Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M311581200 on December 2, 2003

J. Biol. Chem., Vol. 279, Issue 8, 7082-7090, February 20, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/8/7082    most recent
M311581200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, S.-M.
Right arrow Articles by Song, W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, S.-M.
Right arrow Articles by Song, W. K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Modulation of Thr Phosphorylation of Integrin {beta}1 during Muscle Differentiation*

Seon-Myung Kim{ddagger}§, Min Seong Kwon{ddagger}§, Chun Shik Park, Kyeong-Rock Choi, Jang-Soo Chun, Joohong Ahn, and Woo Keun Song

From the Department of Life Science, Kwangju Institute of Science and Technology, Kwangju 500-712, Korea

Received for publication, October 22, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By using transient elevations of cytosolic free calcium levels triggered by integrin antibody or laminin (Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443), we have demonstrated that protein phosphatase 2A (PP2A) is implicated in the regulation of reversible phosphorylation of integrin. In E63 skeletal myoblasts, the treatment of PP2A inhibitors such as okadaic acid and endothall induces an increase of phosphorylation of integrin {beta}1A and thereby inhibits integrin-induced elevation of cytosolic calcium level and formation of focal adhesions. None of these effects were in differentiated myotubes expressing the alternate {beta}1D isoform. In the presence of okadaic acid, PP2A in association with integrin {beta}1A was reduced on myoblasts, whereas {beta}1D on myotubes remained bound with PP2A. Both co-immunoprecipitation and in vitro phosphatase assays revealed that dephosphorylation of residues Thr788-Thr789 in the integrin {beta}1A cytoplasmic domain is dependent upon PP2A activity. Mutational analysis of the cytoplasmic domain and confocal microscopy experiments indicated that substitution of Thr788-Thr789 with Asn788—Asn789 is of critical importance for regulating the function of integrin {beta}1. These results suggest that PP2A may be a primary regulator of threonine phosphorylation of integrin {beta}1A and subsequent activation of downstream signaling molecules. Taken together, we propose that dephosphorylation of residues Thr788-Thr789 in the cytoplasmic domain of integrin {beta}1A may contribute to the linkage of integrins to focal adhesion sites and induce the association with cytoskeleton proteins. The switch of integrin {beta}1A to {beta}1D isoform in myotubes therefore may be a mechanism to escape from phospho-regulation by PP2A and promotes a more stable association of the cytoskeleton with the extracellular matrix.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are heterodimeric transmembrane proteins consisting of {alpha} and {beta} subunits and mediate many of the interactions between adjacent cells or between cells and extracellular matrix (ECM).1 The specificity of ligand binding by integrins is conferred by the particular combination of the cytoplasmic domains of {alpha} and {beta} subunits (2). The sequences of the cytoplasmic domains of {alpha} and {beta} subunits are quite divergent from one another. Alternative forms of cytoplasmic domains that arise from alternate splicing have been found in the integrin {beta}1, {beta}3, {beta}4, {alpha}3, {alpha}6, and {alpha}7 cytoplasmic domains (3-7). Presumably, these diversities within the cytoplasmic domains modulate the capacity of integrins to mediate the transduction of ECM-mediated signals as well as signals arising within the cell that are directed outward via activation of integrins (2, 4, 8-9).

The association of integrins with the cell cytoskeleton and the formation of cell adhesions have been the most widely studied in the aspects of cytoplasmic domain functions. The cytoplasmic domains of the integrin {beta}1, in particular, have been demonstrated to play central roles in many aspects of integrin function such as cell adhesion, cell migration, control of cell differentiation, proliferation, and programmed cell death. In recent studies (10, 11), more cellular proteins have been reported to directly or indirectly interact with the cytoplasmic domain of integrin {beta}1. Cytoskeletal proteins such as {alpha}-actinin, F-actin, skelemin, talin, vinculin, filamin, and tensin are known to bind the integrin {beta}1 cytoplasmic domain (12-15). Also, signaling molecules such as focal adhesion kinase, integrin-linked kinase, phosphoinositide 3-kinase, Src, Cas, and novel proteins such as TAP 20 are targeted to adhesion sites where they come into contact with the integrin cytoplasmic domain (16-24).

The spatial relationships and interactions between either cytoskeletal or signaling molecules and the integrin cytoplasmic domain are highly dependent upon integrin phosphorylation and can alter the binding affinity of integrins for their respective ligands within the ECM. A variety of studies concerning phosphorylation of integrin cytoplasmic domains has been reported (25-32). Phosphorylated integrin {beta}1 in src-transformed fibroblasts does not localize to focal contacts (16). Increased phosphorylation of integrin {beta}3 on Thr residues by addition of calyculin A decreased platelet adhesion and spreading on fibrinogen (34). Mutational analysis of amino acid residues of the {beta}1 cytoplasmic domain has shown that integrin activity is functionally related to the potential phosphorylation sites of the {beta}1 cytoplasmic domain. One of the potential sites is composed of two NPXY motifs in the cytoplasmic domains of integrin {beta}1A, {beta}1D, {beta}2, {beta}3, {beta}5, {beta}6, and {beta}7 (35, 36). Upon phosphorylation of the NPXY motifs by tyrosine kinase(s), the integrin loses its affinity for both extracellular ligand and cytoplasmic components of the focal contacts and exits to focal contact. Mutagenesis of Asn or Pro residues of the NPXY motifs causes the loss of {beta} subunits from focal contacts. Tyr783 mutation of the NPXY motif of the {beta}1 cytoplasmic domain completely inhibits binding of PAC1 that recognizes integrins in the activated state (37).

In particular, S785D, Y783A, and Y795A resulted in an inability to localize to focal adhesion sites (38). Conversion of the unphosphorylated and phosphorylated state of integrins takes place during cell differentiation. Undifferentiated F9 cells are phosphorylated on the serine residue of the cytoplasmic domain of the {beta}1 chain and dephosphorylated during differentiation (37). During muscle cell differentiation, the integrin {beta}1 undergoes isoform switch from {beta}1A to {beta}1D (39, 40). Interestingly, the Thr788-Thr789 residues, potential phosphorylation sites in the integrin {beta}1A cytoplasmic domain, are substituted with Asn788-Asn789 in the {beta}1D splice variant.

Although a number of studies have suggested that phosphorylation of {beta}1 integrin cytoplasmic domains alters their functions, there are still numerous conflicting reports regarding the modulation of integrin function. Moreover, relatively little is known about the potential role of Thr phosphorylation (788-789) of the integrin {beta}1 cytoplasmic domain and the coordinated activity of protein kinases and phosphatases that regulate threonine phosphorylation in modulating the function of the integrin {beta}1 cytoplasmic domain.

Recently, it was shown that binding of integrin antibodies or laminin (LN) to integrin induces extracellular Ca2+ influx through L-type Ca2+ channels (1) and increases the cytosolic free calcium concentration ([Ca2+]i), which can be used as an indicator of integrin activation (1, 41). In this study, we showed that treatment with PP2A inhibitors such as okadaic acid and endothall inhibits the elevation of [Ca2+]i and the formation of focal adhesion complex in E63 myoblasts. Also, we demonstrated that PP2A in association with integrin {beta}1 mediates dephosphorylation of residues Thr788-Thr789 on integrin {beta}1A and thereby induces an increase of integrin association with cytoskeleton. In particular, the isoform switch of {beta}1A to {beta}D may be a mechanism to escape from phosphoregulation by PP2A during skeletal muscle differentiation. The dephosphorylated {beta}1D is likely to provide high tensile strength in association with cytoskeletal proteins for muscle function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—Rabbit anti-integrin {beta}1A and {beta}1D antibodies were generated as described previously (42). Anti-PP2A catalytic subunit (PP2Ac) and anti-PP2A B subunit antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-vinculin antibody, anti-myosin heavy chain (MHC) antibody, and ECL reagent were from Sigma. Anti-paxillin antibody was from Transduction Laboratories (Lexington, KY). Anti-phosphothreonine antibody was from Zymed Laboratories Inc.. Anti-phosphothreonine (Thr(P)788-Thr789)antibody was purchased from BIOSOURCE (Nivelles, Belgium). Anti-PP2A B' subunit antibody against the C terminus of PR56{alpha} was generously provided by Craig Kamibayashi (University of Texas Southwestern Medical Center, Dallas, TX). Anti-PP2A B'' subunit antibody against the C terminus of PR72/130 was obtained from Brian Hemmings (Friedrich Miescher Institute, Basel, Switzerland). Horseradish peroxidase-conjugated goat anti-mouse antibody, horseradish peroxidase-conjugated goat anti-rabbit antibody, and TRITC-conjugated donkey anti-mouse immunoglobulin antibody were from Jackson ImmunoResearch (West Grove, PA). Okadaic acid (OA) and endothall (ETL) were from Calbiochem. Dulbecco's modified Eagle's medium (DMEM) and antibiotic/antimycotic were from Invitrogen. horse serum (HS) was from Gemini Bioproducts (Calabasas, CA). Synthetic phosphopeptides {beta}1A (KWDpTGENPIYKpSAVpTpTVV), {beta}1A1 (KWDpTGEN), {beta}1A2 (IYKpSAVT), {beta}1A3 (SAVpTTVV), {beta}1A4 (AVTpTVVN), {beta}1D1 (KWDpTQEN), and {beta}1D2 (IYKSpPIN) were from Anygen (Kwangju, Korea). Ser/Thr phosphatase assay system was from Promega (Madison, WI). Fluo-3/AM was from Molecular Probes (Eugene, OR).

Mutation of Integrin {beta}1A—Mutant integrin {beta}1A constructs were generated from pHSX-{beta}1X encoding full-length human {beta}1A integrin subunit (56). The integrin {beta}1A was cloned into pDsRed1-N1 (Clontech, Palo Alto, CA) by the unique XhoI and BamHI restriction sites. Mutants were generated by oligonucleotide-primed DNA synthesis using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The following mutants were constructed by PCR: T788N using the forward primer 5'-ATAAGAGTGCCGTAAACACTGTGGTCAATCCG and the reverse primer 5'-CCGATTGACCACAGTGTTTACGGCACTCTTAT; T789N using the forward primer 5'-AGAGTGCCGTAACAAATGTGGTCAATCCGAAG and the reverse primer 5'-CTTCGGATTGACCACATTTGTTACGGCACTCT; T788N/T789N using the forward primer 5'-ATAAGAGTGCCGTAAACAATGTGGTCAATCCGAAG and reverse primer 5'-CTTCGGATTGACCACATTGTTTACGGCACTCTTAT. Plasmids were sequenced to confirm the integrity of the newly constructed mutants.

Cell Culture and Transfections—E63 cells, a myogenic clone of L8 rat skeletal myoblasts, were grown in DMEM supplemented with 10% HS, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 250 µg/ml amphotericin in a humidified atmosphere of 90% air, 5% CO2 at 37 °C as described previously (43). At confluence, differentiation medium (2% HS) was added to induce myotube formation. The expression vectors were transiently introduced into the cells using the LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer's instructions.

Intracellular Calcium Measurement using Confocal Microscopy—Measurement of [Ca2+]i by confocal microscopy was performed according to the methods of Kwon et al. (1). Cells cultured on 0.2% (w/v) gelatin-coated coverslips were rinsed twice with bath solution (140 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 20 mM glucose, 2.5 mM CaCl2, 5.5 mM HEPES, pH 7.4) and then incubated in the dark for 1 h at 25 °C in bath solution containing 5 µM fluo-3/AM. Ca2+ measurements in single cells were made using a Leica (Nussloch, Germany) TCS 4D laser scanning microscope equipped with an argon/krypton laser to excite the dye at 488 nm. Elevation of [Ca2+]i was initiated by adding the appropriate anti-integrin {alpha}7 antibodies (15 µg/ml) to the tissue chamber. Images (512 x 512 pixels) were obtained at a rate of 1 image/3 s. In order to quantify fluorescence, pixel intensities within the selected single cell areas of interest were measured and averaged. The acquired data were analyzed using Microsoft Excel version 4.0 (Redmond, WA). Mean intensity (Imean) was defined as an average of fluorescence intensity obtained from each pixel in the selected area, whereas average Imean was calculated from Imean. Independent experiments were repeated at least five times with the same gain. Cells exhibiting a calcium influx following treatment with the ionophore A23187 [GenBank] (Sigma) were regarded as viable cells.

Immunoprecipitation and Immunoblotting—E63 cells were washed three times in cold phosphate-buffered saline (PBS) and then lysed with extraction buffer containing 200 mM n-octyl-{beta}-D-glucopyranoside (Sigma), 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM PMSF, 20 µg/ml aprotinin, and 12.5 µg/ml leupeptin for 1 h at 4 °C. The cell lysate was centrifuged for 10 min at 12,000 x g, and protein concentrations were determined using BCA-based assay (Bio-Rad). Lysates were either analyzed directly by immunoblotting or used for immunoprecipitation. For immunoprecipitation, 1 mg of total protein was incubated overnight at 4 °C with 20 µg/ml anti-integrin {beta}1A antibody (42) or 20 µg/ml anti-integrin {beta}1D antibody (42) followed by incubation with protein G-Sepharose beads or protein A-Sepharose beads (Amersham Biosciences) for 4 h at 4 °C with constant agitation. Immune complexes were treated with SDS sample buffer (5% glycerol, 100 mM dithiothreitol, 2% SDS, 0.01% bromphenol blue, and 125 mM Tris, pH 6.8) and subjected to SDS-PAGE. Following electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, and the membranes was blocked for 1 h at room temperature with TBS containing 0.1% Tween 20 (TBST) and 5% dry milk or 5% bovine serum albumin. The blots were incubated for 1 h at room temperature with indicated antibodies, diluted TBST. The blots were then washed three times in TBST and incubated an additional hour with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin. The proteins were detected using ECL according to the manufacturer's protocol. In some cases, the membranes were then stripping buffer (100 mM {beta}-mercaptoethanol, 2% SDS, and 62.5 mM Tris, pH 6.7) and reprobed.

To determine the association between PP2A and integrin {beta}1A or {beta}1D in the presence of OA, cells were incubated with 100 nM OA for 2 h at 37 °C. To examine the change in phosphorylation level in the presence of OA, cells were added with 100 nM OA and maintained at 37 °C in serum-free DMEM.

In Vitro Phosphatase Assay—The 4-day-old cultured cells were collected and homogenized in 1 ml of storage buffer (50 mM imidazole, 0.2 mM EGTA, and 0.02% {beta}-mercaptoethanol) containing 1% Triton X-100, 2 mM PMSF, 20 µg/ml aprotinin, and 12.5 µg/ml leupeptin. The lysate was centrifuged for 1 h at 100,000 x g, and the supernatant was passed through a Sephadex G-25 column. The supernatants were centrifuged at 600 x g for 5 min at 4 °C. The supernatant (crude PP2A) was used in an in vitro phosphatase assay.

The 4-day-old cultured cells were washed three times in cold PBS and then lysed with RIPA buffer containing 1% Triton X-100, 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM PMSF, 20 µg/ml aprotinin, and 12.5 µg/ml leupeptin for 1 h at 4 °C. Cell lysate was centrifuged for 10 min at 12,000 x g, and protein concentrations were determined using BCA-based assay. 1 mg of lysates was incubated overnight at 4 °C with anti-B regulatory subunit antibody, followed by incubation with protein G-Sepharose beads or protein A-Sepharose beads for 4 h at 4 °C. Immune complexes were treated with storage buffer and subjected to an in vitro phosphatase assay.

Reaction components (50 µl) including 200 µM substrate (synthetic phosphopeptides; {beta}1A (KWDpTGENPIYKpSAVpTpTVV), {beta}1A1 (KWDpTGEN), {beta}1A2 (IYKpSAVT), {beta}1A3 (SAVpTTVV), {beta}1A4 (AVTpTVVN), {beta}1D1 (KWDpTQEN), and {beta}1D2 (IYKSpPIN)), PP2A reaction buffer (50 mM imidazole, pH 7.2), 0.2 mM EGTA, 0.02% {beta}-mercaptoethanol, 0.1 mg/ml bovine serum albumin, and inhibitors (1 mM vanadate, 50 nM or 1 µM OA) were incubated for 5 min at room temperature followed by incubation for 30 min with crude PP2A or purified PP2A (AC dimer; core enzyme, Upstate Biotechnology, Inc., Lake Placid, NY). After the reaction, 50 µl of molybdate dye was added to stop the phosphatase reaction. The color reaction was analyzed in an enzyme-linked immunosorbent assay reader (BioTek Instrument, Inc., Winooski, VT) with a 600 nm filter. This experiment was repeated at least five times.

Immunofluorescence Microscopy—E63 cells cultured on 0.2% gelatin-coated coverslips were washed twice with PBS and fixed for 10 min at room temperature in 2% paraformaldehyde solution. The cells were then washed with PBS. Cells were permeabilized with 0.1% Triton X-100 and washed again. Cells were washed and incubated for 60 min with anti-vinculin antibody or anti-paxillin antibody, washed three times for 10 min, and incubated for 60 min with TRITC-conjugated donkey anti-mouse immunoglobulin. Cells were mounted with 90% glycerol and 0.1% o-phenylenediamine in PBS. Immunofluorescence was analyzed using a Leica DMRBE microscope equipped with a x63 objective lens and filters for epifluorescence. Images were captured using a CoolSNAP digital camera (RP Photometrics, Tucson, AZ) with and analyzed by Meta Imaging SeriesTM software (version 5.0) from Universal Imaging Corp. (West Chester, PA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PP2A Inhibitors Block the Elevation of [Ca2+]i in E63 Myoblasts—Integrin ligation has been shown to result in activation of a variety of cellular signaling pathways (8, 44, 45). In skeletal muscle cells, we demonstrated previously (1) that binding of integrin {alpha}7 antibody or LN to integrin induced both Ca2+ release from inositol 1,4,5-trisphosphate-sensitive SR Ca2+ stores and extracellular Ca2+ influx through L-type Ca2+ channels. Changes in the cytosolic free calcium concentration ([Ca2+]i) mediated by integrin have been reported in a variety of cell types and can be used as an indicator of integrin activation (1, 41). By utilizing integrin antibody-/LN-induced changes of [Ca2+]i, we investigated the relationship between integrin activation and the relative state of phosphorylation of the integrin cytoplasmic domain. E63 myogenic cells elicited the elevation of [Ca2+]i by the addition of integrin {alpha}7 antibody (O26, 15 µg/ml) (1), which specifically binds to the extracellular domain of the integrin {alpha}7 subunit, or by the addition of LN (100 µg/ml). The elevation of [Ca2+]i in undifferentiated myoblasts (4-day-old culture, Fig. 1, A and B) was completely blocked by pretreatment with OA (100 nM) or ETL (100 µM), Ser/Thr-phosphatase inhibitors (46, 47). In contrast, the pretreatment with OA or ETL on differentiated myotubes (8-day culture) did not inhibit the integrin-mediated [Ca2+]i elevation (Fig. 1, A and B). The inhibitory effect of OA or ETL evident in these experiment, therefore, indicates that integrin activation is closely related to its phosphorylation status (48), and the differential sensitivity of myoblasts and myotubes to these phosphatase inhibitors may be due to the different regulatory mechanisms controlling phosphorylation and/or due to the presence of different integrin isoforms.



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 1.
Elevation of [Ca2+]i elicited by integrin {alpha}7 antibody in E63 muscle cells. A, E63 skeletal muscle cells preloaded with fluo-3/AM were treated with integrin {alpha}7 antibody (O26, 15 µg/ml) or left untreated as a control, and the fluorescence intensity on myoblasts (4-day-old culture) and on myotubes (8-day-old culture) was measured every 3 s using a confocal microscope. Pretreatment of 100 nM OA (n = 20; S.E. = ±0.425-1.336) for 2 h or 100 µM ETL (n = 21; S.E. = ±0.201-0.456) was completely blocked the integrin {alpha}7{beta}1-mediated [Ca2+]i elevation in myoblasts (red dotted line) and did not block the elevation of [Ca2+]i in fully differentiated myotubes (red solid line). Exposure to buffer had no effect on [Ca2+]i. The values depicted are the averages of fluorescence intensity obtained from at least 20 single cells in at least five independent experiments conducted under identical experimental conditions. B, a and d show the elevated [Ca2+]i within 40 s following exposure to integrin {alpha}7 antibody (15 µg/ml); the b and e were obtained in the presence of 100 nM OA, and c and f were obtained in the presence of 100 µM ETL. Cells shown in a-c were grown for 4 days (myoblasts, MB) and in the d-f were grown for 8 days (myotubes, MT). Bar, 50 µm.

 
Okadaic Acid Increases the Level of Phosphorylation of Integrin {beta}1A—The integrin {beta}1 undergoes an isoform switch from {beta}1A to {beta}1D during myogenesis. The integrin {beta}1A isoform is expressed on myoblasts, and the {beta}1D isoform is predominantly expressed on myotubes (Fig. 2A, 49-53). Thr788-Thr789 residues, one of potential phosphorylation sites in the integrin {beta}1A cytoplasmic domain, are substituted with Asn788-Asn789 in the {beta}1D splice variant (Fig. 2B). To assess the effects of OA upon Thr phosphorylation of integrin {beta}1, we preincubated 4-day-old cultured myoblasts with 100 nM OA for 4 h. This treatment had no effect upon either cell viability or the synthesis of the integrin {beta}1 (not shown). As shown in Fig. 2C, integrin {beta}1A seem to be Thr-phosphorylated at a relatively low level in 4-day-old cultured cells. However, OA treatment resulted in a large increase of Thr-phosphorylated integrin {beta}1A. Interestingly, in the presence of OA, the level of Thr phosphorylation of integrin {beta}1D, another splice variant of integrin {beta}1, was unchanged. To identify threonine phosphorylation on integrin {beta}1A, we performed immunoblot analysis with the antibody that specifically recognizes the phosphorylated threonine at 788-789 residues in the cytoplasmic domain of integrin {beta}1A. The level of Thr788-Thr789 phosphorylation of integrin {beta}1A was increased in the presence of OA or ETL (Fig. 2D), suggesting that threonine 788-789 residues could be primarily phosphorylation sites in integrin {beta}1A. The insensitivity of myotubes to OA is likely due to the unphosphorylated state of {beta}1D since the Thr788-Thr789 residues are substituted with Asn788-Asn789 in the {beta}1D splicing variant. Therefore, the alternative splicing that generates alternative integrin {beta}1 cytoplasmic domains may be an important mechanism to facilitate regulation of integrin activity that depends upon its unphosphorylated or phosphorylated state.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 2.
Effect of okadaic acid on integrin {beta}1 phosphorylation. A, during muscle differentiation, the expression of integrin {beta}1A and {beta}1D was examined by immunoblotting (IB). As phenotypic markers indicating myogenic differentiation, muscle-specific protein, myosin heavy chain was detected. B, sequence comparison of integrin {beta}1A and {beta}1D cytoplasmic domains. Potential Ser/Thr phosphorylation sites are indicated by asterisks. C, the 4-day-old cultured cells were incubated with or without 100 nM OA for 4 h, followed by immunoprecipitation with anti-integrin {beta}1A and {beta}1D antibodies, followed by immunoblot analysis with anti-phosphothreonine (P-thr) antibody. To verify equal loading amounts of protein, the blot was reprobed with anti-integrin {beta}1A or {beta}1D antibodies. D, threonine phosphorylation of integrin {beta}1A was analyzed with the antibody that specifically recognizes the phosphorylated threonine at 788-789 residues in the integrin {beta}1A. The 4-day-old cultured cell treated with 100 nM OA and 100 µM ETL were immunoprecipitated with anti-integrin {beta}1A antibody and followed by immunoblotting with anti-phosphothreonine (P-thr788/789) antibody. To verify equal loading amounts of protein, the immunoprecipitates were blotted with anti-integrin {beta}1A antibody.

 
Differential Association of PP2A with Integrin {beta}1A and {beta}1D during Myogenic Differentiation—In order to identify endogenous candidate phosphatases responsible for dephosphorylation of integrin {beta}1A, cell lysates of 4-day-old cultured cells were immunoprecipitated with specific antibodies to the integrin {beta}1A and {beta}1D and then immunoblotted with PP2A catalytic subunit (PP2Ac) antibody. As shown in Fig. 3, integrin {beta}1A and {beta}1D appeared to be associated with PP2Ac. The expression of PP2A was unchanged during E63 muscle cell differentiation (data not shown). The association of PP2Ac with integrin {beta}1A was dramatically decreased in OA-treated myoblasts (Fig. 3A). The association of the integrin {beta}1A with PP2Ac therefore seems to be highly dependent upon the phosphorylated states of the integrin. However, OA did not affect the association of {beta}1D with PP2Ac on either myoblasts or myotubes. These data suggest that dephosphorylation of integrin {beta}1A is regulated, at least in part, by the action of an OA-sensitive phosphatase PP2Ac.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 3.
Association of integrin {beta}1A and {beta}1D with PP2A subunits. A, E63 cells pretreatment with or without 100 nM OA were immunoprecipitated (IP) with anti-integrin {beta}1A or {beta}1D antibodies, followed by immunoblotting (IB) with anti-PP2A catalytic subunit (PP2Ac) antibody. To verify equal loading amounts of protein, the blot was reprobed with anti-integrin {beta}1A or {beta}1D antibodies. B, cells were immunoprecipitated with anti-B, B', or B'' regulatory subunit antibodies, followed by blotting with anti-integrin {beta}1A or {beta}1D antibodies. C, after OA treatment (100 nM, for 2 h), E63 cells were immunoprecipitated with anti-B or B'' subunit antibodies and then immunoblotted with anti-integrin {beta}1A or {beta}1D antibodies.

 
To identify PP2A regulatory subunits in association with integrin {beta}1A or {beta}1D, E63 cell lysates were immunoprecipitated with PP2A regulatory subunits, B, B', or B'' antibodies, and immunoblotted by integrin {beta}1A or {beta}1D antibodies. B, B', and B'' regulatory subunits showed differential affinities for the integrin {beta}1A and {beta}1D. B regulatory subunit was predominantly associated with {beta}1A but not {beta}1D, and B' subunit was weakly interacted with both integrin {beta}1A and {beta}1D.B'' subunit appeared to be associated with both integrin {beta}1A and {beta}1D. In addition, the association of B subunit to integrin {beta}1A was dramatically decreased in the presence of OA, and association of B'' subunit to integrin {beta}1A or {beta}1D remains bound even in the presence of OA. The dissociation of B subunit with integrin {beta}1A by pretreatment of OA is similar to that of PP2Ac with integrin {beta}1A. It suggests that differential association of B subunit of PP2A with integrin {beta}1A and {beta}1D may be related to the myogenic differentiation, and B subunit of PP2A may participate in the dephosphorylation of integrin {beta}1A.

PP2A Dephosphorylates Residues Thr788-Thr789 in the Cytoplasmic Domain of Integrin {beta}1A—To further confirm that PP2A mediates dephosphorylation of the integrin {beta}1 cytoplasmic domain, in vitro phosphatase activity assays were conducted with synthetic phosphopeptides corresponding to the potential O-linked phosphorylation sites in the cytoplasmic domain of integrin {beta}1A and {beta}1D, i.e. Thr777, Ser785, Thr788, and Thr789 (Fig. 4A). Cell lysates of 4-day-old cultured cells were used as a source of crude PP2A. Incubation of {beta}1A, {beta}1A3, and {beta}1A4 phosphopeptides with crude PP2A or purified PP2A AC dimer resulted in the release of phosphate, and addition of OA completely blocked its release in a dose-dependent manner (Fig. 4, B and C). In contrast, crude PP2A and PP2A AC dimer did not exhibit any phosphatase activity toward {beta}1A1, {beta}1D1, or {beta}1D2 phosphopeptides and exhibited only a low phosphatase activity against {beta}1A2 phosphopeptide. In order to inhibit the tyrosine phosphatase activity of PP2A, 1 mM vanadate was included in the incubation with the phosphopeptides. In this reaction, phosphate release was comparable to control (Fig. 4, B and C), indicating that the release of phosphate is due to specific Thr phosphatase activity. To identify PP2A regulatory subunits mediating the dephosphorylation of integrin {beta}1A, cells were immunoprecipitated with anti-B regulatory subunit antibody, and the immunocomplexes were used as an enzyme source for phosphatase activity assay. The immunocomplex with B subunit readily released the phosphate from {beta}1A3 and {beta}1A4 phosphopeptides but did not from {beta}1A1 phosphopeptide. The release of phosphate was completely blocked by OA treatment. The results indicate that PP2A may interact directly with these phosphopeptides and mediate, at least in part, the release of phosphate from residues Thr788-Thr789 in the cytoplasmic domain of integrin {beta}1A, and the B subunit of PP2A seems to participate in this process.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 4.
PP2A mediates dephosphorylation of residues Thr788-Thr789 in integrin {beta}1A. A, schematic representation of integrin {beta}1A and {beta}1D phosphopeptides. B and C, the phosphopeptides were reacted with the crude PP2A (B) or purified PP2A AC dimer (C) in the presence of inhibitors including vanadate (Va) or okadaic acid (OA) as described under "Materials and Methods." D, cells (4-day-old) were immunoprecipitated with anti-B regulatory subunit antibody or without antibody (Con), and the immunocomplexes were used as an enzyme (Enz) source for phosphatase activity assay. To verify the binding of PP2A with integrin {beta}1, the immunocomplexes were probed with anti-PP2Ac antibody. The released phosphates were detected by color reaction with molybdate dye. The control reaction (Rxn) indicates the absence of inhibitors.

 
Okadaic Acid Does Not Block Integrin-mediated [Ca2+]i Elevation in the {beta}1A Mutant-transfected Myoblasts—To test the function of two consecutive threonines in the cytoplasmic domain of integrin {beta}1A, three point mutants of {beta}1A such as T788N/T789N, T788N, and T789N were constructed into RFP vector (pDSRed1-N1) and transfected into E63 myoblasts, and cells expressing {beta}1A mutants were tested for their ability to induce calcium influx by treatment of integrin {alpha}7 antibody (O26) using confocal microscopic analysis. In E63 myoblasts transfected with RFP vector or RFP-{beta}1 wild-type, the elevation of [Ca2+]i was triggered by integrin {alpha}7 antibody, and the elevation of [Ca2+]i was completely inhibited by pretreatment with 100 nM OA. However, the elevation of [Ca2+]i was not affected even in the presence of OA in the three mutants, T788N, T789N, and T788N/T789N (Fig. 5). In addition, the confocal microscopic analysis confirmed that the level of elevated [Ca2+]i in T788N- or T789N-transfected myoblasts was not much different from that in T788N/T789N-transfected myoblasts. Therefore, these results indicate that substitution of Thr788-Thr789 with Asn788-Asn789 in the {beta}1A is at least able to mimic the {beta}1D isoform.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 5.
Comparison of the ability of integrin {beta}1A mutants and {beta}1A wild type to elevate cytosolic free calcium in E63 myoblasts. A series of {beta}1A mutants was constructed in pDsRed1-N1 (RFP) vector. The E63 myoblasts were transiently transfected with RFP vector, RFP vector containing {beta}1A wild-type or {beta}1A mutants as indicated. E63 myoblasts preloaded with fluo-3/AM were treated with integrin {alpha}7 antibody (O26, 15 µg/ml). Cells expressing red fluorescence proteins were selected, and the green fluorescence intensity corresponding to calcium influx (4-day-old culture) was measured every 3 s using confocal microscope and plotted graphically. The graph shows in RFP (n = 24; S.E. = ±0.513-2.701, A) or RFP-{beta}1A wild-type (n = 22; S.E. = ±0.208-1.804, B) transfected myoblasts, pretreatment of 100 nM okadaic acid (dotted line) for 2 h completely blocked the elevation of [Ca2+] and did not block the elevation of [Ca2+]i in T788N (n = 25; S.E. = ±0.424-1.881, C), T789N (n = 20; S.E. = ±0.410-1.385, D) and T788N/T789N (n = 21; S.E. = ±0.731-2.827, E) transfected myoblasts. The values depicted are the averages of fluorescence intensity obtained from at least 20 single cells in at least five independent experiments conducted under identical experimental conditions.

 
PP2A Inhibitors Inhibit the Formation of Focal Adhesions in E63 Myoblasts—It has been reported that the binding of integrin either with antibodies or its ligands promotes its association with the cell cytoskeleton (7), and the formation of focal adhesion is highly dependent upon phosphorylation of integrin {beta}1 (11, 16, 21, 23-32, 37-40). To address the formation of focal adhesions depending upon phosphorylation of integrin {beta}1, cells were treated with PP2A inhibitor such as OA or ETL, and the formation of focal adhesions was visualized by staining with anti-vinculin or anti-paxillin antibodies. Myoblasts treated with OA or ETL exhibited the dramatic reduction of focal adhesions containing vinculin and paxillin (Fig. 6, b, c, e, and f). However, myotubes that predominantly express integrin {beta}1D isoform were unchanged in the formation of focal adhesions even in the presence of OA or ETL (Fig. 6, h, i, k, and l). These data reflect that phosphorylation of residues Thr788-Thr789 in the {beta}1A cytoplasmic domain results in integrin inactivation and promotes dissociation from cytoskeleton proteins. The alternatively spliced integrin {beta}1D (T788N/T789N) may therefore represent a mechanism to escape from the regulation of PP2A activity and thereby display an increased affinity for both ECM and cytoskeleton. These results are consistent with observations in integrin {beta}1D-transfected cells, which exhibit targeting of the transfected integrin {beta}1D into focal adhesions with subsequent displacement of the endogenous {beta}1A and an elevated stability of focal adhesions (40, 56).



View larger version (120K):
[in this window]
[in a new window]
  		FIG. 6.
Effect of okadaic acid or endothall on the focal adhesion formations. The cultured E63 cells were treated for 4 h with 100 nM OA or with 100 µM ETL for 2 h. The formation of focal adhesions was visualized by staining with anti-vinculin (Vin) or anti-paxillin (Pax) antibodies. Formation of focal adhesions was dramatically decreased by OA or ETL treatment of myoblasts (4-day-old culture; b, c, e, and f), and no effects were evident on myotubes (8-day-old culture; h, i, k, and l). Bar, 30 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have analyzed the regulation of integrin {beta}1 phosphorylation in skeletal muscle cells. Our studies demonstrate that pretreatment of protein serine/threonine phosphatase inhibitors such as OA or ETL inhibits the elevation of [Ca2+]i and formation of focal adhesions. Integrin {beta}1A is associated with the PP2A, and its association with PP2A seems to be dependent upon the phosphorylation status of the {beta}1A cytoplasmic domain. Furthermore, an in vitro phosphatase assay indicates that two consecutive residues Thr788-Thr789 in the cytoplasmic domain of integrin {beta}1A are specifically dephosphorylated by PP2A.

Although OA treatment results in increased phosphorylation of integrin {beta}1A, it may also result in Ser/Thr phosphorylation of a variety of proteins as a result of diverse PP2A activity (47, 48, 57, 58). However, the increase of integrin {beta}1A phosphorylation does indicate that integrin {beta}1A has PP2A-sensitive dephosphorylation sites. In addition, in vitro phosphatase assay and immunoblotting data clearly proved that the phosphate of the Thr788-Thr789 residues on integrin {beta}1A is specifically released by the action of PP2A and that dephosphorylation of Thr788-Thr789 residues results in enhanced focal adhesion formation. However, there are conflicting reports that Thr788-Thr789 mutant was defective in mediating cell attachment and did not contribute to fibronectin formation (29), and mutation of the three consecutive threonines in integrin {beta}2 to alanines strongly inhibits the integrin functions (59). Even though several {beta} subunits have been reported to become phosphorylated or unphosphorylated, the cellular functions of the phosphorylation are still controversial. The conservation of the region corresponding to Thr788-Thr789 residues between the cytoplasmic domains of the integrin {beta} subunits, however, indicates that it is important for a general cellular function of the integrin {beta}1 subunits.

Reversible phosphorylation of integrin {beta}1 is known to be involved in a variety of cellular functions such as formation of focal adhesions, association with cytoskeletal components, and signal transduction (11, 16, 21, 23-32, 35, 37-38). Mutation studies of integrin {beta}1 cytoplasmic domains have focused mainly on the possible role of dephosphorylation sites along integrin {beta}1A. It was shown that PP2A is associated with the integrin {beta}1. PP2A can accumulate at focal contact sites due to its interaction with paxillin and a truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation (60). The activation of PP2A may participate in many integrin {alpha}2{beta}1-mediated cellular events, suggesting that PP2A may play a role as a regulator of integrin function (33). Our study demonstrated that PP2A participates in the dephosphorylation of integrin {beta}1A, and in particular, the B subunit is likely to participate in this process. However, PP2A activity capable of dephosphorylation of Thr788-Thr789 residues needs to be further investigated both in vitro and in vivo. The inactive PP2A or PP2A knock out cells would help to understand the integrin regulation, but further work is necessary to elucidate this subject.

Four cytoplasmic domain variants ({beta}1A, {beta}1B, {beta}1C, and {beta}1D) of the integrin {beta}1 subunit have been identified. These variants arise from alternative mRNA splicing on the region encoding the cytoplasmic domain. These splice variants may allow alternative integrin-cytoskeleton interactions and mediate different signaling pathways. In this work, we have analyzed integrin {beta}1A, comparing its properties with those of {beta}1D isoform. {beta}1A isoform is characteristically expressed in many cell types. However, {beta}1D isoform displaces {beta}1A isoform in differentiated skeletal muscle cells (49-52). It was reported that integrin {beta}1D displays an increased affinity for both the extracellular matrix ligands and the actin cytoskeleton. The {beta}1D phenotype includes altered morphology, retarded spreading, enhanced ligand binding, and extracellular matrix assembly, as well as reduced migration and significantly increased contractility. The increased integrin-ligand and integrin-cytoskeleton associations mediated by the {beta}1D isoform cause a significant reinforcement of the entire cytoskeleton-matrix link. Integrin-mediated cytoskeleton-ECM linkage has to be particularly robust in muscle fibers because of the high tensile forces transmitted across the membrane that imposes a requirement for enhanced stability of muscle-adhesive structures. This function appears to be due primarily to the integrin {beta}1D cytoplasmic variant (12, 56). The isoform switch from {beta}1A to {beta}1D may also have additional physiological consequences in signal transduction during myocyte differentiation since {beta}1A but not {beta}1D interacts with the Nck signaling protein (42).

The cytoplasmic domain of integrin {beta}1D is alternatively spliced and substituted with Asn788-Asn789 residues instead of Thr788-Thr789 residues that represent the PP2A dephosphorylation site in the integrin {beta}1A subunit. Transfection of integrin {beta}1D subunits into E63 cells results in an increase in the ligand binding affinity and enhances the association of cytoskeletal proteins such as talin and ECM ligand (56). The PP2A-insensitive integrin {beta}1D is therefore well placed to play a role as a dominant positive role in cytoskeleton-ECM associations that result in the maintenance of muscle tension. Taken together, we propose that dephosphorylation of the Thr788-Thr789 residue in the cytoplasmic domain of integrin {beta}1 may contribute to the linkage of integrins to focal adhesion proteins and cytoskeleton proteins. The substitution of Thr788-Thr789 residues with Asn788—Asn789 residues in the {beta}1D isoform may represent a mechanism to escape from the regulation of PP2A activity and to strengthen the cytoskeleton-matrix link or muscle function. Therefore, this study partly explains why muscle cells undergo an isoform switch from integrin {beta}1A to {beta}1D during muscle cell differentiation.


   FOOTNOTES
 
* This study was supported in part by grants from National Research Laboratory (The Ministry of Science and Technology). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} These authors contributed equally to this work. Back

§ Supported by Brain Korea 21 Program. Back

To whom correspondence should be addressed: Dept. of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Buk-gu, Kwangju 500-712, Korea. Tel.: 82-62-970-2487; Fax: 82-62-970-2484; E-mail: wksong{at}kjist.ac.kr.

1 The abbreviations used are: ECM, extracellular matrix; OA, okadaic acid; ETL, endothall; PP2A, protein phosphatase 2A; PP2Ac, protein phosphatase 2A catalytic subunit; [Ca2+]i, cytosolic free calcium concentration; LN, laminin; MHC, myosin heavy chain; RFP, red fluorescence protein; PMSF, phenylmethylsulfonyl fluoride; HS, horse serum; DMEM, Dulbecco's modified Eagle's medium; TRITC, tetramethylrhodamine isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We are grateful to Craig Kamibayashi (University of Texas Southwestern Medical Center, Dallas, TX) and Brian Hemmings (Friedrich Miescher Institute, Basel, Switzerland) for the generous gift of PP2A B regulatory subunits antibodies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443[Abstract/Free Full Text]
  2. Hynes, R. O. (1992) Cell 69, 11-25[CrossRef][Medline] [Order article via Infotrieve]
  3. Albelda, S. M., and Buck, C. A. (1990) FASEB J. 4, 2868-2880[Abstract]
  4. Helmer, M. E. (1990) Annu. Rev. Immunol. 8, 365-400[CrossRef][Medline] [Order article via Infotrieve]
  5. Damsky, C. H., and Werb, Z. (1992) Curr. Opin. Cell Biol. 4, 772-781[CrossRef][Medline] [Order article via Infotrieve]
  6. Dustin, M. L., Carpen, O., and Springer, T. A. (1992) J. Immunol. 148, 2654-2663[Abstract]
  7. Song, W. K., Wang, W., Sato, H., Bielser, D. A., and Kaufman, S. J. (1993) J. Cell Sci. 106, 1139-1152[Abstract]
  8. Ginsberg, M. H., Du, X., and Plow, E. F. (1992) Curr. Opin. Cell Biol. 4, 766-771[CrossRef][Medline] [Order article via Infotrieve]
  9. Boudreau, N., Sympson, C. J., Werb, Z., and Bissell, M. J. (1995) Science 267, 891-893[Abstract/Free Full Text]
  10. Green, L. J., Mould, P., and Humphries, M. J. (1998) Int. J. Biochem. Cell Biol. 30, 179-184[CrossRef][Medline] [Order article via Infotrieve]
  11. Lewis, J. M., and Schwartz, M. A. (1995) Mol. Biol. Cell 6, 151-160[Abstract]
  12. Pfaff, M., Liu, S., Erle, D. J., and Ginsberg, M. H. (1998) J. Biol. Chem. 273, 6104-6109[Abstract/Free Full Text]
  13. Otey, C. A., Vasques, G. B., Burridge, K., and Erickson, B. W. (1993) J. Biol. Chem. 268, 21193-21197[Abstract/Free Full Text]
  14. Calderwood, D. A., Shattil, S. J., and Ginsberg, M. H. (2000) J. Biol. Chem. 275, 22607-22610[Free Full Text]
  15. Liu, A., Calderwood, D. A., and Ginsberg, M. H. (2000) J. Cell Sci. 113, 3563-3571[Abstract]
  16. Sakai, T., Jove, R., Fassler, R., and Mosher, D. F. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3808-3813[Abstract/Free Full Text]
  17. Kanner, S. B., Grosmaire, L. S., Ledbetter, J. A., and Damle, N. K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7099-7103[Abstract/Free Full Text]
  18. Bastianutto, C., Clementi, E., Codazzi, F., Podini, P., De Giorgi, F., Rizzuto, R., Meldolesi, J., and Pozzan, T. (1995) J. Cell Biol. 130, 847-855[Abstract/Free Full Text]
  19. Camacho, P., and Lechleiter, J. D. (1995) Cell 82, 765-771[CrossRef][Medline] [Order article via Infotrieve]
  20. Lawson, M. A., and Maxfield, F. R. (1995) Nature 377, 75-79[CrossRef][Medline] [Order article via Infotrieve]
  21. Pomies, P., Frachet, P., and Block, M. R. (1995) Biochemistry 34, 5104-5112[CrossRef][Medline] [Order article via Infotrieve]
  22. Hendey, B., Lawson, M., Marcantonio, E. E., and Maxfield, F. R. (1996) Blood 87, 2038-2048[Abstract/Free Full Text]
  23. Wrenn, R. W., Creazzo, T. L., and Herman, L. E. (1996) Biochem. Biophys. Res. Commun. 226, 876-882[CrossRef][Medline] [Order article via Infotrieve]
  24. Bouvard, D., Molla, A., and Block, M. R. (1998) J. Cell Sci. 111, 657-665[Abstract]
  25. Mulrooney, J., Foley K., Vineberg, S., Barreuther, M., and Grabel, L. (2000) Exp. Cell Res. 258, 332-341[CrossRef][Medline] [Order article via Infotrieve]
  26. Sakai, T., Zhang, Q., Fassler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538[Abstract/Free Full Text]
  27. Crowly, E., and Horwitz, A. F. (1995) J. Cell Biol. 131, 525-537[Abstract/Free Full Text]
  28. Hannigan, G. E., Leung-Hagesteijn, C., Fitz-Gibbon, L, Coppolino, M. G., Radeva, G., Filmus, J., Bell, J. C., and Dedhar, S. (1996) Nature 379, 91-96[CrossRef][Medline] [Order article via Infotrieve]
  29. Wennerberg, K., Fassler, R., Warmegard, B., and Johansson, S. (1998) J. Cell Sci. 111, 1117-1126[Abstract]
  30. Wennerberg, K., Armulik, A., Sakai, T., Kaelsson, M., Fassler, R., Schaefer, E. M., Mosher, D. F., and Johansson, S. (2000) Mol. Cell. Biol. 20, 5758-5765[Abstract/Free Full Text]
  31. Chang, D. D., Wong, C., Smith, H., and Liu, J. (1997) J. Cell Biol. 138, 1149-1157[Abstract/Free Full Text]
  32. Burridge, K., and Chrzanowska-Wodnicka, M. (1996) Annu. Rev. Cell Dev. Biol. 12, 463-519[CrossRef][Medline] [Order article via Infotrieve]
  33. Ivaska, J., Nissinen, L., Immonen, N., Eriksson, J. E., Kahari, V. M., and Heino, J. (2002) Mol. Cell. Biol. 22, 1352-1359[Abstract/Free Full Text]
  34. Lerea, K. M., Cordero, K. P., Sakariassen, K. S., Kirk, R. I., and Fried, V. A. (1999) J. Biol. Chem. 274, 1914-1919[Abstract/Free Full Text]
  35. O'Toole, T. E., Ylanne, J., and Culley, B. M. (1995) J. Biol. Chem. 270, 8553-8558[Abstract/Free Full Text]
  36. Filardo, E. J., Brooks, P. C., Deming, S. L., Damsky, C., and Cheresh, D. A. (1995) J. Cell Biol. 130, 441-450[Abstract/Free Full Text]
  37. Barreuther, M. F., and Grabel, L. B. (1996) Exp. Cell Res. 222, 10-15[CrossRef][Medline] [Order article via Infotrieve]
  38. Rezka, A. A., Hayashi, Y., and Horwitz, A. (1992) J. Cell Biol. 117, 1321-1330[Abstract/Free Full Text]
  39. Belkin A. M., Zhidkova, N. I., Balzac, F., Altruda, F., Tomatis, D., Maier, A., Tarone, G., Koteliansky, V. E., and Burridge, K. (1996) J. Cell Biol. 132, 211-226[Abstract/Free Full Text]
  40. Belkin, A. M., Retta, S. F., Pletjushkina, O. Y., Silengo, L., Fassler, R., Koteliansky, V. E., Burridge, K., and Tarone, G. (1997) J. Cell Biol. 139, 1583-1595[Abstract/Free Full Text]
  41. Richardson, A., and Parsons, J. T. (1995) BioEssays 17, 229-236[CrossRef][Medline] [Order article via Infotrieve]
  42. Kim, Y. Y., Lim, C. S., Song, Y. H., Ahnn, J., Park, D., and Song, W. K. (1999) Cell Adhes. Commun. 7, 85-97[Medline] [Order article via Infotrieve]
  43. Kaufman, S. J., and Parks, C. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 3888-3892[Abstract/Free Full Text]
  44. Clark, E. A., and Bruggle, J. S. (1995) Science 268, 233-239[Abstract/Free Full Text]
  45. Diamond, M. S., and Springer, T. A. (1994) Curr. Biol. 4, 506-517[CrossRef][Medline] [Order article via Infotrieve]
  46. Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999) Trends Biochem. Sci. 24, 186-191[CrossRef][Medline] [Order article via Infotrieve]
  47. Mumby, M. C., and Walter, G. (1993) Physiol. Rev. 73, 673-699[Abstract/Free Full Text]
  48. Davare, M. A., Horne, M. C., and Hell, J. W. (2000) J. Biol. Chem. 275, 39710-39717[Abstract/Free Full Text]
  49. de Melker, A. A., and Sonnenberg, A. (1999) Bioessays 21, 499-509[CrossRef][Medline] [Order article via Infotrieve]
  50. Baudoin, C., Goumans, M., Mummery, C., and Sonnenberg, A. (1998) Genes Dev. 12, 1202-1216[Abstract/Free Full Text]
  51. Meredith, J. E., Kiosses, W. B., Takada, Y., and Schwartz, M. A. (1999) J. Biol. Chem. 274, 8111-8116[Abstract/Free Full Text]
  52. Belkin, A. M., and Retta, S. F. (1998) J. Biol. Chem. 273, 15234-15240[Abstract/Free Full Text]
  53. Wu, C. (1999) J. Cell Sci. 112, 4485-4489[Abstract]
  54. Huang, Y., Li, J., Zhang, Y., and Wu, C. (2000) J. Cell Biol. 150, 861-871[Abstract/Free Full Text]
  55. Dedhar, S. (2000) Curr. Opin. Cell Biol. 12, 250-256[CrossRef][Medline] [Order article via Infotrieve]
  56. Retta, S. F., Balzac, F., Ferraris, P., Belkin, A. M., Fassler, R., Humphries, M. J., Leo, G. D., Silengo, L., and Tarone, G. (1998) Mol. Biol. Cell 9, 715-731[Abstract/Free Full Text]
  57. Hiraga, A., and Tamura, S. (2000) Biochem. J. 346, 433-439[Medline] [Order article via Infotrieve]
  58. Bryant, J. C., Westphal, R. S., and Wadzinski, B. E. (1999) Biochem. J. 339, 241-246[CrossRef][Medline] [Order article via Infotrieve]
  59. Hibbs, M. L., Jakes, S., Stacker, S. A., Wallace, R. W., and Springer, T. A. (1991) J. Exp. Med. 174, 1227-1238[Abstract/Free Full Text]
  60. Ito, A., Kataoka, T. R., Watanabe, M., Nishiyama, K., Mazaki, Y., Sabe, H., Kitamura, Y., and Nojima, H. (2000) EMBO J. 19, 562-571[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
K. R. Legate and R. Fassler
Mechanisms that regulate adaptor binding to {beta}-integrin cytoplasmic tails
J. Cell Sci., January 15, 2009; 122(2): 187 - 198.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
H. Takala, E. Nurminen, S. M. Nurmi, M. Aatonen, T. Strandin, M. Takatalo, T. Kiema, C. G. Gahmberg, J. Ylanne, and S. C. Fagerholm
{beta}2 integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding
Blood, September 1, 2008; 112(5): 1853 - 1862.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
P. A. Kiely, D. O'Gorman, K. Luong, D. Ron, and R. O'Connor
Insulin-Like Growth Factor I Controls a Mutually Exclusive Association of RACK1 with Protein Phosphatase 2A and {beta}1 Integrin To Promote Cell Migration.
Mol. Cell. Biol., June 1, 2006; 26(11): 4041 - 4051.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. Zheng and P. J. McKeown-Longo
Cell adhesion regulates Ser/Thr phosphorylation and proteasomal degradation of HEF1
J. Cell Sci., January 1, 2006; 119(1): 96 - 103.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/8/7082    most recent
M311581200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, S.-M.
Right arrow Articles by Song, W. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, S.-M.
Right arrow Articles by Song, W. K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement