Threonine 788 in integrin subunit β1 regulates integrin activation

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Threonine 788 in integrin subunit β1 regulates integrin activation Stina Nilsson a,⁎, Dorota Kaniowska a,1 , Cord Brakebusch b , Reinhard Fässler b , Staffan Johansson a a

Department of Medical Biochemistry and Microbiology, Uppsala University, 751 23 Uppsala, Sweden Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany

b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

In the present study, the functional role of suggested phosphorylation of the conserved

Received 26 August 2005

threonines in the cytoplasmic domain of integrin subunit β1 was investigated. Mutants

Revised version received

mimicking phosphorylated and unphosphorylated forms of β1 were expressed in β1

16 November 2005

deficient GD25 cells. T788 in β1 was identified as a site with major influence on integrin

Accepted 1 December 2005

function. The mutation to A788 strongly reduced β1-dependent cell attachment and

Available online 6 January 2006

exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate integrin

Keywords:

activation, but the talin head domain bound equally well to the wild-type β1 and the

Adhesion

mutants indicating that the T788A mutation caused defect integrin activation by another

Integrin

mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting

Cytoplasmic domain

cell adhesion. GD25 cells expressing β1T788D accumulated increased number of focal

Activation

contacts and migrated slowly compared to GD25 β1 wild-type. An analogous phenotype is

Threonine

seen when focal adhesion kinase activation is abrogated. However, neither the β1T788D nor

Phosphorylation

the β1T788A mutation failed to induce tyrosine phosphorylation of focal adhesion kinase. The results suggest that phosphorylation of T788 in integrin β1 promotes inside-out receptor activation, as well as focal contact accumulation. © 2005 Elsevier Inc. All rights reserved.

Introduction Integrins are dynamic cell adhesion receptors required for various vital adhesive processes in the body. While integrin functions are regulated at several levels, a prerequisite for initial ligand binding and all subsequent intracellular events is the activation of integrins by cytoplasmic signals that confer a large conformational change to the extracellular domain (“inside-out signaling”). A framework for the understanding of integrin inside-out activation is provided by high resolution

data on integrin structures [1–4] and the finding that talin induces active integrin conformations by binding to the cytoplasmic domain [5]. However, the molecular mechanisms regulating the talin-integrin interaction remain unknown. Circulating blood cells express integrins that display a low affinity conformation, which is shifted to the high affinity state upon cellular response to blood vessel injury or infection. This activation can be reversed [6], which for example allows lymphocytes to repeatedly switch between adherent and nonadherent states. In adherent tissue cells, e.g. fibroblasts, most

⁎ Corresponding author. Fax: +46 18 4714244. E-mail address: [email protected] (S. Nilsson). 1 Present address: Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, PA 19122, USA. 0014-4827/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.12.001

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integrins are in the high affinity state, but integrins exposing a low affinity conformation are often found as a minor pool. Dynamic inactivation/activation of integrin conformations in adherent cells has been suggested as a mechanism to facilitate temporary detachment during mitosis and migration. The cytoplasmic domains of integrin β-subunits contain several potential phosphorylation sites, which could provide mechanisms to reversibly modulate integrin conformations, e.g. by regulating access of the β-subunit for talin-binding [7,8]. In the case of the widely expressed integrin subunit β1A, phosphorylation has been demonstrated to occur, but only under special circumstances and at low level [7,9,10]. In cells transformed by v-src, tyrosine 783 and 795 in β1 become phosphorylated with concomitant dissociation of β1-integrins from focal contacts [9,11]. It is unclear whether these residues are targets for phosphorylation in non-transformed cells. However, double mutation of the two tyrosine residues to phenylalanine leads to impaired activation of focal adhesion kinase (FAK) [12] and slow cell spreading and migration [11,13]. Phosphorylation of S785 has been demonstrated and suggested to cause exit of β1-integrins from focal contacts and to serve as mechanism of negative regulation of the association with the cytoskeleton [10]. Mutations mimicking phosphorylation of β1-integrin S785 inhibited cell spreading and directed cell migration, but promoted cell adhesion [14]. The conserved threonine cluster in the cytoplasmic domain of the integrin βsubunits has been implicated to be more directly involved in the inside-out activation of β1, β2, and β3 integrins. The β2 subunit is phosphorylated on one or more of the threonine residues concurrent with activation of integrin ligand-binding ability after stimulation of leukocytes via CD3 or by phorbol ester [15–18]. Similarly, phosphorylation and dephosphorylation of serine and threonine residues in the β3 cytoplasmic domain correlate with exposure and closure of the ligandbinding site of the platelet αIIbβ3 integrin [19]. Replacement of the two threonine residues 788–789 in β1 by alanines was shown to cause a conformation of the extracellular domain that is inactive for ligand binding when expressed in GD25 fibroblasts [20]. Also for the β2 subunit, the corresponding mutation of threonine residues (758–760) abolishes activation of the integrin [21]. These findings suggest that reversible phosphorylation of the threonine cluster may be involved in the regulation of integrin conformations. While phosphorylation of one or more residues in the cytoplasmic threonine cluster has been directly demonstrated for β2, β3, and β7 integrin subunits [16,22,23], it has not been reported for β1. However, evidence that the corresponding modification occurs also in β1 was recently presented by use of site-specific anti-phosphothreonine antibodies [24–26], but the functional role in β1-mediated adhesion is not clear. For these integrin subunits, the described threonine phosphorylations appear to be transient or for other reasons difficult to observe, and potent inhibitors of phosphatase PP2A are used for its detection [15–17,19,22,25]. Since PP2A has broad functions in cells, PP2A inhibitors obviously have a limited value for elucidation of the functional role(s) for integrin threonine phosphorylation. In order to allow specific studies of this issue, we used the alternative approach to express β1 mutants that mimic unphosphorylated or phosphorylated forms of the protein in GD25 cells.

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Materials and methods Antibodies and reagents The following antibodies were purchased from BD Biosciences (USA): hamster anti-β1 (Ha2/5), rat anti-β1 (9EG7), mouse anti-focal adhesion kinase (FAK, clone 77), mouse anti-paxillin (clone 349), and HRP-conjugated anti-phosphotyrosine (RC20H). From Jackson ImmunoResearch Laboratories, Inc. (USA) were rabbit anti-mouse IgG, FITC-conjugated anti-hamster IgG, FITC-conjugated anti-rat IgG, and Cy™3conjugated anti-mouse. HRP-conjugated anti-mouse IgG (NA931) was from Amersham Biosciences (Sweden). GRGDS-peptide was obtained from Bachem (KeLab; Sweden). Soybean trypsin inhibitor was purchased from SIGMA (Germany), Protein A–Sepharose CL-4B from Amersham Biosciences (Sweden), and Pefabloc SC (AEBSF) from Roche (Germany). Fibronectin (FN) and vitronectin were purified from human plasma as described previously [27,28]. A cDNA construct coding for GST fused with an 80 kDa integrinbinding fragment of invasin (GST-invΔ1) was kindly provided by Dr. M. Fällman (Umeå University, Sweden). The GST-invΔ1 fusion protein was purified from E. coli cultures as described [29]. Invasin binds selectively to a subset of β1-integrins and efficiently promotes cell spreading and assembly of focal adhesions [29,30]. Constructs for GST-talin head domain (residues 1–433) and two polyhis-tagged talin FERM-domain fragments (residues 86–410 and 196–400, respectively), kindly provided by Dr. D. Critchley (University of Leicester, UK), were expressed in E. coli and purified according to standard protocols.

Cell culture and cDNA transfections The cell lines GD25, GD25-β1, GD25-β1T788A, GD25-β1T789A, GD25-β1T788/789A, and GD25-β1B have been described previously [20,29,31–33]. The GD25 cells lack the β1 family of integrins due to a disruption of the integrin β1 gene. The GD25-β1T788D cell line was generated as previously described for the GD25-β1T788/789A cell line [32] with the exception that GD25-β1T788D cells were subjected to sorting by flow cytometry to establish a cell population with β1 expression level similar to previously established cell lines used in this study, instead of arising from a single cell-based population. GD25 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin–streptomycin, and Fungizone, whereas transfected cells were cultured in the same medium containing puromycin (20 μg/ml) (selection medium).

Flow cytometry analysis and sorting Integrin subunit expression levels were analyzed by a FACScan (Becton Dickinson) instrument. Analyzed cells were harvested, washed, and subsequently incubated in β1-integrin mAbs Ha2/5 or 9EG7 diluted in TBS containing 5% BSA on ice for 30 min. After subsequent incubations with fluoresceinlabeled secondary antibodies and TBS containing propidium iodide (1 μg/ml), 1 × 104 cells/sample were analyzed. GD25-

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β1T788D cells were subjected to sorting using a FACSVantage (SE DiVa) instrument at the Cell Analysis Core Facility (Uppsala University, Sweden) after labeling with FITC-conjugated anti-β1(Ha2/5) antibody. GD25-β1T788D cells with β1 expression levels similar to previously established cell lines used in this study were collected.

Cell attachment assay Wells of 96-well microtiter plate (Nunclon™ #167008; Denmark) were coated overnight at 4°C with purified human FN (0.5–25 μg/ml), vitronectin (10 μg/ml, as reference extracellular matrix protein), and 1% heat-treated BSA (as negative control) as described [32]. Cells were harvested, suspended in serumfree DMEM medium, and preincubated with GRGDS-peptide (0.2 mg/ml) for 15 min at room temperature (RT) to block the contribution of αVβ3 integrins binding to FN [32]. In each well, 1 × 105 cells were seeded and allowed to attach for indicated times at 37°C. Unattached cells were removed, and the remaining cells were fixed, stained with 0.1% crystal violet, and quantified as described [32]. All samples were assayed in triplicates and background attachment to BSA-coated wells was subtracted from all measurements.

Talin binding assay Subconfluent cultures of cells were lysed in ice-cold PBS buffer containing 1% Triton X-100, 1 μg/ml Pefabloc, 1 μg/ml Pepstatin A. Cell lysates, of equal total protein concentration, were precleared with glutathione–Sepharose and subsequently incubated with 10 μg GST-talin head domain or only GST protein for 2 h at 4°C. Glutathione–Sepharose was added, and after a 30 min incubation, the Sepharose beads were washed with the lysis buffer. The precipitated proteins were subjected to SDS-PAGE and analyzed by Western blotting.

Transfilter migration Cells were detached with trypsin-EDTA and washed twice with the serum-free DMEM. The polycarbonate membrane (8 μm pore size; Neuro Probe, USA) was coated on both sides with FN (50 μg/ml) or GST-invasin (2 μg/ml). The membrane was blocked for 1 h in 1% heat-treated BSA and rinsed in PBS prior to mounting into the Boyden chamber. DMEM/10% FCS was added to the lower wells of the migration chamber. 1 × 105 cells/well were seeded to the upper well of the Boyden chamber in serum-free DMEM. GRGDS-peptide (25 μg/ml) was included in the upper well to block the integrin αVβ3 contribution when cells were seeded on FN. The assay was carried out at 37°C for 15 h. The membrane was then rinsed in PBS, cells remaining on the upper surface of the membrane were removed by scraping and the membrane was fixed in 96% ethanol for 10 min followed by staining with 1% crystal violet. The excess color was washed away, and the intensity of cell staining was scanned and quantified using the Molecular Analyst 2.1 software (BioRad Laboratories, Inc.; USA). All samples were assayed in triplicates and background staining from BSA-coated wells was subtracted from all measurements.

Wound assay 24-well plates (Nunclon™ #143982; Denmark) were coated with FN (50 μg/ml) or GST-invasin (2 μg/ml). Cells were harvested, suspended in normal growth medium, and 5 × 105 cells were seeded in each well. The cells were incubated at 37°C overnight. Wounds were scratched in the confluent cell layer using a 100 ml-pipette tip. Wounds were recoated with the FN or GST-invasin in serum-free medium for 30 min at 37°C and further blocked for 30 min at 37°C with 0.5% heat-inactivated BSA in serum-free medium. Cells were then cultured in normal growth medium and migration was monitored over time.

Immunocytochemistry 8-well chamber slides (BD Falcon CultureSlide #354108; USA) were coated with FN (50 μg/ml) or GST-invasin (2 μg/ml). The cells were harvested, washed once with serum-free DMEM, and plated onto FN in the presence of 0.1 mg/ml GRGDSpeptide, or onto GST-invasin. The cells were incubated at 37°C for 2 h, fixed with 2% paraformaldehyde at RT for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 20 min at RT, and blocked with 10% goat serum in PBS for 1 h at 37°C. The samples were subsequently incubated with anti-paxillin and anti-mouse-Cy™3-conjugated antibodies to visualize focal contacts. The samples were mounted in Vectashield® Mounting medium (Vector Laboratories Inc.; USA) and examined for Cy™3 staining by use of a Zeiss 510 confocal microscope at the Cell Analysis Core Facility (Uppsala University, Sweden).

FAK activation (immunoprecipitation and Western blotting) 6-well plates (Nunclon™ #150229; Denmark) were coated with 10 μg anti-β1 (Ha2/5) or 1% heat-inactivated BSA. Serum starved cells (24 h) were detached with trypsin-EDTA, trypsin was inactivated with soybean trypsin inhibitor (1 mg/ml). Cells (5 × 106 per well) were seeded in serum-free media and allowed to attach for 1 h at 37°C. The cells were lysed in ice-cold RIPA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 50 mM NaF, 30 mM Na4P2O7, 2 mM EDTA, 1% NP-40, 0.1% SDS, 1% (w/v) Na-DOC, and inhibitors (2 mM PMSF, 2 mM NEM, 1 μg/ml Pepstatin A, 1 μg/ml Leupeptin, and 0.2 mM Na3VO4)) and the lysates were aspirated through 27G-injection needle to sheer the DNA. After preclearing the lysates with Protein A–Sepharose CL-4B, FAK was immunoprecipitated by incubation with 3 μg of monoclonal antibody overnight and subsequent incubation with 1.5 μg of rabbit-anti-mouse IgG for 30 min. The immune complexes were recovered by 30 min incubation with Protein A–Sepharose CL-4B and centrifugation. The Sepharose–immune complex was washed once in triton washing buffer (10 mM Tris pH7.5, 0.5 M NaCl, 50 mM NaF, 30 mM Na4P2O7, 1% Triton X-100, and inhibitors) and boiled in SDS-sample buffer containing 0.1 M DTT. The immunoprecipitated proteins were separated by SDS-PAGE followed by transfer to Protran® nitrocellulose transfer membrane (Schleicher and Schuell; Germany). The membranes were blocked with 5% BSA in TBS-Tween (150 mM NaCl, 10 mM Tris pH 7.4, and 0.1% Tween 20) and incubated with anti-phosphotyrosine HRP-conjugated antibodies. Reactive bands were detected with enhanced chemiluminescence (Amersham Pharmacia Biotech; Sweden). As loading control,

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the membranes were stripped and reprobed with antibodies towards total FAK-protein. The intensities of pTyr-signals were scanned and integrated using ImageJ 1.34s (NIH software) and correlated to total lysate protein concentration of each sample (instead of FAK protein due to partial antigen blocking by incomplete removal of phosphotyrosine antibody).

Results Mutations of threonine 788 in β1 cytoplasmic domain affect cell attachment

Fig. 1 – GD25-β1T788A showed impaired cell attachment to fibronectin. The cells were incubated in fibronectin-coated wells in the presence of 0.2 mg/ml GRGDS-peptide. Attachment is expressed as % of cells bound to wells coated with vitronectin in the absence of GRGDS-peptide. Cells, GD25 ( ), GD25-β1 (×), GD25-β1T788A (E), GD25-β1T788D (■), GD25-β1T789A ( ), were seeded in microtiter-plate and left to adhere (A) for 1 h on 0–25 μg/ml of fibronectin, or (B) for 15–180 min on 10 μg/ml of fibronectin.

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The double mutation of threonines 788 and 789 to alanines in the cytoplasmic tail of integrin subunit β1 was previously found to impair the ligand-binding ability of the receptor [20]. In order to investigate in more detail the role of these threonines in the regulation of integrin activation, single amino acid mutations of the β1 subunit were generated and stably expressed in GD25 cells. Cell attachment assays revealed that integrin α5β1T789A mediated attachment equally well to FN as the wild-type integrin, whereas integrin α5β1T788A mediated poor attachment (Fig. 1A). The defect attachment of GD25-β1T788A cells was not overcome by increasing the incubation time (Fig. 1B), while GD25-β1T789A cells reached maximum binding level within the same time as wild-type β1 expressing cells (not shown). The dramatic effect caused by the minor structural change of replacing a threonine with alanine suggests that T788 may represent one regulatory phosphorylation site in the β1 subunit. Therefore, T788 was mutated to D788 in an attempt to mimic the phosphorylated residue, the β1 mutant was expressed in GD25 cells, and its ability to mediate cell attachment was analyzed. GD25-β1T788D attached efficiently

Fig. 2 – Cell surface expression levels of 9EG7-epitope in β1 mutant cells. (A) Integrin β1 cytoplasmic domain amino acid sequences. A vertical line marks the membrane–cytoplasm interface for integrins in the active conformation, and an arrowhead marks the site where the amino acid sequence of splice variant β1B begins to differ from β1A. (B and C) Cell surface expression levels of total (Ha2/5, in grey) and active (9EG7, in white) integrin β1 epitopes, presented as normalized data (B) with respect to total integrin expression level for cell lines expressing wild-type β1A, Thr-mutants, and the β1B splice-variant and in arbitrary units (C) for cells expressing wild-type β1A, β1T788A, and β1T788D, respectively. Levels of bound primary antibodies were determined by flow cytometry after incubation with FITC-conjugated secondary antibodies.

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Fig. 3 – Threonine mutant β1 integrins bind the talin head domain. Cell lysates of equal total protein concentration prepared from GD25 cell expressing wild-type β1, β1T788A, and β1T788D, respectively, were incubated in the presence of excess talin head domain fused with GST, or with GST only. After precipitation with glutathione–Sepharose, the recovered proteins were subjected to SDS-PAGE, transferred to nitrocellulose filters and blotted for β1 integrin.

to FN, indicating that phosphorylation of T788 is compatible with ligand-binding function of the integrin (Fig. 1A).

Threonine 788 influences the exposure of the extracellular 9EG7 epitope The double mutant β1T788/789A is known to expose a reduced level of the conformation-sensitive epitope of

mAb 9EG7 [20,34], which is located in the extracellular domain of the β1 subunit. In this study, we found that this effect was mainly linked to the T788A substitution (Figs. 2B, C). As shown by flow cytometry, the level of 9EG7 reactivity was slightly lower (91%) than that of the reference mAb (directed towards a non-regulated β1 epitope) in GD25 cells expressing wild-type β1, while the 9EG7 level in GD25β1T788A cells was reduced to a similar extent as in GD25β1T788/789A (25 and 29% of the reference values, respectively). Intermediate levels of the 9EG7 epitope were monitored for T788D (63%) and T789A (61%) integrins. The cytoplasmic splice variant β1B, which is completely unable to bind FN under physiologic conditions [33,35], exposed essentially no 9EG7 epitope (8%) (Fig. 2B). Thus, the level of 9EG7 epitopeexpression in the different β1 mutants in some cases correlated closely to their ability to mediate cell attachment, whereas two mutants moderately affected the epitope without disturbing the ligand-binding ability.

Substitution of threonine 788 does not interfere with talin head domain interaction Since the T788A mutation appeared to shift the equilibrium between inactive and active conformation of the integrin, and the interaction of talin with the cytoplasmic part of βsubunits has been identified as a critical step for the

Fig. 4 – Focal contact assembly and distribution are differently affected by two β1T788-substitutions. Cells were allowed to spread for 2 h at 37°C on fibronectin (FN) in the presence of 0.1 mg/ml GRGDS-peptide (A, B, C) and on GST-invasin (D, E, F). The cells were immunostained with anti-paxillin and secondary Cy™3-tagged antibody to visualize focal contacts. Original magnification was 63×; the bar corresponds to 10 μm.

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T788D mutants (Fig. 3). Identical results were obtained with polyhis-tagged FERM-domain fragments of the talin head (not shown).

Threonine 788 is involved in the regulation of focal contacts

Fig. 5 – Transfilter cell migration is moderately affected by threonine substitutions. Cells were stimulated to migrate in a transfilter chamber towards a serum gradient. Filters were precoated with GST-invasin (2 μg/ml) or fibronectin (not shown), cells were seeded in serum free medium and migration was allowed for 15 h. Membranes were stained and the intensity was quantified by scanning and Molecular Analyst 2.1 software.

activation, we investigated whether the ability bind the β1 was altered by the mutations. protein of the talin head domain was found equally well with the wild-type β1 as with the

of talin to GST-fusion to interact T788A and

The importance of T788 for the assembly of focal contacts was examined by immunofluorescent staining for paxillin (Fig. 4) in the mutant cell lines after spreading on FN (A, B, C) and invasin (D, E, F). As expected, relatively few GD25-β1T788A cells (B, E) adhered strongly enough to resist the sheer force of washing; notably, the cells that remained attached had been able to spread but entirely lacked focal contacts. In contrast, GD25-β1T788D cells (C, F) typically had increased numbers of focal contacts compared to GD25-β1 wild-type cells (A, D). While the focal contacts were confined to the cell edges in wild-type β1 expressing cells, the GD25-β1T788D cells also accumulated distinct focal contacts centrally under the cells.

Phosphorylation of threonine 788 is not required for β1-mediated cell migration To investigate the effect of the phospho-mimetic mutations on cell migration, the above cell lines were tested in a transfilter migration assay. GD25-β1T788D cells migrated significantly less efficiently than GD25-β1 wild-type cells through filters coated with invasin (Fig. 5) and FN (not

Fig. 6 – Wound healing migration is moderately affected by threonine substitutions. Confluent monolayers of cells were “wounded” by scratching across the plate with a pipette tip. The wounds were recoated with substrate (fibronectin (FN) 50 μg/ml or GST-invasin 2 μg/ml) and the extent of wound closure was monitored over time.

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shown). Surprisingly, β1T788A integrins were found to promote cell migration through the coated filters (Fig. 5) in spite of the poor adhesive properties of the mutant cell line. These unexpected observations were confirmed in an in vitro “wound” assay of cell migration (Fig. 6). Thus, while the weak adhesion observed for GD25-β1T788A cells is sufficient to support migration, the reduced migration speed of GD25β1T788D cells may be due to too firm adhesion. The results demonstrate that phosphorylation of T788 in integrin subunit β1 is not required for cell migration, and suggest that such phosphorylation could represent a mechanism to modulate migration.

Mutations of threonine 788 do not interfere with FAK activation The reduction in migration of cells expressing β1T788D is likely related to the elevated numbers of focal contacts. Reduced turnover of focal contacts can cause such effects, as shown for cells where FAK activity is impaired [12,36,37]. The ability of the mutant integrins to induce FAK phosphorylation was therefore analyzed. Anti-β1 antibodies were used as receptor substrate to measure post-ligand signals regardless of ligand binding properties of the receptor mutants. However, all the investigated β1-integrin mutants possessed the ability to induce tyrosine phosphorylation of FAK after stimuli and clustering by anti-β1 antibodies (Fig. 7), suggesting that the T788D mutation either negatively affected another step in the turnover of focal

contacts or enhanced their assembly. Increase in intensity of pTyr-signal in β1-mAb stimulated cells over signal from cells in suspension is summarized in Fig. 7B.

Discussion The cytoplasmic threonine cluster is present in six of the eight integrin β-subunits. In the cases of β2, β3, and β7, phosphorylation at this site has been shown to correlate with inside-out activation of the integrins [16,17,19]. In the case of β1, cytoplasmic threonine phosphorylation has been suggested as a mechanism to disrupt focal contacts and integrin–actin connections, as concluded from data obtained with antibodies specific for a β1 peptide with both threonines phosphorylated [24,25]. In the present study, data are presented which oppose the latter view and instead are consistent with a role of the cytoplasmic threonines in β1 that is similar to those in β2 and β3. Initially, T788 in β1 was identified as a site with major influence on integrin function. The mutation T788A markedly reduced β1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. A critical final step in the activation of β1, β2, and β3 integrins is carried out by the head domain of talin by binding to the cytoplasmic domain of the β-subunit and thereby disrupting the interaction between the cytoplasmic membrane proximal parts of α and β-subunits [38,39]. T788 is located close to, but

Fig. 7 – Threonine mutant β1 integrins induced tyrosine phosphorylation of FAK. FAK was immunoprecipitated from cell lysates prepared from suspended cells (S) and from cells adhering to anti-β1 mAb-coated plates (Ab). Tyrosine phosphorylation of FAK (pTyr) (A, upper panel) was detected by SDS-PAGE and Western blot. After stripping, the filter was reprobed with anti-FAK mAb to monitor loading of immunoprecipitated FAK in all lanes (A, lower panel). The β1-mAb-induced increase in pTyr-signal over cells in suspension is summarized in panel B (for details see Materials and methods).

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outside of, the identified interaction surfaces on β1 for the talin head domain [40], and our data show that the inactive conformation of the T788A mutant is not due to inability to bind the talin head domain. In spite of the weak binding of the GD25-β1T788A cells to FN (in the presence of soluble RGD-peptide to block αVβ3) and invasin, the cells were able to spread on these ligands over time and to migrate efficiently in two different assays. Interestingly, β1T788A integrin-induced focal contacts were not seen in these cells. It is possible that the reduced number of receptors in the active conformation in the mutant cells, due to the shifted equilibrium among integrin conformations, prevented formation of focal contacts. Alternatively, the mutation may have a direct effect on focal contact formation (“post-ligand effect” [21]) beside the inhibition of inside-out integrin activation. The double T788/789A mutation was previously reported to not interfere with focal adhesion localization [41]. However, those observations are not in conflict with our results since they can be attributed to ligand-independent association of the transfected mutant integrin to focal contacts formed on fibronectin by endogenously expressed αVβ3 and α5β1 in 3T3 cells. β1-integrins, carrying the phospho-mimetic T788D mutation, were fully active in mediating cell attachment, indicating that phosphorylation of T788 is compatible with the active receptor conformation. The finding that the interaction between the β1 in cell lysates and exogenously added talin head domain was undisturbed by the T788D mutation supports this conclusion. Somewhat surprisingly, the conformation-sensitive extracellular epitope 9EG7 was exposed in a significantly smaller fraction of the cell surface β1-integrins carrying T788D compared to the wild-type receptor. As was found for β1B, β1T789/789A, and β1T788A integrins, the exposure of 9EG7 epitope often correlates well with ligand-binding activity [34,42]; however, exceptions to this rule have been reported [43]. The 9EG7 epitope alone is therefore not a reliable marker for a specific integrin conformation, but in this study, it served as a valuable indicator for transmembrane conformational changes in response to modifications at amino acid 788. GD25 cells expressing β1T788D migrated slowly compared to GD25-β1 wild-type, probably as a result of the observed accumulation of focal contacts. A similar cellular phenotype is obtained by deletion of FAK [36] or by preventing FAK activation through mutation of the two tyrosines in β1 to phenylalanine [12]. However, none of the mutations of T788 or T789 lack the ability to induce tyrosine phosphorylation of FAK. The mechanism by which the phospho-mimicking mutation affected the formation or turnover of focal contacts is presently not known. Integrin cytoplasmic domain-associated protein-1 (ICAP-1) has been shown to negatively modulate β1-integrin function by binding to the six amino acid region AVT788T789VV in the C-terminal part of β1 [44,45,46]. However, ICAP-1 is not expressed at detectable levels in GD25 cells [45] and can therefore not contribute significantly to the regulation of integrins in these cells. Although the mechanism of β1 threonine phosphorylation and its regulation is still unclear, our results indicate that phosphorylation of T788 in β1 will promote inside-out receptor activation, as well as focal contact accumulation. Thus, they provide evidence against the suggested role of

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threonine phosphorylation in destabilizing focal contacts and integrin–actin connections [24,25]. Consistent with our conclusion, threonine phosphorylation in β2 was reported to correlate with increased cytoskeletal association [47]. Our data support a common model for β1, β2, and β3 activation in which binding of the talin head domain to the β-subunit is a central event [38] and threonine phosphorylation has an additional, yet unidentified regulatory role [23]. This phosphorylation may have to be transient in order to maintain appropriate numbers of focal contacts. The present results provide a plausible working model on the role of the threonine cluster in integrins, specifically for T788 in β1.

Acknowledgments We thank Dr. D. Critchley for kindly providing the talin head constructs. Dr. M. Thuveson and B. Wärmegård are gratefully acknowledged for excellent technical support. This study was supported by grants to S.J. from the Swedish Research Council (no. 7147), Swedish Cancer Foundation, and King Gustaf V:s 80-års Fond. R.F. is funded by the Max Planck Society and C.B. by the German Research council (DFG).

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