JBC

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


     

Originally published In Press as doi:10.1074/jbc.C600039200 on August 25, 2006

J. Biol. Chem., Vol. 281, Issue 40, 29426-29430, October 6, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/40/29426    most recent
C600039200v1
Right arrow Alert me when this article is cited
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xi, X.
Right arrow Articles by Du, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xi, X.
Right arrow Articles by Du, X.
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?

Tyrosine Phosphorylation of the Integrin beta3 Subunit Regulates beta3 Cleavage by Calpain*

Xiaodong Xi{ddagger}§1, Panagiotis Flevaris{ddagger}1, Aleksandra Stojanovic{ddagger}1, Athar Chishti{ddagger}, David R. Phillips, Stephen C. T. Lam{ddagger}, and Xiaoping Du{ddagger}2

From the {ddagger}Department of Pharmacology, University of Illinois, Chicago, Illinois 60612, Portola Pharmaceuticals, Inc., South San Francisco, California 94080, and the §Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China

Received for publication, February 21, 2006 , and in revised form, August 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Outside-insignalingofbeta3integrinsinducesandrequiresphosphorylation at tyrosine 747 (Tyr747) and tyrosine 759 (Tyr759) of the beta3 subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta3 cytoplasmic domain. Here we show that beta3 tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta3 to calpain cleavage. Furthermore, phosphorylation at Tyr747 and Tyr759 of beta3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr759 is associated with calpain cleavage at Tyr759. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta3 cytoplasmic domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Integrins mediate cell adhesion and transduce signals that are critical in the dynamic regulation of cell adhesion, spreading, migration, and proliferation (1, 2). Integrin signaling is a two-way process exemplified by inside-out and outside-in signaling of the platelet integrin, {alpha}IIbbeta3. Inside-out signaling is believed to be transduced by talin binding to the cytoplasmic domain of {alpha}IIbbeta3 (3–7), and consequent conformational changes (5, 6), which propagate to the ligand binding domain of {alpha}IIbbeta3, activating ligand binding function (8, 9). Ligand binding to {alpha}IIbbeta3 not only forms adhesive bond but also induces outside-in signaling, leading to cell spreading, secretion, stabilization of platelet adhesion, and amplification of platelet aggregation (10, 11).

The cytoplasmic domain of beta3 is critical in bidirectional signaling (12–15). Inside-out signaling requires the membrane proximal region and the two NXXY motifs in the beta3 cytoplasmic domain (3–6, 15–19). Outside-in signaling requires the intact cytoplasmic domain of beta3 (15) and also requires tyrosine phosphorylation in NXXY motifs (20, 21). However, the mechanism responsible for the role of tyrosine phosphorylation of beta3 in outside-in signaling is unclear. On the other hand, the cytoplasmic domain of beta3 is cleaved by the calcium-dependent proteases (calpain) at sites flanking two NXXY motifs, preferentially at the C-terminal side of Tyr759 (15, 22, 23). A consequence of calpain cleavage of beta3 at Tyr759 is the inhibition of beta3-dependent cell spreading, which is an outside-in signaling event (15). In studying the relationship between these two seemingly unrelated beta3 modifications that regulate the function of the cytoplasmic domain of beta3, we found that tyrosine phosphorylation in beta3 cytoplasmic domain inhibits cleavage of beta3 by calpain. Since calpain cleavage negatively regulates outside-in signaling-mediated cell spreading, our finding provides a mechanism by which tyrosine phosphorylation of beta3 promotes integrin outside-in signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Peptides—Peptides were synthesized by Protein Chemistry Laboratory, University of Illinois at Chicago, purified by reverse phase-high performance liquid chromatography, and correct molecular weights verified by electrospray ion-trap mass spectrometry. Double tyrosine-phosphorylated beta3 cytoplasmic domain peptides were verified by SDS-PAGE and immunoblots with anti-beta3 cytoplasmic domain antibodies.

Antibodies—To generate anti-peptide antibodies, synthetic peptides conjugated to keyhole limpet hemocyanin were used to immunize rabbits (24). The antibody pY759 was generated using the peptide CTpYRGT with a linker cystein and a 5-residue sequence corresponding to tyrosine-phosphorylated beta3 C terminus. The anti-serum was absorbed (three times) with Sepharose 4B coupled with a non-phosphorylated CTYRGT peptide to remove phosphorylation-independent reactivity. Rabbit antibodies specific for beta3 with a phospho-tyrosine at Tyr747 or Tyr759 were also purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A rabbit antibody, 8053, and a mouse monoclonal antibody, mAb15, against the extracellular domain of beta3, were generous gifts from Dr. Mark Ginsberg, University of California, San Diego, CA. The antibody against the beta3 C-terminal TYRGT sequence, Ab762, or antibodies recognizing the calpain cleavage-generated new C terminus at each of the calpain cleavage sites, Ab759, Ab754, Ab747, and Ab741, were described previously (15, 22). Purified µ-calpain and an antibody specific for calpain-cleaved fodrin were generous gifts from Dr. T. Saido (25).

Calpain Cleavage of Synthetic beta3 Cytoplasmic Domain Peptides—The beta3C-pY peptide (1 mg/ml, 0.1 ml) solubilized in 0.05 M Tris, 0.15 M NaCl, 1 mM CaCl2, and 1 mM dithiothreitol (pH 7.4) were incubated with 1 µg of purified µ-Calpain at 30 °C for 30 min. After adding an equal volume of 2 x SDS-PAGE sample buffer containing 5 mM EDTA and 0.1 mM E64 (a calpain inhibitor), the samples were subjected to SDS-PAGE using 10–20% gradient gels and immunoblotted with antibodies recognizing intact beta3 C terminus or calpain cleaved fragments of beta3.

Detection of Tyrosine Phosphorylation and Calpain Cleavage of Integrin beta3 in Platelets—Blood from healthy human donors or from wild type and knock-in mice with both Tyr747 and Tyr759 mutated to phenolalanine (20, 21) was anticoagulated with 1/7 volume of ACD (2.5% trisodium citrate, 2.0% D-glucose, 1.5% citric acid) (26). Washed platelets in Tyrode's buffer were allowed to stay at 25 °C for 1 h (26). Platelet aggregation was induced by 0.1 unit/ml of {alpha}-thrombin in a Chronolog aggregometer stirring at 1000 rpm for 3 min. In some cases, 1 µM calcium ionophore A23187 [GenBank] was added 2 min after adding {alpha}-thrombin. In phosphatase inhibition experiments, platelets were incubated with 0.5 mM sodium vanadate at 37 °C for 5 min prior to addition of agonists. Platelets were solubilized in SDS-PAGE sample buffer containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM E64; analyzed by SDS-PAGE using 4–15% gradient gels; and immunoblotted with various antibodies. Results were visualized with the enhanced chemiluminescence reagent (Amersham Biosciences).

Localization of Calpain-cleaved or Tyrosine-phosphorylated beta3 Molecules in Spreading Platelets—Lab-Tek® chamber slides (Nalgen Nunc, Naperville, IL) were precoated with 10 µg/ml fibrinogen followed by incubation with 5% bovine serum albumin. Platelet suspension in Tyrode's buffer (100 µl, 108/ml) was added to the wells and incubated at 37 °C for 90 and 180 min. The plates were rinsed, and adherent platelets were fixed with 4% paraformaldehyde and permeabilized with 0.1 M Tris, 10 mM EGTA, 0.15 M NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM E64, 0.1% Triton X-100, 1% bovine serum albumin (pH 7.5). The samples were incubated with a mouse anti-beta3 antibody, mAb15, and one of the rabbit anti-beta3 cytoplasmic domain antibodies. After washing, platelets were stained with Alexa Fluor® 488-conjugated goat anti-mouse IgG and Alexa Fluor® 594-conjugated goat anti-rabbit IgG. Data were collected using a Zeiss confocal microscope. The area of beta3 colocalized with tyrosine phosphorylation or calpain cleavage was quantitated in randomly chosen fields by using the colocalization tool in Zeiss LSM 5 software and expressed as average number of pixels/platelet. Statistical significance was determined using a t test.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
To determine whether tyrosine phosphorylation of the integrin beta3 cytoplasmic domain regulates calpain cleavage of beta3, a 43-residue phosphopeptide (beta3C-pY) corresponding to the sequence of the beta3 cytoplasmic domain was synthesized with both Y747 and Y759 phosphorylated (Fig. 1A). As a control, we also synthesized a non-phosphorylated peptide with the sequence identical to beta3C-pY (beta3C). These peptides were treated with purified human µ-calpain and then immunoblotted with antibodies that recognize the beta3 cytoplasmic domain only when beta3 is cleaved at the previously characterized calpain cleavage sites (cleavage indicator antibodies Ab754 and Ab759) (22), and with Ab762, an antibody that recognizes the beta3 C-terminal TYRGT sequence (15). Ab762 still reacts with the beta3 cytoplasmic domain when Tyr759 is phosphorylated but at a reduced affinity (Fig. 1B). However, the reactivity of this antibody is totally abolished with calpain-cleaved beta3 (15) (Fig. 1B). Treatment of non-phosphorylated beta3C peptide with µ-calpain caused the loss of reactivity with Ab762 and gain of reactivity with cleavage indicator antibodies, indicating that calpain cleaved the peptide at specific sites. In contrast, the tyrosinephosphorylated beta3 cytoplasmic domain peptide, beta3C-pY, showed no loss of reactivity with Ab762 following calpain treatment and no gain of reactivity with the cleavage indicator antibodies. Thus, tyrosine phosphorylation protects beta3 cytoplasmic domain from calpain cleavage in the in vitro assays using purified calpain and beta3 peptides.


Figure 1
View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 1.
The effect of tyrosine phosphorylation on calpain cleavage of synthetic beta3 cytoplasmic domain peptides. A, the integrin beta3 cytoplasmic domain peptide sequence with tyrosine phosphorylation and calpain cleavage sites indicated. B, synthetic peptides corresponding to beta3 cytoplasmic domain with (beta3CpY) or without (beta3C) phosphorylation at Tyr747 and Tyr759 were incubated with purified µ-calpain at 30 °C for 30 min and analyzed by SDS-PAGE and Western blotting with various anti-beta3 antibodies (Ab762 specific for intact integrin beta3 C terminus, Ab759 specific for calpain cleavage site at Tyr759, Ab754 specific for calpain cleavage site at Phe754, and Ab pY759, a phosphotyrosine-specific anti-beta3 antibody.

 
To detect beta3 tyrosine phosphorylation in vitro and in platelets, we developed an anti-peptide antiserum (Ab pY759) specific for the phosphopeptide, C-TpYRGT, corresponding to the beta3 C-terminal sequence. The Ab pY759 reacted with the phosphorylated beta3C-pY peptide but not the nonphosphorylated beta3C peptide, further verifying the beta3C-pY phosphopeptide (Fig. 1B). These results also show that the reactivity of Ab pY759 with beta3 is phosphorylation-dependent and thus specifically indicates beta3 tyrosine phosphorylation. In immunoblot, Ab pY759 had essentially no reactivity with beta3 from resting platelets but reacted strongly with beta3 from platelets aggregated in response to thrombin, which induces beta3 tyrosine phosphorylation. Prevention of platelet aggregation by the integrin inhibitor, RGDS, inhibited the reaction of Ab pY759 with beta3, confirming that beta3 phosphorylation is induced by integrin outside-in signaling (20).

To investigate whether tyrosine phosphorylation protects integrin from cleavage by calpain in platelets, platelets were treated with thrombin to induce tyrosine phosphorylation of beta3. Platelets were also treated with tyrosine phosphatase inhibitor, sodium vanadate (Fig. 2) to prevent dephosphorylation of beta3 cytoplasmic domain by tyrosine phosphatases and thus further enhance beta3 phosphorylation. Phosphorylation at Tyr747 and Tyr759 induced by thrombin and their further enhancement by sodium vanadate were indicated by immunoblotting with Ab pY759 and an anti-pY747 antibody (Fig. 2). Platelets were then treated with the calcium ionophore, A23187 [GenBank] , to induce cleavage of beta3. Consistent with our previous results, A23187 [GenBank] induced cleavages of beta3 at the C-terminal side of residues 747, 754, and 759 in platelets. However, A23187 [GenBank] -induced beta3 cleavage is substantially reduced in thrombin-treated platelets and further reduced in platelets treated with both thrombin and sodium vanadate, in correlation with the increased beta3 tyrosine phosphorylation. The effects of thrombin and sodium vanadate are unlikely to be related to changes in expression levels of beta3 because immunoblotting with the anti-beta3 extracellular domain antibody, 8053, showed similar levels of beta3 in platelets treated with or without thrombin or/and sodium vanadate. To exclude the possibility that the effects of thrombin and sodium vanadate in inhibiting calpain cleavage of beta3 may be caused by nonspecific effect of thrombin or sodium vanadate on calpain activity, we also examined whether thrombin or/and sodium vanadate may inhibit calpain cleavage of another calpain substrate, fodrin. A23187 [GenBank] induced calpain cleavage of fodrin as indicated by reactivity with a calpain cleavage-specific antibody against fodrin (25) (Fig. 2). Since this cleavage was not affected by the treatment of platelets with thrombin and/or sodium vanadate, it is unlikely that thrombin and/or sodium vanadate had a nonspecific effect on calpain activity. Rather, their effect on beta3 cleavage is likely to be specifically caused by reducing beta3 susceptibility to calpain cleavage. Since we showed that thrombin induced tyrosine phosphorylation at Tyr759 and Tyr747 of beta3, which is enhanced by sodium vanadate, our results suggest that tyrosine phosphorylation of beta3 inhibited A23187 [GenBank] -induced calpain cleavage of beta3 cytoplasmic domain in platelets.


Figure 2
View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 2.
Effects of tyrosine phosphorylation on A23187-induced calpain cleavage of beta3 in platelets. Washed platelets (3 x 108/ml) were pretreated with or without thrombin or thrombin plus a tyrosine phosphatase inhibitor, sodium vanadate (0.1 mM), to induce beta3 tyrosine phosphorylation for 2 min. The platelets were then treated with 1 µM calcium ionophore A23187 to induce calpain activation and cleavage of beta3. After 5 min, platelets were solubilized and analyzed by immunoblotting with antibodies specific for intact beta3 C terminus (Ab762), calpain-cleaved forms of beta3 (Ab759, Ab754, and Ab747), and tyrosine-phosphorylated beta3 (pY759 and pY747). As controls, platelet lysates were also immunoblotted with an antibody against the extracellular domain of beta3, 8053, and with an antibody recognizing the calpain cleavage-dependent epitope on a calpain substrate, fodrin.

 
It is known that calpain is activated following platelet aggregation induced by thrombin. However, unlike A23187 [GenBank] that induces cleavage of ~70% of beta3 molecules in 5 min, thrombin only induces calpain cleavage of a small population of beta3 during platelet aggregation (15). Fig. 2 shows that thrombin, but not A23187 [GenBank] , induced tyrosine phosphorylation of beta3. To further examine whether thrombin-induced tyrosine phosphorylation protects beta3 from calpain cleavage without adding calcium ionophore, platelets were pretreated with or without sodium vanadate and then stimulated with thrombin. Fig. 3 shows that thrombin-induced calpain cleavage of a population of integrin molecules mainly occurs at Tyr759 site, and this cleavage is inhibited by sodium vanadate. To exclude the possible nonspecific effect of sodium vanadate on calpain activity, we show that sodium vanadate failed to affect thrombin-induced calpain cleavage of fodrin. These results suggest that thrombin-induced phosphorylation of beta3 inhibited thrombin-induced calpain cleavage of beta3 during platelet aggregation. To further support the protective effect of tyrosine phosphorylation against calpain cleavage, we show that mouse beta3 (DiYF) with both Tyr747 and Tyr759 mutated to phenylalanine is more susceptible to calpain cleavage than wild type during thrombin-induced platelet aggregation (Fig. 3B).


Figure 3
View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 3.
Effects of tyrosine phosphorylation on thrombin-induced calpain cleavage of beta3 cytoplasmic domain in platelets. A, washed human platelets (3 x 108/ml) were pretreated with or without sodium vanadate and then exposed to thrombin to induce platelet aggregation for 5 min. Platelets were then solubilized and immunoblotted with antibodies specific for calpain-cleaved forms of beta3 (Ab747, Ab754, and Ab759), intact beta3 C terminus (Ab762), tyrosine-phosphorylated beta3 (Ab pY759, Ab pY747), and the extracellular domain of beta3 (8053). Platelet lysates were also immunoblotted with the calpain cleavage-specific anti-fodrin antibody to determine whether sodium vanadate affects calpain activity nonspecifically. B, platelets (5 x 108/ml) from wild type mice or knock-in mutant mice (DiYF) with both tyrosine residues corresponding to human pY747 and pY759 of beta3 replaced by phenylalanine were stimulated with thrombin for 5 min and immunoblotted with Ab754 to indicate calpain cleavage and anti-beta3 antibody 8053 to indicate loading. Note that cleavage at Phe754 only occurred in DiYF platelets.

 
Tyrosine phosphorylation of beta3 also occurs in platelets spread on fibrinogen surfaces (Fig. 4) as indicated by staining with antibodies specifically recognizing beta3 molecules that are phosphorylated at Tyr759 or Tyr747 (Fig. 4). At an earlier time point (90 min), staining of both pY759 and pY747 is strong and forms punctate focal adhesion-like structures mainly in the leading edge of spreading platelets particularly at the tips of pseudopods (arrows). At a later time point, while pY747 staining becomes even stronger and forms focal adhesion-like structures ringing the platelets, pY759 staining becomes decreased and more diffuse, and its focal adhesion-like structures are almost lost, suggesting pY759 dephosphorylation at focal adhesion sites and leading edge. These data indicate that phosphorylation at Tyr759 and Tyr747 is differentially regulated in spreading platelets, and that pY759 is preferentially dephosphorylated at a later stage of platelet spreading. To determine whether tyrosine phosphorylation affected calpain cleavage of beta3 in spreading platelets, we also stained spreading platelets with the antibody Ab759 that recognizes the calpain cleavage site at Tyr759 (the major calpain cleavage site in platelets, see Fig. 3A). At 90 min, Ab759 staining was weak in spreading platelets. Interestingly, Ab759 stain was even weaker in the margin of platelets where the punctated ring of tyrosine-phosphorylated b3 was strong. These results are consistent with the above observation that tyrosine phosphorylation inhibits calpain cleavage of beta3. At 180 min, more beta3 molecules are cleaved at Tyr759, which correlated well with the decrease in pY759 staining. Calpain cleavage at sites near pY747 was barely detectable in spreading platelets (data not shown). These results suggest that calpain cleavage mainly occurs to a population of dephosphorylated integrin molecules. The differential dephosphorylation of pY747 and pY759 also explains why calpain preferentially cleaves beta3 at the Tyr759 site in platelets. Furthermore, we previously reported that cells expressing a beta3 truncation mutant mimicking calpain cleavage at Tyr759 showed reduced spreading on fibrinogen, suggesting that the calpain-cleaved form of beta3 is defective in mediating spreading (15). Thus, our results suggest that one mechanism by which tyrosine phosphorylation of beta3 facilitates outside-in signaling and platelet spreading is its inhibition of calpain cleavage of beta3.


Figure 4
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 4.
beta3 tyrosine phosphorylation and calpain cleavage in platelets spreading on fibrinogen. A, platelets were allowed to spread on fibrinogen-coated slides for 90 and 180 min, fixed, and permeabilized. The slides were stained with a mouse anti-beta3 extracellular domain antibody, mAb15 (green) and one of the tyrosine phosphorylation-specific (pY747 and pY759) or calpain cleavage-specific (Ab759) anti-beta3 antibodies (red). Affinity-depleted Ab 759 antiserum was used as negative control (NC). Data were collected with a Zeiss confocal microscope (63x lens). B, quantitation of area (pixel number/platelet) of beta3 tyrosine phosphorylation and calpain cleavage in six random fields of 6 slide wells from three experiments (mean ± S.E., platelet numbers are marked above each column; *, p < 0.001). Quantitation of negative control (NC) is from 4 slide wells from two separate experiments.

 
It is known that the intact beta3 C terminus is required for outside-in signal leading to cell spreading (15). This requirement is consistent with the findings that c-Src binds to the beta3 C-terminal domain and that inhibition of Src family of protein kinases inhibited cell spreading (27–29). Calpain cleavage of beta3 cytoplasmic domain disrupts the c-Src binding site in the C-terminal domain of beta3, which potentially explains why calpain cleavage of integrins plays important roles in detaching the rear end of a cell during migration (30), and in cell detachment during apoptosis (31). Calpain is abundant in the cell focal adhesion sites (32) and is activated by integrin-mediated calcium elevation (33). Thus, cell spreading and firm adhesion would not be possible without protection of beta3 cytoplasmic domain from calpain cleavage. Furthermore, calpain also regulates the functions of several other focal adhesion proteins including talin (34), focal adhesion kinase (35), protein-tyrosine phosphatase IB (36), and Rho A (37). Thus, phosphorylation-dependent regulation of integrin cleavage by calpain allows calpain to differentially and dynamically regulate the functions of integrins and other focal adhesion proteins at different locations as well as during different stages of cell adhesion, spreading, and migration. We show that tyrosine phosphorylation of beta3 serves as a molecular mechanism that directly protects integrins from calpain cleavage. It is possible that phosphorylation-dependent beta3 binding to cytoplasmic proteins also has a protective effect. Thus, our results provide an important mechanism by which tyrosine phosphorylation of beta3 promotes integrin outside-in signaling and by which the timing and location of calpain cleavage of integrins are dynamically regulated.


   FOOTNOTES
 
* This work was supported in part by Grants HL62350 and HL68819 from NHLBI/National Institutes of Health (to X. D.) and by a grant-in-aid from the American Heart Association (to X. D.). 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

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Pharmacology, University of Illinois at Chicago, 835 S. Wolcott Ave., Rm. E403, Chicago, IL 60612. Tel.: 312-355-0237; Fax: 312-996-1225; E-mail: xdu{at}uic.edu.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Hynes, R. O. (2002) Cell 110, 673–687[CrossRef][Medline] [Order article via Infotrieve]
  2. Ginsberg, M. H., Partridge, A., and Shattil, S. J. (2005) Curr. Opin. Cell Biol. 17, 509–516[CrossRef][Medline] [Order article via Infotrieve]
  3. Calderwood, D. A., Zent, R., Grant, R., Rees, D. J., Hynes, R. O., and Ginsberg, M. H. (1999) J. Biol. Chem. 274, 28071–28074[Abstract/Free Full Text]
  4. Tadokoro, S., Shattil, S. J., Eto, K., Tai, V., Liddington, R. C., de Pereda, J. M., Ginsberg, M. H., and Calderwood, D. A. (2003) Science 302, 103–106[Abstract/Free Full Text]
  5. Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E., and Qin, J. (2002) Cell 110, 587–597[CrossRef][Medline] [Order article via Infotrieve]
  6. Vinogradova, O., Vaynberg, J., Kong, X., Haas, T. A., Plow, E. F., and Qin, J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 4094–4099[Abstract/Free Full Text]
  7. Yuan, W., Leisner, T. M., McFadden, A. W., Wang, Z., Larson, M. K., Clark, S., Boudignon-Proudhon, C., Lam, S. C., and Parise, L. V. (2006) J. Cell Biol. 172, 169–175[Abstract/Free Full Text]
  8. Du, X., Gu, M., Weisel, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H. (1993) J. Biol. Chem. 268, 23087–23092[Abstract/Free Full Text]
  9. Leisner, T. M., Wencel-Drake, J. D., Wang, W., and Lam, S. C. (1999) J. Biol. Chem. 274, 12945–12949[Abstract/Free Full Text]
  10. Shattil, S. J., Kashiwagi, H., and Pampori, N. (1998) Blood 91, 2645–2657[Free Full Text]
  11. Parise, L. V. (1999) Curr. Opin. Cell Biol. 11, 597–601[CrossRef][Medline] [Order article via Infotrieve]
  12. Chen, Y. P., Djaffar, I., Pidard, D., Steiner, B., Cieutat, A. M., Caen, J. P., and Rosa, J. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10169–10173[Abstract/Free Full Text]
  13. Chen, Y. P., O'Toole, T. E., Leong, L., Liu, B. Q., Diaz-Gonzalez, F., and Ginsberg, M. H. (1995) Blood 86, 2606–2615[Abstract/Free Full Text]
  14. Wang, R., Shattil, S. J., Ambruso, D. R., and Newman, P. J. (1997) J. Clin. Investig. 100, 2393–2403[Medline] [Order article via Infotrieve]
  15. Xi, X., Bodnar, R. J., Li, Z., Lam, S. C.-T., and Du, X. (2003) J. Cell Biol. 162, 329–339[Abstract/Free Full Text]
  16. Hughes, P. E., O'Toole, T. E., Ylanne, J., Shattil, S. J., and Ginsberg, M. H. (1995) J. Biol. Chem. 270, 12411–12417[Abstract/Free Full Text]
  17. Hughes, P. E., Diaz, G. F., Leong, L., Wu, C., McDonald, J. A., Shattil, S. J., and Ginsberg, M. H. (1996) J. Biol. Chem. 271, 6571–6574[Abstract/Free Full Text]
  18. Knezevic, I., Leisner, T. M., and Lam, S. (1996) J. Biol. Chem. 271, 16416–16421[Abstract/Free Full Text]
  19. Patil, S., Jedsadayanmata, A., Wencel-Drake, J. D., Wang, W., Knezevic, I., and Lam, S. C. (1999) J. Biol. Chem. 274, 28575–28583[Abstract/Free Full Text]
  20. Jenkins, A. L., Nannizzi-Alaimo, L., Silver, D., Sellers, J. R., Ginsberg, M. H., Law, D. A., and Phillips, D. R. (1998) J. Biol. Chem. 273, 13878–13885[Abstract/Free Full Text]
  21. Law, D. A., DeGuzman, F. R., Heiser, P., Ministri-Madrid, K., Killeen, N., and Phillips, D. R. (1999) Nature 401, 808–811[CrossRef][Medline] [Order article via Infotrieve]
  22. Du, X., Saido, T. C., Tsubuki, S., Indig, F. E., Williams, M. J., and Ginsberg, M. H. (1995) J. Biol. Chem. 270, 26146–26151[Abstract/Free Full Text]
  23. Pfaff, M., Du, X., and Ginsberg, M. H. (1999) FEBS Lett. 460, 17–22[CrossRef][Medline] [Order article via Infotrieve]
  24. Bodnar, R. J., Gu, M., Li, Z., Englund, G. D., and Du, X. (1999) J. Biol. Chem. 274, 33474–33479[Abstract/Free Full Text]
  25. Saido, T. C., Yokota, M., Nagao, S., Yamaura, I., Tani, E., Tsuchiya, T., Suzuki, K., and Kawashima, S. (1993) J. Biol. Chem. 268, 25239–25243[Abstract/Free Full Text]
  26. Du, X., Plow, E. F., Frelinger, A. L. I., O'Toole, T. E., Loftus, J. C., and Ginsberg, M. H. (1991) Cell 65, 409–416[CrossRef][Medline] [Order article via Infotrieve]
  27. Arias-Salgado, E. G., Lizano, S., Sarkar, S., Brugge, J. S., Ginsberg, M. H., and Shattil, S. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 13298–13302[Abstract/Free Full Text]
  28. De Virgilio, M., Kiosses, W. B., and Shattil, S. J. (2004) J. Cell Biol. 165, 305–311[Abstract/Free Full Text]
  29. Arias-Salgado, E. G., Lizano, S., Shattil, S. J., and Ginsberg, M. H. (2005) J. Biol. Chem. 280, 29699–29707[Abstract/Free Full Text]
  30. Huttenlocher, A., Palecek, S. P., Lu, Q., Zhang, W., Mellgren, R. L., Lauffenburger, D. A., Ginsberg, M. H., and Horwitz, A. F. (1997) J. Biol. Chem. 272, 32719–32722[Abstract/Free Full Text]
  31. Meredith, J. J., Mu, Z., Saido, T., and Du, X. (1998) J. Biol. Chem. 273, 19525–19531[Abstract/Free Full Text]
  32. Beckerle, M. C., Burridge, K., DeMartino, G. N., and Croall, D. E. (1987) Cell 51, 569–577[CrossRef][Medline] [Order article via Infotrieve]
  33. Fox, J. E., Taylor, R. G., Taffarel, M., Boyles, J. K., and Goll, D. E. (1993) J. Cell Biol. 120, 1501–1507[Abstract/Free Full Text]
  34. Fox, J. E., Goll, D. E., Reynolds, C. C., and Phillips, D. R. (1985) J. Biol. Chem. 260, 1060–1066[Abstract/Free Full Text]
  35. Cooray, P., Yuan, Y., Schoenwaelder, S. M., Mitchell, C. A., Salem, H. H., and Jackson, S. P. (1996) Biochem. J. 318, 41–47[Medline] [Order article via Infotrieve]
  36. Frangioni, J. V., Oda, A., Smith, M., Salzman, E. W., and Neel, B. G. (1993) EMBO J. 12, 4843–4856[Medline] [Order article via Infotrieve]
  37. Kulkarni, S., Goll, D. E., and Fox, J. E. (2002) J. Biol. Chem. 277, 24435–24441[Abstract/Free Full Text]

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. Biol. Chem.Home page
B. Hu, L. L. Kong, R. T. Matthews, and M. S. Viapiano
The Proteoglycan Brevican Binds to Fibronectin after Proteolytic Cleavage and Promotes Glioma Cell Motility
J. Biol. Chem., September 5, 2008; 283(36): 24848 - 24859.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
X. Su, J. Mi, J. Yan, P. Flevaris, Y. Lu, H. Liu, Z. Ruan, X. Wang, N. Kieffer, S. Chen, et al.
RGT, a synthetic peptide corresponding to the integrin {beta}3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin {alpha}IIb{beta}3 with Src kinase
Blood, August 1, 2008; 112(3): 592 - 602.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
P. Flevaris, A. Stojanovic, H. Gong, A. Chishti, E. Welch, and X. Du
A molecular switch that controls cell spreading and retraction
J. Cell Biol., November 5, 2007; 179(3): 553 - 565.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/40/29426    most recent
C600039200v1
Right arrow Alert me when this article is cited
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xi, X.
Right arrow Articles by Du, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xi, X.
Right arrow Articles by Du, X.
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 © 2006 by the American Society for Biochemistry and Molecular Biology.