Oncogene (2002) 21, 96 ± 107 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Paxillin null embryonic stem cells are impaired in spreading and of kinase

Ramon Wade1, Joanna Bohl1 and Scott Vande Pol*,1

1Case Western Reserve University School of Medicine, Institute of Pathology, Cleveland, Ohio, OH 44106, USA

Paxillin is a focal-adhesion associated implicated 1999; Turner, 2000a). cytoplasmic domains in the regulation of integrin signaling and organization of lack known enzymatic activity and initiate signaling the actin cytoskeleton. Paxillin associates with numerous through association with non- kinases including signaling molecules including adaptor molecules Integrin-linked Kinase, Focal Adhesion Kinase (FAK) (p130Cas, CRK), kinases (FAK, Pyk2, PAK and and SRC family members, as well as a number of SRC), tyrosine phosphatases (PTP ± PEST), ARF ± regulatory adaptor molecules including paxillin (Buck- GAP (p95pkl, PAG3) and papillomavirus E6 ley et al., 1998; Glenney and Zokas, 1989; Turner et al., oncoproteins. Although paxillin is tyrosine phosphory- 1990). Paxillin is a 68 kDa focal adhesion protein ®rst lated in cellular processes such as cell attachment and identi®ed as a prominent tyrosine phosphorylated spreading, little direct evidence is available about protein in v-src transformed cells, and is one member paxillin's role in these events. Targeted disruption in a family of three related adaptor proteins including was used to generate paxillin null mouse embryonic stem the hematopoietic protein Leupaxin and Hic-5 (Buckley (ES) cells and paxillin null di€erentiated cells. Paxillin et al., 1998; Glenney and Zokas, 1989; Shibanuma et al., null ES cells exhibit delayed spreading on integrin 1994; Turner, 2000a; Lipsky et al., 1998). binding substrates ®bronectin and laminin, and there is Paxillin is a regulatory molecule that interacts with a reduced tyrosine phosphorylation of Focal Adhesion variety of molecules that regulate the cytoskeleton, and Kinase (FAK). Both of these phenotypes are recovered is expressed at low levels compared to structural in paxillin knockout cells upon exogenous re-expression proteins such as and that couple of paxillin. The individual LD motifs of paxillin that are to the actin cytoskeleton (Turner et al., 1990). The binding sites for FAK, vinculin and ARF ± GAP proteins, amino terminus of paxillin contains ®ve protein- as well as tyrosine residues that when phosphorylated binding rich motifs (LDXLLXDL), known as create binding sites for CRK family members, are LD motifs, that are conserved among mammalian, dispensable for FAK phosphorylation and early cell avian, and Drosophila paxillin molecules (Brown et al., spreading. These results demonstrate that paxillin 1998a; Wheeler and Hynes, 2001). LD1 can interact contributes to attachment-dependent tyrosine phosphor- with a recently described protein called actopaxin that ylation of FAK and early cell spreading in ES cells. is implicated in cytoskeletal remodeling as well as Oncogene (2002) 21, 96 ± 107. DOI: 10.1038/sj/onc/ ILK3, a threonine kinase homologous to 1205013 Integrin Linked Kinase (Nikolopoulos and Turner, 2000, 2001). LD2 and LD4 interact with FAK in cell Keywords: cytoskeleton; paxillin; FAK; integrin; lysates and mutation of both LD motifs is required to kinases; actin abrogate the interaction of paxillin with FAK (Brown et al., 1996; Thomas et al., 1999). LD1, LD2 and LD4 can associate with the papillomavirus E6 protein and Introduction this interaction can compete for the binding sites on paxillin for FAK and vinculin (Tong et al., 1997; our Cell adhesion to extra cellular matrix (ECM) through unpublished observations; Vande Pol et al., 1998). the binding of cellular integrin receptors with ECM Paxillin has recently been found to associate with a proteins initiates a signaling cascade number of ARF ± GAP proteins known to regulate that regulates multiple functions including cell attach- both actin rearrangement and membrane tracking ment, spreading, migration, di€erentiation, survival and (Kondo et al., 2000; Mazaki et al., 2001; Norman et (reviewed in Giancotti and Ruoslahti, al., 1998). The ARF ± GAP protein p95PKL binds to LD4, thereby linking paxillin to the serine/threonine kinase PAK1 that can regulate actin organization (Lu and Mayer, 1999; Turner et al., 1999). Overexpression *Correspondence: S Vande Pol, Case Western Reserve University of activated ARF proteins causes co-localization of School of Medicine, Institute of Pathology, 10900 Euclid Avenue, paxillin with speci®c ARF family members, and can Cleveland, Ohio, OH 44106, USA; E-mail: [email protected] Received 16 July 2001; revised 12 September 2001; accepted 10 cause paxillin recruitment to focal adhesions (Norman October 2001 et al., 1998; Kondo et al., 2000). A poly- rich Paxillin in cell spreading and FAK phosphorylation R Wade et al 97 domain in the amino terminus of paxillin may be a mechanism for generating this phenotype is unclear binding site for the SRC protein (Turner, 1998, 2000b). (Liu et al., 1999). Paxillin binds many signaling Upon cell attachment to the ECM, the FAK/SRC molecules and may therefore be involved in integrin complex phosphorylates tyrosine residues at positions signaling and membrane tracking. Adherent prolif- 31 and 118 providing binding sites for the CRK family erative cell lines contain paxillin and determining the of SH2-containing adaptor molecules (Igishi et al., e€ect of overexpressed paxillin mutants is dicult due 1999; Petit et al., 2000; Schaller and Parsons, 1994; to the presence of the native protein. In this report, a Turner, 2000a). paxillin null mouse ES cell was generated by targeted The carboxyl terminus of paxillin contains four zinc gene disruption. We ®nd that paxillin null ES cells have ®nger motifs known as LIM domains, one of which a defect in the early events of spreading on ®bronectin (LIM3) is necessary for the localization of paxillin to and laminin coated surfaces. Analysis of FAK tyrosine focal adhesions (Brown et al., 1996). Paxillin LIM phosphorylation revealed that the paxillin null ES cells domains contain binding sites for proteins such as are de®cient for FAK tyrosine phosphorylation. These PTP ± Pest and ! and g (Angers-Loustau et al., results demonstrate that paxillin facilitates the early 1999; Brown et al., 1996, 1998b; Brown and Turner, events of cell spreading and FAK phosphorylation in 1999; Cote et al., 1999; Herreros et al., 2000; Shen et ES cells. al., 1998, 2000; Turner, 1998, 2000a,b). The LIM domains contain a number of serine/threonine residues that are phosphorylated upon cell adhesion to Results ®bronectin and stimulation. Serine and threonine phosphorylation of residues in LIM3 by as Targeted disruption of paxillin in ES cells yet unidenti®ed kinases may regulate paxillin localiza- tion to focal adhesions (Brown et al., 1996, 1998b; In order to better understand the role of paxillin in Brown and Turner, 1999; Turner, 1994). integrin signaling, we created a cell line in which both Paxillin associates with the Focal Adhesion Kinase alleles of paxillin were deleted by targeted gene (FAK), a 125 Kda non- that is disruption as detailed in the Materials and methods. activated upon integrin ligation to immobilized ECM Oligonucleotides used to isolate paxillin genomic components (reviewed in Guan, 1997; Schaller and fragments and to screen ES cell colonies for homo- Parsons, 1994; Schlaepfer et al., 1999). Overexpression logous recombination between cellular paxillin gene of the carboxy terminal domain of FAK blocks the and the targeting plasmid are shown in Figure 1. interaction of FAK and paxillin and inhibits FAK The paxillin targeting plasmid was electroporated activation and cell spreading that is initiated upon cell into ES cells and drug resistant colonies were selected attachment to the ECM (Richardson and Parsons, and screened in a process consisting of two separate 1996). For this reason, the association of FAK with PCR reactions. Oligonucleotide pair C/G generates a paxillin has been implicated in the regulation of FAK 2.8 kb band in any cell that has a wild type paxillin activation. FAK contains an amino terminal regulatory allele; this reaction serves as a positive control for the domain, a central kinase domain and the c-terminal DNA preparation. Oligonucleotide pair F/G only focal adhesion-targeting (FAT) domain that is respon- generates a 2.4 kb product in ES cell colonies where sible for FAK localization to focal adhesions (Guan, the targeting vector has recombined homologously with 1997; Schlaepfer et al., 1999; Schlaepfer and Hunter, the genome so that the neomycin gene is in proximity 1996; Shen and Schaller, 1999). The FAT domain of to oligonucleotide G (Figure 1). FAK is also responsible for the association of FAK with paxillin, although the association of FAK with Clone 17 is paxillin null paxillin may not be required for FAK localization to focal adhesions (Guan, 1997; Thomas et al., 1999). Using oligonucleotide pair F/G to screen for homo- FAK contains several tyrosine residues that are logous recombination between the targeting plasmid phosphorylated in response to cell spreading and and genomic DNA, clone 31 cells are haploid for growth factor signaling (Guan, 1997; Schlaepfer et paxillin (Figure 2a). PCR reactions with oligonucleo- al., 1999). Tyrosine 397 is the major autophosphoryla- tide pair C/G showed that each of the colonies tion site and this phosphorylated tyrosine can serve as contained DNA that yielded a product in a PCR a binding site for c-src with subsequent tyrosine reaction. To con®rm the previous result, oligonucleo- phosphorylation of paxillin and FAK by the FAK/c- tides H, I, F, and G (Figure 1) were used in src complex. Phosphorylated tyrosine 925 is a binding combination reactions with wild type and clone 31 site for the adaptor protein Grb2 thereby linking cells. The wild type cells did not make product with integrin signaling to the activation of ERK (Guan, any of the oligonucleotide pairs; however, the clone 31 1997; Schlaepfer et al., 1999). cell line produced products with all the oligonucleotide Despite the association of FAK and paxillin, there is pairs (Figure 2b). By this analysis clone 31 cells contain little direct evidence for the function(s) of paxillin in one paxillin allele deleted by targeted gene disruption. the cell. Paxillin has been shown to interact with a4 In order to select for disruption of the second paxillin integrins and thereby reduce cell spreading of mouse allele, clone 31 cells were placed in high G418 to select embryonic ®broblasts on VCAM1; however, the exact for gene conversion and the resulting colonies were

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 98

Figure 1 Diagram of the knockout strategy. At the top is an illustration of the human paxillin gene exon structure (not drawn to scale). The lines between exon 1 and 2 indicate an approximately 40 kb intron in the human gene structure. The mouse paxillin gene segments ampli®ed in this study had identical exon-intron boundaries as the human gene. Genomic fragments of the paxillin gene were ampli®ed using oligonucleotides A/B and D/E as detailed in Materials and methods and cloned into the pPNT targeting vector (middle diagram). Segment A/B contains exon 2 ± 6 and segment D/E is exon 10 ± 3'UTR. TK refers to the thymidine kinase gene in the targeting plasmid. After homologous recombination between the targeting vector and the mouse paxillin gene the neomycin resistance gene is incorporated into the genome and exons are deleted, corresponding to paxillin a cDNA amino acids 225 ± 404 (bottom diagram). The HSV ± TK gene is lost during homologous recombination, rendering cells resistant to both G418 and gancyclovir

screened for a paxillin null genotype (see Materials and approximately equal levels for each protein investigated methods); after an event that disrupts both paxillin (Figure 3). ES cells and in vitro di€erentiated cells alleles, the binding site for oligonucleotide C should be derived from embryoid bodies were investigated for the lost, but the binding site for oligonucleotide F should presence of the paxillin related protein Hic-5. Wild remain. Screening of colonies after high-level drug type and paxillin null undi€erentiated ES cells show no selection by PCR revealed that clone 17 was paxillin detectable Hic-5 by Western blot analysis, even after null (Figure 2c). The null phenotype was con®rmed by prolonged exposure (Figure 3). The in vitro di€er- screening for paxillin protein expression on blots with entiated cells derived from the wild type, haploid and equalized cell lysates from wild type, haploid and null paxillin null cells show similar levels of Hic-5 (Figure cells; no truncated paxillin products resulted from gene 3). A 46 kDa paxillin band, which may represent a targeting (Figure 2d) while vinculin expression was the proteolytic, or internally initiated paxillin molecule is same from all samples (Figure 2d). Clone 43 (paxillin absent in the paxillin null cells (Figure 3 bottom panel). +/7) cells were subjected to similar selection and growth conditions as clone 17 (paxillin 7/7) cells but Spreading and attachment of ES cells on fibronectin remained +/- for paxillin, and therefore clone 43 and clone 17 cells were used in all subsequent assays for Wild type and paxillin null ES cells were evaluated for paxillin functions. attachment and spreading. Clone 43 and clone 17 cells were plated onto ®bronectin-coated glass coverslips for 24 h. Compared to most di€erentiated cells, ES cells Western blot analysis of paxillin null ES cells spread slowly after attachment. The clone 43 haploid Disruption of the talin gene resulted in the loss of cells were partially spread 24 h after plating; however, integrin b1 expression in ES cells (Priddle et al., 1998). the paxillin null clone 17 cells were mostly unspread at We therefore wanted to characterize the paxillin null 24 h (compare Figure 4a to b). In ES cell cultures there cells for the loss of focal adhesion proteins. Equalized are two morphologically distinct populations. Most ES lysates from wild type, haploid clone 43 and paxillin cells are small and spread in an angular shape, while a null clone 17 were fractionated on SDS ± PAGE and second small subpopulation of ES cells spread to a very blotted to PVDF membrane. The blots were probed large and irregular shape (Figure 4a,b arrow heads). with the indicated antibodies and all blots showed Both the paxillin null and haploid ES cell lines contain

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 99

Figure 2 ES clone 17 is null for paxillin. (a) Screening ES colonies for targeted disruption of a single paxillin allele. A PCR reaction with oligonucleotides C/G generates a 2.8 kb fragment in any cell with a wild type paxillin gene. A PCR reaction with oligonucleotides F/G generates a 2.4 kb fragment only if homologous recombination replaces part of the paxillin gene with the PGK ± Neo cassette. This result is consistent with clone 31 being haploid for paxillin. (b) Con®rmation of clone 31 cells as haploid for paxillin. Additional oligonucleotides outside of the F/G pair were used together and in combination to con®rm the screening results shown in a. Oligo F and H bind in the neomycin cassette and oligo G and I bind in the paxillin genome. PCR reactions with oligo pairs F/G, F/I, H/G and H/I only generate the 2.4 kb product in a colony where a homologous recombination replaces paxillin exons 6 ± 9 with the PGK neo gene. Clone 31 is haploid for paxillin by this analysis. The lanes marked + in Figure 2 are positive control reactions utilizing a cell line speci®cally created for this purpose as described in the methods. (c) Selection of paxillin null ES cells from clone 31 ES cells. Paxillin haploid clone 31 cells were cultured in ES media containing 1 mg/ml G418 to select for duplication of the neomycin resistance gene and deleting the remaining paxillin allele (Wiles, 1993), resulting in the loss of the binding site oligonucleotide C. ES cells null for both alleles of paxillin will not make the 2.8 kb product with the C/G oligo pair since both copies of the wild type gene have been removed, but will make the expected 2.4 kb product for the F/G PCR reaction. This result is consistent with clone 17 cells being null for paxillin. (d) Paxillin protein expression in ES cell clones. Cell lysates from ES clones genotypicaly characterized by PCR as wild type +/+, haploid +/7 (31, 43, 55) and null 7/7 (17) were equalized for protein concentration, separated by SDS ± PAGE, and blotted to PVDF membrane. Duplicate blots were probed with antibodies to paxillin (top) and vinculin (bottom). Clone 17 cells do not express paxillin

these infrequent but large spreading cells that contain type and paxillin haploid ES cells spread similarly after prominent actin stress ®bers. In the paxillin null clone attachment, however the clone 17 cells were delayed in 17 cells the large cells retain the ability to spread spreading (Figure 5b). Stable re-expression of wild type quickly, while the small cells are delayed in spreading paxillin in the clone 17 null cells restored spreading to compared to the clone 43 cells (compare clone 43 Figure levels similar to that of wild type and haploid cells 4c and clone 17 cells 4d). Similar results were obtained (Figure 5b). Similar results were observed with clone on surfaces coated with laminin or collagen (data not 43, clone 17, and paxillin reconstituted clone 17 cells shown). The spreading defect of small clone 17 ES cells plated onto laminin-coated plastic surfaces (data not on matrix coated glass coverslips is apparent at 8 to shown). We used stably transfected clone 17 cells 16 h and continues for 24 h. After 48 h the small clone expressing paxillin mutants to recover spreading of the 43 and clone 17 cells appear similar (data not shown). A paxillin null cells plated onto ®bronectin-coated plastic growth curve for clone 43 and clone 17 cells is shown in surfaces for 1 h. All of the mutants that we tested were Figure 4e. Clone 43 cells grew at a slightly faster rate able to restore spreading (Figure 6b). The level of and to a slightly higher density than the clone 17 cells paxillin protein in the stable paxillin-transfected clone (Figure 4e). 17 cell lines is slightly less than that of the clone 43 The attachment and spreading di€erence between haploid cells (Figure 5c, bottom). In all of our clone 17 and clone 43 cells was quanti®ed in Figure 5. experiments, ES cells spread more quickly on matrix- While both paxillin null and paxillin expressing cell coated plastic surfaces than on matrix-coated glass lines appeared to have approximately equal attachment coverslips, possibly due to the higher matrix binding to a ®bronectin-coated plastic surface (Figure 5a), there capacity of plastic compared to glass (our unpublished were marked di€erences in cell spreading. The wild observations).

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 100 In order to determine if transient paxillin expression in clone 17 cells would result in enhanced cell spreading, clone 43 and paxillin-null clone 17 cells were transiently cotransfected with plasmids to express wild type paxillin or paxillin mutants together with green ¯uorescent protein (GFP). Twenty-four hours after transfection, cells were trypsinized and replated onto ®bronectin- coated glass coverslips for an additional 8 h. Approxi- mately 60% of clone 43 cells were spread at 8 h (Figure 6a). Less than 10% of the clone 17 paxillin null cells were spread at 8 h post plating (Figure 6a). The spreading defect was substantially restored in clone 17 cells transfected with plasmids expressing the 46 kDa mole- cule (paxillin amino acids 133 ± 559) or wild type paxillin (Figure 6a). The 46 kDa paxillin molecule is deleted of LD1, the amino-terminal poly-proline region and SH2 binding motifs at amino acids 31, and 118. In contrast, neither paxillin amino acids 1 ± 331 (deleted of the LIM domains) nor the paxillin LIM domains either alone or together restored cell spreading (Figure 6a). Expression of paxillin was con®rmed by Western blot (Figure 6b); the LIMS only domain was expressed at very low levels Figure 3 Western blot analysis of paxillin null ES cells. (and is not evident in the Figure 6 Western blot), and the Equalized ES lysates for wild type (+/+), null (7/7, clone 1 ± 331 domain of paxillin is not recognized by the 17), and haploid cells (+/7, clone 43) were separated by SDS ± PAGE and blotted to PVDF membrane, and probed with the monoclonal antibody used in the experiment. indicated antibody. The designations S and P refer to soluble and pellet fractions of the lysates. Lysates from in vitro di€erentiated ES cells (Dif wt, Dif 43, Dif 17) and undi€erentiated ES cells were Reduced tyrosine phosphorylation of FAK in analysed with the Hic-5 and paxillin antibodies. MDBK cell paxillin null cells lysate was used as a positive control for Hic-5 expression. The position of molecular weight markers in kDa is indicated to the Because paxillin binds a FAK/SRC complex and paxillin right of the lower blots has been implicated in the regulation of FAK and SRC

Figure 4 Delayed spreading of paxillin null clone 17 cells on ®bronectin coated cover slips, and reduced growth rate of paxillin null ES cells. (a) Clone 43 ES (a and c) and Paxillin null clone 17 cells (b and d) were plated onto ®bronectin coated glass cover slips for 24 h. Cells were ®xed and stained with FITC conjugated phalloidin (Sigma). Arrows indicate small cells and arrowhead indicate the large cells. (e) Equal numbers of paxillin +/7 clone 43 and paxillin 7/7 clone 17 cells were seeded onto gelatin coated plastic surfaces and cell numbers ascertained daily as described in the Materials and methods

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 101

Figure 5 Time course of ES cell attachment and spreading on a ®bronectin coated surface, and recovery of cell spreading upon re- expression of paxillin. Wild type ES cells (^), haploid clone 43 (&), paxillin null clone 17 (D) and a clone 17 cell line stably transfected with wild type paxillin (*) were used in attachment (a) and spreading (b) assays as described in the Materials and methods on ®bronectin coated plastic surfaces. All points are the average of three separate experiments with triplicate samples per experiment. Standard deviations did not exceed 20% of the mean values. The clone 17 paxillin cells are delayed for spreading and this phenotype is recovered upon re-expression of paxillin. (c) Spreading assay with clone 43 cells and clone 17 cells stably re- expressing paxillin or paxillin mutants. Transfected plasmids and paxillin expression levels in the stably transfected clone 17 paxillin 7/7 cells is indicated below. Cells were plated onto ®bronectin-coated plastic surfaces and assessed for cell spreading 1 h later. Paxillin re-expression restores cell spreading

functions, we examined cell lysates for tyrosine phos- recover FAK tyrosine phosphorylation (Figure 8b). phorylation. Equalized protein lysates from wild type, Expression of paxillin mutants deleted of individual clone 17 and clone 43 cells were separated by SDS ± LD motifs LD1, LD3, LD4, and LD5 restored FAK PAGE, transferred to PVDF membrane, and probed tyrosine phosphorylation (Figure 8c and data not with an antibody against phosphotyrosine. The wild type shown). Our studies indicate that paxillin tyrosine and haploid lanes showed similar banding patterns; residues 31, 33, 40 and 118, as well as LD1, LD3, LD4 however, the paxillin null cells showed reduced tyrosine and LD5 and the amino terminal proline rich region phosphorylation of bands at approximately 125 kDa are individually dispensable for FAK tyrosine phos- and 205 kDa (Figure 7). phorylation in ES cells (Figure 9). Paxillin molecules The loss of tyrosine phosphorylated bands at deleted of LD2 were unstable in ES cell expression 125 kDa suggested that FAK might be reduced for (data not shown). Comparison of immunoprecipitated phosphotyrosine; therefore equalized protein lysates pp120, p130 CAS, CRK, SRC and PYK2 from clone from clone 43 and 17 ES cells were immunoprecipi- 43 and clone 17 ES cell lysates revealed equal protein tated for FAK and separated by SDS ± PAGE. levels and phosphotyrosine content by Western blot Duplicate gels were transferred to PVDF, one probed analysis (data not shown). for phosphotyrosine, the other for FAK. The wild type, clone 43, and clone 17 cells had very similar levels of FAK protein; however, the clone 17 cells showed Discussion markedly decreased levels of FAK tyrosine phosphor- ylation (Figure 8a). The decreased FAK phospho- To further our understanding of the role of paxillin in tyrosine in clone 17 cells was reversed by stable integrin signaling, we used targeted gene disruption to re-expression of wild type paxillin (Figure 8a). FAK create a paxillin null cell line. Clone 17 ES cells do not tyrosine phosphorylation was restored in clone 17 cell express detectable levels of paxillin or Hic-5 by lines expressing the paxillin L7A mutant and the Western blot. This is in contrast to the di€erentiated amino-terminally truncated 46 kDA molecule (amino cells derived from the clone 17 ES cells that express acids 133 ± 559) (Figure 8b). Expression of the amino Hic-5 (Figure 3 and our unpublished observations on terminus alone (amino acids 1 ± 331) was not able to di€erentiated cell lines derived from clone 17 ES cells).

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 102

Figure 6 Recovery of spreading defect in paxillin null cells expressing paxillin and paxillin mutants. ES clone 43 and clone 17 cells were cotransfected with GFP and paxillin expressing vectors. The bars in order are clone 43, clone 17, 17+wt paxillin, 17+ paxillin 133 ± 559 (46 kDa molecule), 17+ paxillin 1 ± 331, 17+ paxillin LIM domain (aa310 ± 559), 17+ paxillin 1 ± 331 + paxillin LIMS. Twenty-four hours post transfection the cells were plated onto ®bronectin coated coverslips for an additional 8 h and the GFP positive cells were scored for spreading as described in the Materials and methods. The results from three independent experiments are shown (Top). The expression of paxillin in the clone 43 and clone 17 cells transfected with paxillin vectors is shown (Bottom). The monoclonal antibody used in the experiment does not recognize the 1 ± 331 paxillin molecule, and expression of the LIMS only domain was low and not seen in this exposure

gene from ES cells did not result in reduced levels of any of the proteins that we investigated. The paxillin null cells attach to ECM protein coated surfaces similar to wild type cells but they exhibit delayed spreading on those coated surfaces. The di€erence in the kinetics of cell spreading observed between Figures 4 and 6 versus Figure 5 is due to the use of glass versus plastic matrix coated surfaces; spreading is consider- ably faster on plastic surfaces. In addition to delayed kinetics of cell spreading, the paxillin null cells show a reduction in FAK tyrosine phosphorylation. Signaling from integrins can feed back and alter the anity of integrins for the matrix (reviewed in Tozer et al., 1996). The clone 17 paxillin null cells attach equally as well as wild type cells, suggesting that signaling needed by ES cells for initial attachment to a surface does not require paxillin. Once the cell has engaged the surface, tyrosine kinase dependent signaling results in Figure 7 Paxillin null ES cells are altered in tyrosine phosphor- reorganization of the actin cytoskeleton and cell ylation. Wild type, clone 43 and clone 17 cells were lysed in SDS spreading (reviewed in Feller et al., 1998; Giancotti, sample bu€er; proteins were separated by SDS ± PAGE, and blotted to PVDF. The membranes were probed with the anti- 1997; Lewis and Schwartz, 1998). The paxillin null phosphotyrosine antibody 4G10. Paxillin null clone 17 cells have clone 17 cells show a delayed spreading onto reduced tyrosine phosphorylated bands at approximately 120 and ®bronectin and laminin coated surfaces. This result 205 kDa indicates that paxillin is involved in generating or transmitting the signals that coordinate cell spreading. Forty-eight hours after plating, paxillin null cells The absence paxillin and Hic-5 from the clone 17 cells appear fully spread and similar to the wild type cells. allows direct observations on the function of paxillin In the ES cell culture a subpopulation of cells are large reintroduced into clone 17 cells. Deletion of the paxillin and spread quickly in both the wild type and clone 17

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 103

Figure 8 FAK phosphotyrosine levels are reduced in paxillin null ES cells. (a) Wild type clone 43, clone 17 and clone 17 stable transfected with paxillin-expressing vectors were lysed in NP40 lysis bu€er and equal amounts of protein immune-precipitated with antibodies to FAK. Immune complexes were solublized with SDS sample bu€er, separated by SDS ± PAGE and duplicate gels blotted to PVDF. The upper blot was probed with the anti-phosphotyrosine antibody 4G10; the lower blot was probed with anti- FAK mAb. (b) Wild type ES cells and clone 17 cells stably expressing wild type and mutant paxillin proteins were analysed for FAK phosphorylation as in (a)

Figure 9 Summary of paxillin mutants and FAK phosphorylation. The major features and protein binding sites of the paxillin molecule are shown at the top. Below, the ability of the paxillin mutants to restore FAK tyrosine phosphorylation in ES cells is indicated

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 104 ES cells. The antibody that recognizes Hic-5 stains members on di€erent sites on paxillin may be able to focal adhesion like structures in these cells by substitute for the loss of the paxillin/p95PKL complex. immuno¯uorescence, however the identity of the FAK is comprised of three primary domains, an protein(s) observed by immune ¯uorescence is un- amino terminal regulatory, a central catalytic, and a certain as there are no detectable 55 kDa bands on carboxyl terminus Focal Adhesion Targeting (FAT) Western blots of ES cell lysates with the paxillin or domain. The FAT domain is the region necessary for Hic5 antibodies, even after very long exposure times targeting to focal adhesions and binding to talin and (Figure 3 and data not shown). paxillin (Hildebrand et al., 1993, 1995). FAK targeted These results indicate that there may be di€erent to the membrane using either a FAK/vinculin chimera mechanisms that can promote cell spreading in ES or a myristoylation tag is tyrosine phosphorylated cells. The large cells can spread rapidly in a paxillin (Igishi et al., 1999; Shen and Schaller, 1999). FAK can independent manner, possibly due to the expression of be activated in response to growth factors as well as Hic-5 or another compensatory molecule. The small ES integrin signaling, resulting in the phosphorylation of cells require paxillin for ecient cell spreading; site 397 as well as the phosphorylation of however given sucient time the paxillin null cells 576/577 and 925 by the SRC proteins (Guan, 1997; spread on the ®bronectin coated surface. The delay in Schlaepfer et al., 1999). Activation of FAK results in spreading of paxillin null cells indicates paxillin may an increase in cell spreading, reduced motility and act as a catalyst in transmitting the signal for a cell to protection from (Schlaepfer et al., 1999; Shen reorganize the actin cytoskeleton and spread onto a and Schaller, 1999). The phosphorylation of FAK may surface. Another possibility is that the attached but as not coincide with the kinase activity of FAK and yet unspread paxillin null ES cells may adapt to permit discerning between the e€ects of kinase activity of paxillin independent cell spreading over the subsequent FAK and FAK-associated SRC in the cell can be 48 h. dicult (Schlaepfer et al., 1999). This is observed in Re-expression of paxillin by transient or stable serum-starved ®broblasts where tyrosine phosphory- transfection restores the spreading defect in paxillin lated FAK has a low in vitro kinase activity. Once the null cells. The wild type and 46 kDa paxillin mutant starved cells are placed in serum containing media and were both able to facilitate spreading to a similar plated onto ®bronectin there is an increase in FAK extent when re-expressed in paxillin null cells. The kinase activity as well as an association of FAK with 46 kDa mutant is deleted of LD1, a proline-rich tract, SRC (Schlaepfer et al., 1999). One model of FAK and tyrosines 31, 33, 40 and 118. This mutant cannot activation suggests that FAK complexes with the associate with CRK proteins through the known CRK integrin intracellular domain, but FAK may be folded binding sites at tyrosines 31 and 118 (Birge et al., in an inactive conformation (Guan, 1997; Schlaepfer et 1993). This would imply that interaction with SH2 al., 1999). After integrin activation, a conformational proteins at these tyrosine residues is not necessary for change in FAK would enable kinase activation, paxillin facilitated cell spreading in ES cells. We autophosphorylation, and association with SRC. The observed equal levels of CRK protein and tyrosine role of paxillin in such a model of FAK activation is phosphorylated CRK protein in the paxillin null versus not clear. wild type ES cells, indicating that overall basal tyrosine We show that FAK tyrosine phosphorylation occurs phosphorylation of CRK is not paxillin dependent in without the individual motifs LD1, 3, 4 and 5. The ES cells (data not shown). The LIMS only domain was anity of FAK for the individual LD motifs 2 and 4 is expressed at very low levels by Western blot (data not approximately equal with a cooperative e€ect when shown). The paxillin 1 ± 331 mutant was unable to both LD motifs are present (Thomas et al., 1999). Our restore cell spreading in ES cells. This mutant is data indicate that, if the interaction of FAK with missing the LIM domains and may be unable to paxillin is required for FAK activation, then the properly localize to focal adhesions. Future studies association of FAK with paxillin LD2 alone may with additional paxillin mutants will be needed to support tyrosine phosphorylation of FAK. Paxillin elucidate the regions of paxillin that facilitate cell molecules deleted in LD2 or containing point muta- spreading. Since clone 17 cells are reduced for both tions in LD2 were unstable in our experiments in ES early cell spreading and FAK tyrosine phosphorylation cells. Further mutational analysis of LD2 will be it is possible that tyrosine phosphorylation of FAK needed to generate a paxillin mutant that does not bind mediates paxillin induced cell spreading in ES cells; FAK at either LD2 or LD4 and can be expressed however this is not proven in our studies. Interestingly, stably in these cells. Such a paxillin molecule will be deletion of paxillin LD4 did not abrogate paxillin necessary to determine if FAK binding to paxillin LD dependent cell spreading. In addition to binding FAK, motifs is necessary for FAK tyrosine phosphorylation LD4 is the binding site for the ARF ± GAP protein and cell spreading in ES cells. p95PKL that can complex paxillin with the GEF FAK activation may involve FAK binding to protein PIX and thereby the serine threonine kinase paxillin, thereby inducing a conformational change in PAK1 (Turner et al., 1999). Other ARF ± GAP FAK that facilitates FAK tyrosine phosphorylation. proteins bind the paxillin amino terminus; however, The association of FAK with paxillin may be required the exact binding sites have not been described (Kondo to correctly localize FAK in focal adhesions. Another et al., 2000). Association of other ARF family possibility would be that paxillin brings substrates to

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 105 activated FAK, and that tyrosine phosphorylation of ES cell culture FAK may not be sucient to support cell spreading. If paxillin brings substrates to FAK, it is conceivable that The R1 line of ES cells (Nagy et al., 1993) was grown in mutants of paxillin could be made that might restore Iscove media supplemented with 20% FBS (Gibco BRL), FAK phosphorylation but would not support spread- 1mM Amino Acids (Life Technologies), 10 mM b Mercap- toethanol (Sigma) and Penicillin/Streptomycin (Life Technol- ing. The phosphorylation status of individual tyrosines ogies) essentially as described (Matise et al., 2000). Fetal calf of FAK in clone 17 and clone 43 cells is under serum lots were screened to exclude serum that induced investigation and may help distinguish among these di€erentiation markers. A Leukemia Inhibitory Factor (LIF) possibilities. Further mutational analysis of FAK and GST fusion plasmid was expressed in E. Coli. and puri®ed paxillin will be necessary to elucidate mechanism used using GSH beads and the LIF cleaved from the GST using by paxillin in cell spreading. thrombin. The LIF preparation was sterile ®ltered and tested for the ability to suppress ES cell di€erentiation before use. ES cells were grown on tissue culture plates coated with 0.1% gelatin (Sigma). ES cells were di€erentiated in vitro to form embryoid bodies as described (Wiles, 1993). Materials and methods

Paxillin targeting construct Electroporation and selection of colonies in ES cells Mouse paxillin genomic DNA regions that were used for Linearized targeting vector DNA was prepared as described homologous recombination were isolated using long range and electroporated into ES cells (Matise et al., 2000). PCR and the fragments were cloned into the pPNT targeting Twenty-four hours after plating, G418 (Life Technologies) vector (Matise et al., 2000; Tybulewicz et al., 1991). Portions at 200 mg/ml and Gancyclovir at 0.2 mM were added to the of the mouse paxillin gene were isolated by PCR and cloned media. Ten days after selection a sterile cotton applicator 5' and 3' to the neomycin resistance gene to generate a wetted with trypsin was used to transfer drug resistant ES paxillin gene targeting plasmid. Sequence from a mouse colonies from the plate into wells of a 24 well plate. Each paxillin partial cDNA in the EST database was used to plate was duplicated and one plate used to prepare genomic generate oligonucleotides C and D (Figure 1). This DNA. To select for a duplication of the neomycin gene and oligonucleotide and a poly dT primer were used in a reverse deletion of the remaining wild type paxillin allele, haploid transcription PCR reaction to isolate a partial mouse paxillin cells were placed in a range of G418 concentrations from cDNA. Oligonucleotides E, I, and G (Figure 1) were 0.8 mg/ml to 2 mg/ml as described (Mortensen et al., 1992). synthesized based on the sequence from the 3' end of the Colonies selected from 1 mg/ml wells were isolated as above paxillin cDNA. Oligonucleotide pair D/E was used in a PCR and screened by PCR to determine that both alleles of reaction with mouse ES cell DNA to amplify a 2.2 kb band paxillin had been disrupted and screened by Western blot for containing exons 10 through the 3'UTR from 129Sv genomic loss of paxillin protein expression. DNA. This band was cloned 3' to the neomycin resistance gene in the targeting plasmid pPNT (Tybulewicz et al., 1991). Screening for paxillin haploid ES colonies Oligonucleotide B was based on the 5' end of the mouse paxillin EST sequence. In order to amplify a region of Drug resistant ES clones were screened in two separate PCR genomic DNA within the 5' portion of the paxillin gene, reactions. Oligonucleotides F and H were generated using oligonucleotide A was designed based on a region of human sequence from the neomycin cassette in the pPNT targeting and chicken sequence that was well conserved at the nucleic vector. A PCR reaction using oligonucleotide pair F/G yields acid level. Oligonucleotide pair A/B was used to amplify a a 2.4 kb product only in a colony where the homologous 2 kb segment of the paxillin gene from mouse ES cell recombination event between the targeting vector and the genomic DNA encoding exon 2 to exon 6. This band was paxillin gene inserts the neomycin resistance cassette in cloned into the targeting plasmid containing the paxillin 3' proximity to oligonucleotide G. No product will be made genomic DNA segment D/E (Figure 1). Homologous in a non-homologously recombined colony. A PCR reaction recombination between the targeting plasmid and genomic with oligonucleotide pair C/G yields a 2.8 kb fragment from DNA is predicted to delete exons corresponding to amino all colonies. PCR reactions were performed in a 96 well plate acids 225 ± 404 of the paxillin a protein. using Expand Long Template PCR kits (Boehringer Oligonucleotides B, C and D were generated against mouse Mannheim) in a Hybaid PCR express machine. For all genomic DNA based on sequence available from mouse EST PCR reactions with oligos F, G, H and I, a positive control sequence (Genbank AA644797). Oligonucleotide D (Figure 1) plasmid (+ lanes in Figure 2a,c) was made by cloning the and a poly dT primer were used in a reverse transcription mouse paxillin cDNA fragment generated by the oligo D/dT reaction (Superscript, Gibco BRL) to generate a mouse cDNA PCR reaction into the pPNT targeting vector. This plasmid of the 3'UTR of paxillin; oligonucleotides E, G and I were was electroporated into ES cells and a cell line containing the generated from that sequence. The 2.4 kb 3' homology region + plasmid was selected. Genomic DNA preparations were in Figure 1 was generated using oligonucleotide pair D/E in a prepared as above and samples of this DNA was used as a PCR reaction (Expand Long Template PCR system, Boehrin- positive control (indicated by + marking above the lane). ger Mannheim) with mouse 129Sv genomic DNA as template. The PCR product from oligos F/G from this + positive To generate the 5' homology region, oligonucleotide A control DNA will be around 1.4 kb since it is generated from (AGAGGTACCgccctgctggcggacttggagtctaccacc) was created a spliced cDNA. This control cell line was created as a using a combination of chicken and human sequence. The positive control for the PCR reaction itself, and to insure that oligonucleotide pair A/B were used in a PCR reaction with PCR products observed from ES colonies with putative mouse 129Sv genomic DNA to generate the 2.2 kb 5' homology knockout recombination events were truly a result of a region. This PCR fragment was cloned into the pPNT vector homologous recombination knockout and not due to that had the 3' homology region cloned in previously. contamination of the ES colony DNA preparation with

Oncogene Paxillin in cell spreading and FAK phosphorylation R Wade et al 106 either the positive control DNA sample ('+' DNA sample) To assess attachment and spreading of transiently or paxillin cDNA plasmids present in the laboratory. transfected cells, ES cells were transiently cotransfected with the indicated paxillin expressing vectors and the pCX ± EGFP plasmid. Twenty-four hours after transfection cells were Paxillin vectors and transfection trypsinized and replated on puri®ed matrix coated cover The vector pCX-EGFP with EGFP replaced by a multiple slips for 8 h. Cells were ®xed, nuclei were DAPI stained, and cloning site was used for paxillin expression in ES cells GFP positive cells were scored for spreading by ¯uorescence (Okabe et al., 1997). The wild type paxillin and 1 ± 331 microscopy. mutant (expresses paxillin amino acids 1 ± 331) were gifts of Chris Turner (Brown et al., 1996). Oligonucleotide directed Antibodies and Western analysis mutagenesis was used to generate all point mutants and LD deletion mutants. LD1-, LD3-, LD4- and LD5- are deleted of Cells were lysed on ice in NP40 lysis bu€er (150 mM NaCl; amino acids 4 ± 11, 218 ± 225, 267 ± 274 and 302 ± 310 50 mM Tris pH 7.5; 50 mM NaF; 5 mM NaPPi; 1% IPEGAL; respectively. DNA was introduced in the ES cells using the 0.01% phenylmethylsulfonyl ¯uoride; 1 mM Sodium Vana- Lipofectamine Plus reagent (Life Technologies). For stable date; 1 mg/ml Leupeptin/Aprotinin). Lysates were equalized expression, paxillin plasmids were cotransfected with the for protein content using the Bio-Rad Protein Assay solution PGK-Puro plasmid (Tucker et al., 1996) and cells were (Bio-Rad). Cells used for phosphotyrosine assays were plated selected in 2 mg/ml puromycin (Life Technologies). Stable cell onto gelatin coated plates 24 h before lysis, no morphological lines were screened for paxillin expression by Western blot. di€erences were observed under these conditions. Immuno- Attachment and spreading assays were performed essen- precipitations were prepared using 0.5 mg of protein and 1 mg tially as described (Humphries, 1998). Twenty-four well tissue of puri®ed antibody. The complex that formed on ice for 1 h culture plates were coated with puri®ed matrix proteins for was precipitated using Rabbit anti mouse IgG and protein A 5 min and allowed to dry overnight before being blocked sepharose beads (Repligen), and rocked at 48C for 1 h. Beads with a 1 mg/ml treated killed BSA solution. Trypsinized, were washed three times with 1.5 ml lysis bu€er and washed and resuspended ES cells were counted and 5x105 complexes were eluted in SDS loading bu€er (2% SDS, cells were plated in each of six wells. After incubation, the 60 mMTris pH 6.8, 100 mM dithiothreitol, 2.5% glycerol, media from three wells for each cell line was aspirated, and 0.01% bromophenol blue). the wells were ®xed in 10% formaldehyde for 10 min. The Antibodies to paxillin, integrin b1, pp120, Pyk2, Hic5 were remaining three un®xed wells were washed three times to all from Transduction Laboratories. The antibodies to remove unattached cells with phosphate bu€ered saline and vinculin and phosphotyrosine (clone 4G10) were from Sigma then ®xed for 10 min. After ®xation, all wells were rinsed and Upstate Biotechnology respectively. Integrin alpha V with PBS and stained in a 0.1% crystal violet in MES (2(N- antibody was a rabbit polyclonal from Chemicon. Antibodies morpholino) ethanesulfonic acid) bu€er for 16 h at 48C. The for FAK Western blots and immunoprecipitations were from stained wells were washed three times with water, allowed to Transduction Labs and Upstate Biotechnology respectively. dry, and cells were observed under the microscope and Primary antibodies on Western blots were detected utilizing counted as spread if the total cell body width was at least horseradish peroxidase conjugated secondary antibodies and twice that of the nucleus. This same standard we used for all Supersignal substrate solution (Pierce). spreading assays. After enumeration of spread cells, the crystal violet was dissolved in 10% acetic acid and the absorbance read on a Beckman DU 64 Spectrophotometer at 570 nm to determine the relative number of attached cells Acknowledgments (Humphries, 1998). The absorbance of the three washed wells The authors wish to thank Dr Chris Turner for providing was averaged and divided by the average value for the the paxillin wild type cDNA and the 1 ± 331 mutant of unwashed wells to give the per cent retained after washing. paxillin, and Drs Ron Conlon and David LePage for the Each spreading assay was repeated three times. generous gifts of the ES cells, targeting vectors, and helpful The growth curves for clone 43 and clone 17 cells were advise. The authors also thank Drs Lloyd Culp and Susann performed on gelatin coated plates. 1x105 cells were plated Brady-Kalnay for critical reading of this manuscript. This onto ®ve identical 6 cm tissue culture plates. Plates were ®xed work was supported by NIH grant CA69292 to S Vande and stained with crystal violet as above. Pol.

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Oncogene