3 withdrawal, 10 ng/ml IL-3 was resupplied for 10 minutes. Immunoprecipitated paxillin (IP-Pax) was analyzed on western blots with a mixture of anti-phosphotyrosine antibodies 4G10 and PY20 (IB-PY) or anti-GFP antibody (IB-GFP). (B) Immunoprecipitated paxillin (IP:Paxillin) was treated in vitro with non-specific potato acid phosphatase (PAP) or Protein Tyrosine Phosphatase (PTP), which specifically dephosphorylates tyrosine residues and was then analyzed on western blots using anti-paxillin antibody (IB:Paxillin).

View larger version (25K): [in a new window]   Fig. 3. PMA inhibits phosphorylation of paxillin tyrosine residues 31 and 118. Baf3 cells were stably transfected with EGFP (vector) or EGFP-conjugated wild-type -paxillin or the -paxillin YF tyrosine phosphorylation mutants 31, 118, 31/118, 40/181, 31/40/118/181, 31/40/181/434/488 and 40/118/181/434/488. Cells were grown on 10 ng/ml IL-3 and aliquots were treated with 100 nM PMA for 30 minutes. After immunoprecipitation with anti-paxillin antibody (IP:Pax), lysates were analyzed on western blots with a mixture of anti-phosphotyrosine antibodies 4G10 and PY20 (IB:PY) or with anti-GFP (IB:GFP) antibody.

View larger version (60K): [in a new window]   Fig. 4. Phosphorylation of paxillin tyrosine residues 31 and 118 in endogenous paxillin is stimulated by IL-3 and inhibited by PMA. After a 6-hour IL-3 deprivation in Baf3 cells, an aliquot was treated for 30 minutes with 10 ng/ml IL-3, 100 nM PMA or their combination. Paxillin was immunoprecipitated (IP Paxillin) and analyzed on western blots with anti-paxillin antibody (IB Paxillin), mixture of anti-phosphotyrosine antibodies 4G10 and PY20 (IB:PY) or antibody specific to phosphorylated paxillin tyrosine residues 31 or 118 (IB pY31Paxillin or IB pY118Paxillin, respectively). The same blot was developed with polyclonal antibodies that recognized the paxillin-binding protein vinculin (IB Vinculin) to demonstrate equal levels of protein loading.

View larger version (50K): [in a new window]   Fig. 5. PMA inhibits the IL-3-induced tyrosine phosphorylation of FAK and FAK-binding proteins. Cells were deprived of IL-3 for 6 hours or grown on IL-3 and treated with 4 µM cytochalasin D (CytD), 1µM GF109203X (GF), 100 nM PMA alone or in combination for 30 minutes. (A) After immunoprecipitation with monoclonal anti-FAK antibody (IP:FAK) or non-immune mouse IgG1 (IP:IgG1) in denaturing conditions, lysates were analyzed on western blots with a mixture of anti-phosphotyrosine 4G10 and PY20 antibody (IB:PY), a phosphospecific anti-FAK(pY397) antibody (IB:FAKpY397) or polyclonal anti-FAK antibody (IB:FAK). (B) In another series of experiments, after immunoprecipitation with anti-FAK antibody (IP:FAK) or non-immune mouse IgG1 (IP:IgG1) in non-denaturing conditions lysates were analysed on western blots with phosphotyrosine PY20 antibody (IB:PY), anti-paxillin antibody (IB:paxillin) or anti-FAK antibody (IB:FAK).

View larger version (41K): [in a new window]   Fig. 6. IL-3-induced phosphorylation of paxillin tyrosine residues 31 and 118 is required for Baf3 cell polarization and motility, and PMA abolishes the effects of IL-3. (A) Baf3 cells expressing EGFP (vector) were deprived of IL-3 for 6 hours, maintained on 10 ng/ml IL-3, or treated with 100 nM PMA for 30 minutes in the presence of IL-3. Nomarski images (DIC) obtained by confocal microscopy are presented in the lower panel; bar, 10 µm. Quantitative analysis of cell morphology was performed by forward light scatter (FSC-H) FACS scans (upper panel). To demonstrate spectrum changes, the silhouette of the broad pattern of EGFP-expressing cells maintained on IL-3 was superimposed on the shaded patterns of exogenous paxillin-expressing cells or EGFP-expressing cells, treated as indicated. (B) Baf3 cells expressing EGFP-conjugated wild-type paxillin or YF tyrosine phosphorylation mutants 31, 118 or 31/118 were maintained on 10 ng/ml IL-3. FACS scan patterns of FSC-H (top panel) and Nomarski images (DIC) (bottom panel) are presented as above. The same control, i.e., FACS profiles of Baf3 cells maintained on IL-3 was used for all panels. The changes in FACS profiles induced by IL-3 deprivation, PMA treatment (A) as well as overexpression of the paxillin YF mutants 31, 118 or 31/118 (B) were statistically significant (Kolmogorov-Smirnov test) with P0.001 and D=0.56; 0.62; 0.19; 0.18 and 0.40, respectively. The figure shows that overexpression of YF 31/118 paxillin mutant rounds the cells in a manner resembling IL-3 deprivation or PMA addition. (C) Spontaneous cell motility was analyzed as described in the Materials and Methods. Results are expressed relative to the motility of Baf3 cells that express EGFP vector growing in IL-3. The average and s.e.m. from six experiments are presented. The motility changes induced by IL-3 deprivation (t=7.5, P=0.0006), cytochalasin D (t=9.8, P=0.0002) or PMA treatment (t=6.62, P=0.0012), as well as overexpression of wild-type paxillin (t= –3.157, P=0.02) or tyrosine phosphorylation mutants 31 (t=3.37, P=0.0198), 118 (t=2.5, P=0.05) and 31/118 (t=8.2, P=0.0004) were assessed by t-test and were statistically significant. Thus, expression of the YF 31/118 paxillin mutant inhibits cell motility in a manner similar to IL-3 deprivation or PMA treatment of Baf3 cells.

View larger version (58K): [in a new window]   Fig. 7. Tyrosine phosphorylation of paxillin residues 31 and 118 does not affect its intracellular distribution in Baf3 cells. Parental Baf3 cells (A) and Baf3 cells expressing EGFP (vector), EGFP-conjugated wild-type paxillin or YF tyrosine phosphorylation mutant 31/118 (B) were maintained on 10 ng/ml IL-3. Endogenous paxillin was visualized by the consecutive staining with monoclonal anti-paxillin and fluorescein-conjugated goat anti-mouse secondary antibody (A). DIC images were collected using transmitted light; intracellular distribution of endogenous paxillin (A) and EGFP-paxillin (B) was analyzed by fluorescent confocal microscopy. Bars, 10 µm. Thus, we show similar distribution of endogenous paxillin, EGFP-conjugated wild-type paxillin and its mutants.

View larger version (36K): [in a new window]   Fig. 8. Effect of PMA on tyrosine phosphorylation and motility of several non-adherent (32D, CCRF CEM, Daudi) and adherent (Swiss 3T3, COS-1) cell lines maintained in complete medium. (A) After a 15-minute treatment with 100 nM PMA, paxillin was immunoprecipitated and analyzed by western blotting with a mixture of anti-PY20 and 4G10 antibody (IB pY) or with anti-paxillin antibody (IB Paxillin). (B) Spontaneous cell motility was analyzed as described in the Materials and Methods. The mean and s.e.m. from six experiments are presented. PMA inhibited the motility of 32D cell line (t=9.2, P=0.0003) and CCRF CEM cell line (t=8.4, P=0.0004), increased the motility of Swiss 3T3 (t=–4.16, P=0.0088) but did not significantly affect the motility of Daudi (t=1.1, P=0.33) or Cos1 cells (t=–0.49, P=0.64).

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