©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Tyrosine Phosphorylation and Enhanced Expression of Paxillin during Neuronal Differentiation in Vitro(*)

(Received for publication, November 13, 1995; and in revised form, January 12, 1996)

Phillip S. Leventhal Eva L. Feldman (§)

From the Department of Neurology, University of Michigan, Ann Arbor, Michigan 48109-0588

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tyrosine phosphorylation has been implicated as a means by which neurite outgrowth is regulated. Because paxillin is a tyrosine-phosphorylated protein that may play a role in regulating cell morphology, we examined its expression in neuronal cells and how its tyrosine phosphorylation is related to neurite outgrowth. Paxillin was identified in several neuronal cell lines with an increased level upon differentiation. In SH-SY5Y cells, paxillin was localized along with actin filaments where processes extended from the cell body and in neuritic growth cones. Furthermore, paxillin was tyrosine-phosphorylated in SH-SY5Y cells upon adhesion to laminin. Paxillin tyrosine phosphorylation paralleled that of focal adhesion kinase and occurred as cell spreading, and neurite formation was initiated. Colchicine blocked neurite outgrowth but had no effect on cell spreading or on paxillin or focal adhesion kinase tyrosine phosphorylation. In contrast, cytochalasin D eliminated neurite outgrowth, cell spreading, and the tyrosine phosphorylation of paxillin and focal adhesion kinase. These results show that paxillin is tyrosine-phosphorylated upon integrin ligand binding in neuronal cells. Our findings suggest that paxillin tyrosine phosphorylation is linked to a remodeling of the actin cytoskeleton that leads to cell spreading and neurite formation and thus a differentiated neuronal phenotype.


INTRODUCTION

Interaction of neuronal cells with the extracellular matrix (ECM) (^1)and soluble neurotrophic factors regulates formation and guidance of neurites (1) by directing changes in microtubules and the actin cytoskeleton(2) . This control of neurite growth is mediated, in part, by protein tyrosine phosphorylation. For example, tyrosine kinase inhibitors can both inhibit and promote neurite outgrowth depending on the growth substrate (3, 4) . Also, neuronal growth cones contain high levels of Src family tyrosine kinases(5, 6) , which play a role in neurite formation(7, 8) . Additionally, tyrosine kinase activity is associated with growth cone glycoproteins(9) , and tyrosine-phosphorylated proteins are concentrated in the tips of growth cone filopodia(10) . Finally, focal adhesion kinase (FAK) is tyrosine-phosphorylated during laminin (LN)-stimulated neurite outgrowth in SH-SY5Y human neuroblastoma cells (11) . This tyrosine phosphorylation of FAK is not unexpected given that laminin-stimulated neurite outgrowth in SH-SY5Y cells is mediated by alpha(1)beta(1) and alpha(3)beta(1) integrins (12) and that ECM-integrin binding results in the tyrosine phosphorylation of FAK in non-neuronal cells such as fibroblasts (13) .

Paxillin is another protein that is tyrosine-phosphorylated upon ECM-integrin binding in fibroblasts(13) . Paxillin binds to vinculin, alpha-actinin, and talin and therefore, like FAK, may help direct actin filament (F-actin)-membrane interactions(13, 14) . Based on this background and the fact that changes in the actin cytoskeleton are required for neurite formation and growth cone guidance(2) , we suspected that paxillin might be tyrosine-phosphorylated during laminin-stimulated neurite outgrowth. Indeed, we found that adhesion to laminin induced the tyrosine phosphorylation of paxillin in SH-SY5Y cells. Paxillin tyrosine phosphorylation appears to be linked to F-actin redistribution during cell spreading and neurite formation and thus to the generation of a differentiated neuronal phenotype. We also found that, compared with undifferentiated cells, differentiated neuronal cells possess an increased level of paxillin protein.


EXPERIMENTAL PROCEDURES

Cell Culture

SH-SY5Y human neuroblastoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum and were differentiated for 48-72 h with 10 µM retinoic acid(15) . Neuro-2a mouse neuroblastoma cells were grown in minimal essential medium containing 10% fetal bovine serum and 1% non-essential amino acids and were differentiated for 48 h with 5 mM dibutyryl cAMP(16) . N1E-115 rat neuroblastoma cells were grown in DMEM containing 10% fetal calf serum and differentiated for 72 h with 1.5% Me(2)SO(17) .

Analysis of LN-stimulated Tyrosine Phosphorylation and Neurite Outgrowth

LN-coated tissue culture plates were obtained from Collaborative-Becton Dickinson (Bedford, MA). SH-SY5Y cells were removed with trypsin-EDTA and resuspended in DMEM (serum free). Cells were then collected immediately or added to tissue culture plates. After 15-120 min, non-adherent cells were removed, and neurite outgrowth (percent of cells extending the process >10 µm) was determined in five random fields. As described previously(18) , cell lysates were then directly analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), or proteins were first immunoprecipitated from lysates (500 µg of protein/sample) using 4 µg/ml anti-paxillin monoclonal antibody (mAb) (Transduction Laboratories), 10 µg/ml anti-FAK mAb 2A7 (gift of Dr. J. T. Parsons, University of Virginia), or 10 µg/ml polyclonal anti-FAK antibody BC3 (Upstate Biochemicals, Inc., Lake Placid, NY). Proteins separated by SDS-PAGE were electrophoretically transferred to nitrocellulose. Anti-phosphotyrosine immunoblotting was performed as described previously (18) using a combination of 1 µg/ml mAb PY20 (Transduction Laboratories) and 0.4 µg/ml mAb 4G10 (Upstate Biotechnology, Inc.). For anti-paxillin immunoblotting, nitrocellulose membranes were incubated with 25 ng/ml anti-paxillin mAb (Transduction Laboratories). Molecular mass was estimated by comparison with prestained molecular weight standards.

Indirect Immunofluorescence and Staining of F-actin

SH-SY5Y cells were grown for 2 h on LN/polylysine-coated coverslips (Collaborative-Becton Dickinson). The cells were fixed with 4% paraformaldehyde, permeabilized with 0.15% Triton X-100, and costained for F-actin and paxillin using 25 µg/ml anti-paxillin mAb and 3 units/ml rhodamine-phalloidin (Molecular Probes). Cells were imaged with a Nikon Diaphot microscope in conjunction with a Bio-Rad MRC-600 laser scanning confocal system.


RESULTS

To explore the role of paxillin in neurons, we first examined the expression of this protein in neuronal cell lines. As shown in Fig. 1A, paxillin was expressed in the neuroblastoma cell lines SH-SY5Y (human), N1E-115 (rat), and Neuro-2a (mouse). On immunoblots, paxillin migrated as a doublet of 70-kDa proteins. This agrees with the characteristics of paxillin in non-neuronal cells(13, 14) . In each cell line, neuronal differentiation increased the level of immunodetectable paxillin (Fig. 1B).


Figure 1: Expression of paxillin in neuronal cell lines. Undifferentiated(-) and differentiated (+) neuroblastoma cell lines SH-SY5Y (human), N1E-115 (rat), and Neuro-2a (mouse) were grown in plastic tissue culture plates under conditions described under ``Experimental Procedures.'' A, 40 µg of total cellular protein was separated by SDS-PAGE and analyzed by immunoblotting for paxillin. B, the percent increase in paxillin expression in differentiated versus undifferentiated cells was assessed from densitometric analyses of autoluminographs. Bars represent mean ± range (n = 2).



We also examined the subcellular localization of paxillin in SH-SY5Y cells. Shown in panels A and B of Fig. 2is a cell that is beginning to extend neurites and in panels C and D is a cell that has already extended neurites. In both cells, staining for paxillin (panels A and C) and F-actin (panels B and D) was most intense at the periphery of SH-SY5Y cells, especially in neuritic growth cones (panels C and D) or where processes extended from the cell body (panels A and B).


Figure 2: Localization of paxillin and F-actin in SH-SY5Y cells. Retinoic acid-differentiated SH-SY5Y cells were plated on LN/polylysine-coated coverslips for 2 h. Cells were fixed, permeabilized, and costained for paxillin (A, C) and F-actin (B, D). In A and B is a cell that is beginning to extend neurites and in C and D is a cell that has already extended neurites.



Because paxillin is tyrosine-phosphorylated during ECM-integrin binding in fibroblasts(13) , we suspected that it would be tyrosine-phosphorylated in SH-SY5Y cells during the rapid outgrowth of neurites on LN(12, 15) . Analyses of whole cell lysates revealed two groups of proteins that were tyrosine-phosphorylated in LN adherent cells but not in suspension cells (Fig. 3). One set of tyrosine-phosphorylated proteins migrated in the 110-135-kDa range. Another group migrated at 70 kDa, consistent with the characteristics of paxillin that we observed (see Fig. 1). An identical pattern of tyrosine phosphorylation was induced by adhesion to fibronectin (not shown).


Figure 3: Adhesion of SH-SY5Y Cells to LN stimulates cellular tyrosine phosphorylation. Retinoic acid-differentiated SH-SY5Y cells in suspension were immediately collected(-) or were added for 1 h to a LN-coated tissue culture plate (+). Tyrosine phosphorylation was assessed in lysates of suspension or LN-adherent cells by anti-phosphotyrosine immunoblotting. Arrow denotes position of the 70-kDa group of proteins.



We next immunoprecipitated paxillin from SH-SY5Y cells in suspension and from LN-adherent SH-SY5Y cells to analyze its tyrosine phosphorylation more directly. We also compared the effect of cellular adhesion to uncoated plastic tissue culture plates wherein there is little cell spreading or neurite outgrowth (Fig. 4A). Finally, we examined the tyrosine phosphorylation of FAK, which typically accompanies paxillin tyrosine phosphorylation (13, 14) and has been reported to be tyrosine-phosphorylated in SH-SY5Y cells during LN-stimulated neurite outgrowth(11) . In cells left in suspension, there was almost no detectable tyrosine-phosphorylated paxillin or FAK (Fig. 4B). Adhesion to LN for 1 h greatly enhanced the tyrosine phosphorylation of paxillin as well as FAK. Adhesion to plastic resulted in a lower level of FAK and paxillin tyrosine phosphorylation. Finally, addition of 10 µg/ml soluble LN to SH-SY5Y cells did not stimulate these tyrosine phosphorylations (not shown).


Figure 4: Tyrosine phosphorylation of paxillin: substrate specificity. Retinoic acid-differentiated SH-SY5Y cells in suspension were collected immediately (Susp) or were added for 1 h to LN-coated or uncoated plastic tissue culture plates. A, images of cells adhering to plastic or LN. B, tyrosine phosphorylation of paxillin was assessed in suspension and substrate-adherent cells by immunoprecipitation of paxillin followed by anti-phosphotyrosine immunoblotting.



Paxillin tyrosine phosphorylation was enhanced within 15 min after addition of the cells to LN-coated tissue culture plates (Fig. 5A). The tyrosine phosphorylation of paxillin reached a maximum 30-60 min after addition to the plates, and in some experiments, a slight decrease in the level of paxillin tyrosine phosphorylation was observed at the 2-h time point. This time course of paxillin tyrosine phosphorylation paralleled that of FAK. Time points earlier than 15 min were not analyzed because of weak cell adhesion. The level of paxillin and FAK in SH-SY5Y cells did not change during the course of these experiments (not shown). Rapid changes in morphology were readily observable by neurite outgrowth measurements (Fig. 5B). After 30 min, measurable neurite outgrowth (processes > 10 µm) was detected, and within 2 h over 80% of the cells had neurites.


Figure 5: Tyrosine phosphorylation of paxillin in SH-SY5Y cells: time course and sensitivity to cytoskeletal inhibitors. Retinoic acid-differentiated SH-SY5Y cells were added to LN-coated or uncoated tissue culture plates. Nonadherent cells were removed after the indicated times. A and C, paxillin was immunoprecipitated from adherent cells and analyzed by anti-phosphotyrosine immunoblotting. Susp represents cells in suspension that were collected at the start of the experiment. B and D, cells were analyzed for the percent extending neurites. A and B, cells were added to LN-coated tissue culture plates for 15-120 min. C and D, cells were incubated for 30 min in DMEM containing no addition, 10 µM colchicine (COL), or 0.5 µM cytochalasin D (CD) and were then added to LN-coated tissue culture plates for 1 h.



Adhesion of neuronal cells to LN results in a remodeling of the actin cytoskeleton as well as polymerization and redistribution of microtubules(2) . Therefore, to address the relationship between paxillin and these cytoskeletal changes, we examined paxillin tyrosine phosphorylation in the presence of colchicine, which inhibits microtubule polymerization(2) . We also assessed the effects of cytochalasin D, which disrupts actin filaments(2) . Addition of 10 µM colchicine had no effect on the tyrosine phosphorylation of paxillin or FAK (Fig. 5C). However, 0.5 µM cytochalasin D eliminated the LN-stimulated tyrosine phosphorylation of both paxillin and FAK. In contrast, both colchicine and cytochalasin D blocked LN-stimulated neurite outgrowth (Fig. 5D). Additionally, cytochalasin D reduced spreading of the cells on LN, whereas colchicine had no effect on cell spreading (not shown).


DISCUSSION

Paxillin is a cytoskeletal protein that is tyrosine-phosphorylated during growth factor-stimulated and integrin-mediated remodeling of the actin cytoskeleton in non-neuronal cells such as fibroblasts(13, 14) . In the current studies, we demonstrate that paxillin is also tyrosine-phosphorylated upon ECM-integrin binding in neuronal cells. This tyrosine phosphorylation of paxillin is not simply due to cell adhesion because cell attachment to plastic resulted in a much lower level of tyrosine phosphorylation than attachment to LN. In general, paxillin tyrosine phosphorylation appears to be related to both cell spreading and neurite formation, morphological changes that are necessary for the generation of a differentiated neuronal phenotype. Finally, consistent with a role for paxillin tyrosine phosphorylation in neurite formation and cell spreading, we found an enhanced level of paxillin in several chemically differentiated neuronal cell lines.

Reorganization of the actin cytoskeleton is necessary for neurite formation (2) and cell spreading (13) while microtubule polymerization is required for elongation of neurites(2) . The fact that colchicine blocked neurite outgrowth but had no effect on paxillin tyrosine phosphorylation suggests that the phosphorylation of paxillin is not dependent on later stages of neurite outgrowth that require microtubule polymerization. Rather, the tyrosine phosphorylation of paxillin appears to be associated with the redistribution of F-actin. First, cytochalasin D, a disrupter of F-actin(2) , prevented laminin-stimulated neurite outgrowth and cell spreading and eliminated the tyrosine phosphorylation of paxillin. Second, we found that paxillin was colocalized with F-actin in SH-SY5Y cells. Third, paxillin tyrosine phosphorylation occurred as the cells spread on the substrate and began to form neurites, processes that are known to be driven by remodeling of F-actin(2, 13) . Finally, in fibroblasts, paxillin tyrosine phosphorylation is thought to help direct reorganization of F-actin(13) . FAK appears to play a related role in these cells because the time course and cytochalasin D sensitivity of FAK phosphorylation paralleled that of paxillin. Such a relationship between FAK and paxillin tyrosine phosphorylation has been identified in several non-neuronal cell lines(13, 14) .

Because laminin-stimulated neurite outgrowth in SH-SY5Y cells is mediated by alpha(1)beta(1) and alpha(3)beta(1) integrins(12) , these findings implicate paxillin tyrosine phosphorylation in integrin signaling by neuronal cells. It is clear, though, that paxillin tyrosine phosphorylation is not limited to a role in integrin-dependent neuronal differentiation because nerve growth factor has also been shown to stimulate the tyrosine phosphorylation of paxillin in PC12 cells(19) . In agreement with our findings, the tyrosine phosphorylation of paxillin in response to nerve growth factor is associated with F-actin redistribution(19, 20) . These studies also support the idea that paxillin tyrosine phosphorylation is not strictly related to neuronal cell adhesion.

In the SH-SY5Y cells, paxillin colocalized with F-actin in the growth cones, suggesting a possible role for paxillin in growth cone guidance. Likewise, vinculin, a protein that binds to paxillin and F-actin(14) , is concentrated in growth cones of PC12 cells(21) . We therefore suspect that paxillin is one of the tyrosine-phosphorylated proteins that has been identified in growth cones of Aplysia neurons (10) .

In summary, paxillin expression is increased during neuronal differentiation. In parallel, paxillin is colocalized with F-actin in growth cones and becomes tyrosine-phosphorylated upon adhesion to laminin. Laminin-stimulated paxillin tyrosine phosphorylation corresponded to the initiation of cell spreading and neurite formation. Tyrosine phosphorylation of paxillin is clearly not only an integrin-mediated response as nerve growth factor also stimulates the tyrosine phosphorylation of paxillin in PC12 cells(12) . Collectively, these results suggest a connection between paxillin tyrosine phosphorylation and F-actin remodeling which, in turn, leads to cell spreading and neurite outgrowth and, thus, the expression of a differentiated neuronal phenotype.


FOOTNOTES

*
This work was supported by Grant R29 NS32843 from the National Institutes of Health (to E. L. F.) and Individual National Research Service Award 1F32NS09912-01 and Training Grant T5T32NW0722 from the National Institutes of Health (to P. S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Neurology, University of Michigan, 4420 Kresge III, 200 Zina Pitcher Place, Ann Arbor, MI 48109-0588. Tel.: 313-763-7274; Fax: 313-763-7275.

(^1)
The abbreviations used are: ECM, extracellular matrix; FAK, focal adhesion kinase; DMEM, Dulbecco's modified Eagle's media; F-actin, actin filaments; LN, laminin; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Dr. Yeoash Raphael and Jackie Kaufman for their extensive help with immunocytochemistry, Walter Meixner and Jim Beals for help in confocal image processing, Ann Randolph, Dr. K. Sue O'Shea, and Dr. Akhilesh Pandey for assistance in manuscript preparation, and Tom Vesbit for expert technical assistance.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.