©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Matrix/Integrin Interaction Activates the Mitogen-activated Protein Kinase, p44 and p42(*)

(Received for publication, August 8, 1994; and in revised form, October 6, 1994)

Noritsugu Morino (1) Toshihide Mimura (1) Ken Hamasaki (1) Kazuyuki Tobe (1) Kohjiro Ueki (1) Kanako Kikuchi (2) Kazuhiko Takehara (2) Takashi Kadowaki (1) Yoshio Yazaki (1) Yoshihisa Nojima (1)(§)

From the  (1)Third Department of Internal Medicine and the (2)Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cell adhesion to extracellular matrix proteins is a dynamic process leading to dramatic changes in the cell phenotype. Integrins are one of the major receptor families that mediate cell-matrix contact. Evidence that integrins can act as signal transducing molecules has accumulated over the past few years. We report here that p44 and p42 mitogen-activated protein (MAP) kinases are rapidly phosphorylated on tyrosine residues upon adhesion of human skin fibroblasts to fibronectin or upon cross-linking of beta1 integrins with antibody. The tyrosine phosphorylation of both kinases is associated with increased enzymatic activity. Pretreatment of the cells with cytochalasin D, which selectively disrupts the network of the actin filaments, completely inhibits this adhesion-mediated MAP kinase activation. Thus, our findings indicate that ligation of beta1 integrins induces an increase in both tyrosine phosphorylation and enzymatic activity of p44 and p42 MAP kinases, and that the integrity of the actin cytoskeleton is essential in this process. Since MAP kinase behaves as a convergence point for diverse receptor-initiated signaling events at the plasma membrane, this serine/threonine kinase plays a key role and helps to account for the diversity of integrin-dependent cell functions.


INTRODUCTION

Integrins are transmembrane, heterodimeric glycoproteins consisting of an alpha and a beta subunit. They function as cell surface receptors for extracellular matrix proteins (ECM) (^1)and also mediate cell-to-cell interaction(1, 2) . It has been well established that cell adhesion to ECM or to opposing cells through integrins results in marked alterations of cell shape, motility, growth, and differentiation (2, 3, 4, 5, 6, 7, 8, 9) . These facts have strongly suggested that integrins could transmit signals into the cell interior and hence have urged researchers in various biological fields to investigate signal transduction pathways triggered by cell adhesion(2, 3, 10) . Such efforts have lead to the notion that protein tyrosine phosphorylation plays a critical role in signal transduction via integrins. A number of reports (11, 12, 13) have documented that engagement of beta1 integrins stimulated tyrosine phosphorylation of several proteins in a variety of cell types. Focal adhesion kinase pp125, originally identified as a putative substrate for a retroviral oncogene product, pp60, has been shown to be tyrosine-phosphorylated in response to the ligation of beta1 or beta3 integrins(14, 15, 16, 17) . pp125 itself is a protein tyrosine kinase which is specifically located in the focal adhesion. Recent studies demonstrated that autophosphorylation of pp125 creates a site having strong affinity for the Src homology 2 (SH2) domain of Src family tyrosine kinases such as pp60 and pp59(18, 19) . It is proposed that binding of pp60 and pp59 to pp125 may result in their enzymatic activation, and additionally provides a mechanism for the recruitment of these Src family kinases to specific sites within the cell(19) . Thus, the focal adhesion is highly abundant in interacting signaling molecules, which would provide a structural and biochemical basis for the diversity of integrin-dependent cell function. However, links between biochemical events occurring just beneath the cell membrane and the nucleus in the integrin-mediated signaling cascade are still largely unknown(3) .

Mitogen-activated protein kinase (referred to as MAP kinase or extracellular signal-regulated kinase (ERK)) is a serine/threonine protein kinase whose activity is rapidly stimulated by a number of external stimuli through mechanisms mediated by tyrosine kinase-encoded receptors, non-receptor type tyrosine kinases, and G protein-coupled receptors(20, 21, 22) . MAP kinase is activated by phosphorylation on its threonine and tyrosine residues, a process which is carried out by a dual-specificity protein kinase, MAP kinase kinase(23) . MAP kinases have been shown to phosphorylate and thereby activate many well studied regulatory proteins located in diverse cellular compartments, including nuclear transcriptional factors(20, 21, 22, 24, 25) . A function of MAP kinases may therefore be to provide a link between transmembrane signaling and the nucleus.

We report here that two isoforms of MAP kinase, p44 and p42, are activated in response to the adherence of human skin fibroblasts (HSF) to fibronectin (FN) or by cross-linking of beta1 integrins with antibody. Our findings indicate that MAP kinases may act as a key molecule connecting cell surface integrins to the expression of genes associated with a variety of adhesion-dependent cell functions.


EXPERIMENTAL PROCEDURES

Reagents

Human FN was purchased from Telios (San Diego, CA). Anti-phosphotyrosine antibody (4G10) was obtained from UBI laboratories (Lake Placid, NY). Rabbit polyclonal anti-MAP kinase antibody, termed alphaY91, was prepared as described previously(26) . Myelin basic protein (MBP), and poly-L-lysine (PLL), trypsin inhibitor, protein kinase inhibitor (rabbit sequence), and cytochalasin D were obtained from Sigma. Monoclonal antibodies against human beta1 integrin subunit (4B4) and MHC class I (W6/32) were kindly provided by Dr. C. Morimoto, Dana-Farber Cancer Institute, Boston, MA.

Cell Culture

Human skin fibroblasts (HSF) were obtained by skin biopsy from dorsal forearms of healthy donors. Primary explant cultures were established in 25-cm^2 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 50 µg/ml streptomycin. Subcultures were established by trypsinization and maintained at 37 °C in 5% CO(2) air. Cells from the second to sixth passages were used in this study.

Cell Adhesion and Preparation of Cell Lysates

Preparation of culture dishes, coated with FN, PLL, and monoclonal antibodies, was as described previously(4, 13) . In brief, dishes were incubated with PBS containing 5 µg/ml of FN, PLL, or monoclonal antibodies, at 4 °C overnight. After washing three times with PBS, dishes were coated with 1% bovine serum albumin (BSA)-PBS by incubating for 1 h at 37 °C. Before plating cells on dishes, confluent cells were serum-depleted by incubating in 0.4% FCS-DMEM for 48 h, and then in serum-free DMEM for 4 h. Cells were detached by treating with 0.05% trypsin-EDTA. Trypsin inhibitor (1.5 mg/ml) was immediately added to the cell suspension, followed by washing three times with serum-free DMEM. Cells were plated onto dishes coated with different reagents and incubated at 37 °C for the indicated time periods in serum-free DMEM. Bound cells were lysed in situ with 1% Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetamide, 10 mM NaF, 10 mM sodium pyrophosphate, and 0.4 mM sodium vanadate) for detection of protein tyrosine phosphorylation by immunoblotting or with buffer A (25 mM Tris-HCl, pH 7.4, 25 mM NaCl, 1 mM sodium vanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 10 nM okadaic acid, 0.5 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) for MBP kinase assays. After removing insoluble materials by centrifugation at 14,000 rpm for 10 min, protein concentrations in the supernatant were determined using micro BCA protein assay kit (Pierce). Cell lysates were stored at -70 °C until use.

Immunoblotting and Immunoprecipitation

Cell lysates were loaded on 7.5 or 10% SDS-polyacrylamide gel in reducing conditions. Proteins on the gel were electrotransferred to nitrocellulose membranes. After blocking nonspecific reaction by incubating the membrane with 3% BSA-PBS, protein tyrosine phosphorylation was detected by anti-phosphotyrosine antibody 4G10 plus alkaline phosphatase-conjugated anti-mouse Ig. Immunoprecipitations were carried out as described previously(26) . Cell extracts were incubated with 1/100 dilution of alphaY91 (anti-MAP kinase) antiserum in the presence of 0.15% of SDS for 1 h at 4 °C. The mixtures were further incubated with protein-A Sepharose beads for 1 h at 4 °C. Thereafter, beads were washed five times with 1% Nonidet P-40 lysis buffer to remove unbound proteins. Immune complexes were treated with sample buffer, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) for anti-phosphotyrosine immunoblotting or kinase assays in MBP-containing gels described below.

MBP Kinase Assay

Aliquots of the supernatants of the cell extracts with buffer A were incubated in 40 µl of kinase buffer (25 mM Tris-HCl, pH 7.4, 10 mM MgCl(2), 1 mM dithiothreitol, 40 µM ATP, 2 µCi of [-P]ATP, 2 µM protein kinase inhibitor peptide, 0.5 mM EGTA), and substrates (25 µg MBP). After 10 min at 25 °C, the reaction was stopped by adding 10 µl of stopping solution containing 0.6% HCl, 1 mM ATP, 1% BSA. Aliquots of the supernatants (15 µl) were spotted on 1.5 times 1.5-cm squares of P81 paper (Whatman), washed five times for at least 10 min each in 0.5% phosphoric acid, washed in acetone, dried, and counted by the Cerenkov technique(26) .

Kinase Assays in MBP-containing Gels after SDS-PAGE

MAP kinases were immunoprecipitated from Nonidet P-40 lysates of HSF cells and were electrophoresed in an SDS-polyacrylamide gel containing 0.5 mg/ml MBP(26) . SDS was removed from the gel by washing the gel with two changes of 20% 2-propanol in 50 mM Tris-HCl (pH 8.0) for 1 h and then with 50 mM Tris-HCl, containing 5 mM 2-mercaptoethanol for 1 h at room temperature. The enzyme was first denatured by treating the gel with two changes of 6 M guanidine HCl at room temperature for 1 h and then renatured with five changes of 50 mM Tris-HCl (pH 8.0), containing 0.04% Tween 20 and 5 mM 2-mercaptoethanol at 4 °C for 3 h each. After renaturation, the gel was preincubated at 25 °C for 1 h with 5 ml of 40 mM HEPES (pH 8.0), containing 2 mM dithiothreitol, and 10 mM MgCl(2). Phosphorylation of MBP was carried out by incubating the gel at 25 °C for 1 h with 5 ml of 40 mM HEPES (pH 8.0), 0.5 mM EGTA, 10 mM MgCl(2), 2 µM protein kinase inhibitor, 40 µM ATP, and 25 µCi of [-P]ATP. After incubation, the gel was washed with a solution containing 5% trichloroacetic acid and 1% sodium pyrophosphate until the radioactivity of the solution became negligible. The washed gel was dried and then subjected to autoradiography.


RESULTS AND DISCUSSION

Recent studies have shown that the engagement of beta1 integrins with components of ECM stimulated tyrosine phosphorylation of several cellular proteins(11, 12, 13, 15, 16, 27, 28, 29) . Some of these phosphoproteins have been identified as tensin, paxillin, and focal adhesion kinase pp125, whereas others (such as pp105 and pp130) have not been fully characterized. In an attempt to determine molecules which are involved in the signaling cascade of beta1 integrins, we chose HSF cells, since adhesion-induced tyrosine phosphorylation could be clearly detected in this cell type. As shown in Fig. 1A (lanes 1 and 2), culturing HSF cells in dishes that had been coated with human FN-induced increased tyrosine phosphorylation of 190-kDa (pp190), 130-kDa (pp130), 120-kDa (pp120), 70-kDa (pp70), and 42-kDa (pp42) proteins. Tyrosine phosphorylation of these proteins was detectable 5 min after starting cell culture, and reached maximal levels in 20-40 min (Fig. 1B, lanes 1-4). HSF cells cultured in dishes coated with PLL failed to exhibited this response, suggesting that enhanced phosphorylation was not induced by binding to nonspecific substrates (Fig. 1B, lanes 5-8). Moreover, pretreatment of cells with soluble anti-beta1 integrin antibody (4B4) inhibited tyrosine phosphorylation as well as cell adhesion to FN (data not shown). To further verify the involvement of beta1 integrin in this response, we cultured HSF cells on dishes coated with anti-4B4 to cross-link the beta1 integrins, and examined protein tyrosine phosphorylation by anti-phosphotyrosine immunoblotting. As shown in Fig. 1A (lanes 3 and 4), adhesion of HSF cells to immobilized anti-beta1 integrin but not to anti-MHC class I antibodies resulted in enhanced tyrosine phosphorylation of the same set of proteins found in FN-adherent cells. These results indicate that engagement of beta1 integrins with antibody stimulates signaling pathways closely related to those triggered by cell adhesion to FN. While this is consistent with the notion that tyrosine phosphorylation induced by adherence to FN is at least partially dependent on beta1 integrins, we have not ruled out the possibility that other classes of integrins or other non-integrin molecules contribute to this response. We examined no fewer than five strains of HSF cells derived from different donors, and obtained essentially identical results (data not shown). By immunoprecipitation using anti-pp125 (2A7), we have confirmed the identity of pp120 as pp125 (data not shown).


Figure 1: Tyrosine phosphorylation induced by engagement of beta1 integrins. A, HSF cells were allowed to adhere to plates that had been coated with PLL (lane 1), FN (lane 2), anti-MHC class I (lane 3), or anti-beta1 integrin (lane 4) antibodies. After 30 min of incubation, bound cells were lysed and processed for anti-phosphotyrosine immunoblotting. B, HSF cells were cultured on plates coated with FN (lanes 1-4) or PLL (lanes 5-8) at 37 °C for indicated periods. Bound cells were lysed and then processed for anti-phosphotyrosine immunoblotting.



p44 and p42 MAP kinases are serine/threonine kinases whose enzyme activity can be stimulated by phosphorylation on tyrosine as well as threonine residues of this molecule(20, 21, 22, 23) . Similar molecular size and a crucial role of MAP kinases in gene regulation prompted us to examine whether pp42 in the present study was identical to MAP kinase. We used alphaY91 to immunoprecipitate MAP kinase from lysates prepared from HSF cells plated on FN. alphaY91 antiserum was raised against a synthetic peptide corresponding to residues 307-327 of the amino acid sequence deduced from the p44 cDNA, and has been shown to recognize both p44 and p42 MAP kinases in the presence of 0.15% of SDS(26) . Immunoprecipitates were then subjected to immunoblotting with anti-phosphotyrosine antibodies. As shown in Fig. 2A, upper panel, enhanced tyrosine phosphorylation of 42- and 44-kDa proteins was induced by adherence to FN in a time-dependent manner. Duplicate filters were analyzed by immunoblotting with alphaY91, to verify that the same amount of the MAP kinases were loaded (Fig. 2A, lower panel). pp42 identified in the whole cell lysates was found to comigrate with p42 in alphaY91 immunoprecipitates (data not shown). Fig. 2B shows the results obtained by densitometric scanning of the transblot bands in Fig. 2A. Tyrosine phosphorylation of MAP kinases in the immunoprecipitates occurred as early as 5 min after plating cells onto FN-coated dishes, and the overall kinetics were identical to that of pp42 in the total cell lysates (Fig. 1B and Fig. 2, A and B). Maximal response, obtained at 20 min after cell adhesion, was 4-5-fold increased from the basal levels. Similar results were obtained after anti-beta1 integrin cross-linking (data not shown). Taken together, tyrosine phosphorylation of both isoforms of MAP kinase were significantly enhanced by cell adherence to immobilized FN or anti-beta1 integrin antibody.


Figure 2: Adherence-dependent tyrosine phosphorylation of MAP kinases. A, MAP kinases were immunoprecipitated with alphaY91 antiserum in the presence of 0.15% SDS from lysates of non-adherent fibroblasts (lane 1) and fibroblasts adhered to plates coated with FN for indicated periods (lanes 2-5). Immunoprecipitates were separated on SDS-PAGE and probed with anti-phosphotyrosine antibody (upper panel). Duplicate filters were probed with alphaY91 to show that the same amount of the MAP kinases were loaded into each lane (lower panel). B, densitometric scanning of transblot bands, shown in Fig. 2A, of p44 (open circle) and p42 (closed circle). Data represent the percentage of control (non-adherent cells, 0 min).



Next, we examined whether adhesion-induced tyrosine phosphorylation of MAP kinases was associated with an increase in kinase activity. First, we measured the enzyme activity of the MAP kinases in whole cell extracts using MBP as a substrate. Fig. 3A shows the kinetics of MBP kinase activity detected in cell extracts from HSF cells adhered to FN or PLL. MBP kinase activity was significantly increased in HSF cells plated on FN compared with those bound to PLL. The time course was again comparable to that of tyrosine phosphorylation of MAP kinases described above ( Fig. 2and 3A). To further verify the activation of MAP kinases, we performed MAP kinase assays in MBP-containing gels after SDS-PAGE. Extracts of HSF cells that had adhered to FN or PLL for 30 min were immunoprecipitated with alphaY91 antiserum. The immunoprecipitates were then electrophoresed in an SDS-polyacrylamide gel containing MBP. After denaturation and renaturation, the gel was incubated with [-P]ATP and Mg. We observed increased MBP kinase activity migrating at both 42 and 44 kDa after cell adhesion to FN but not to PLL (Fig. 3B, lanes 1 and 2). Clear distinction between these two bands, readily apparent on the original autographs, is more difficult to resolve after reproduction. Duplicate filters were immunoblotted with alphaY91 to confirm that the same amounts of MAP kinases were loaded (data not shown). MBP kinase activity was significantly higher in HSF cells adhered to immobilized anti-beta1 integrin than those attached to anti-MHC class I antibodies (Fig. 3B, lanes 3 and 4). Densitometric analysis revealed that the MBP kinase activity of both kinases were increased by 3.5- and 2.4-fold after cell adhesion to dishes coated with FN and anti-4B4, respectively. These results indicated that the kinase activities of both p44 and p42 MAP kinases were increased after cell adhesion to FN, and that this response could be duplicated by the ligation of beta1 integrins with antibody.


Figure 3: MAP kinase activation induced by cell adhesion to FN. A, fibroblasts were allowed to adhere to plates coated with PLL (closed circles) or FN (open circles) for the indicated time periods. MBP phosphotransferase activity in the cell extracts was measured. Data represent the average percentage of control (non-adherent cells) from three independent experiments (*p < 0.05,**p < 0.01 versus adherence to PLL). B, fibroblasts adhered to PLL (lane 1), FN (lane 2), anti-MHC class I (lane 3), and anti-beta1 integrin monoclonal antibodies (lane 4) for 30 min were lysed, and the supernatants were immunoprecipitated with alphaY91 in the presence of 0.15% SDS. The immunoprecipitates were electrophoresed in an SDS-polyacrylamide gel containing MBP, and kinase assays were performed as described under ``Experimental Procedures.'' Note that the enzyme activity was detected in both the 42- and 44-kDa bands in the gel.



The association of integrin subunits with cytoskeletal proteins is thought to contribute to the formation of focal adhesions and actin stress fiber organization(2, 3, 30) . In accordance with this model, cytochalasin D, an agent which disrupts actin polymerization, has been shown to prevent adhesion-dependent tyrosine phosphorylation(28, 29) . Therefore, we next examined whether cytochalasin D inhibited the MAP kinase activation induced by adhesion of HSF cells to FN. HSF cells in suspension were pretreated for 5 min with the indicated concentrations of cytochalasin D prior to being added to FN-coated dishes. The viability of HSF cells was not affected over this dose-range of cytochalasin D. Cells were then allowed to adhere to FN for 30 min in the continuous presence of cytochalasin D. Bound cells were lysed with Nonidet P-40 lysis buffer, and processed for anti-phosphotyrosine immunoblotting or MAP kinase assays. As shown in Fig. 4A, the adherence-induced tyrosine phosphorylation of pp130, pp120, and pp42 was inhibited by treatment with cytochalasin D in a dose-dependent manner. Three µM cytochalasin D, which was capable of suppressing tyrosine phosphorylation to basal levels (Fig. 4A, lane 6), was also sufficient to depolymerize the network of actin filaments in HSF cells and thereby inhibit cell spreading (data not shown). Along with the failure of tyrosine phosphorylation and cell spreading, adherence-induced MAP kinase activation detected by the kinase assay in MBP-containing gels was completely prevented by treating cells with 3 µM of cytochalasin D (Fig. 4B). Immunoblotting of duplicate filters with alphaY91 confirmed that the same amount of MAP kinases were loaded in each lane (data not shown). These results indicate that the integrity of actin fibers is essential for adhesion-induced MAP kinase activation as well as tyrosine phosphorylation of other proteins.


Figure 4: Inhibition of MAP kinase activation by cytochalasin D. A, Cells bound to PLL (lane 1) or FN (lane 2) in the absence of cytochalasin D, and cells bound to FN (lane 3-6) in the presence of the indicated concentrations of cytochalasin D were lysed, and the supernatants were subjected to SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody. B, extracts from cells adhered to PLL (lane 1), and extracts from cells adhered to FN in the absence (lane 2) and in the presence (lane 3) of cytochalasin D (3 µM) were immunoprecipitated with alphaY91. The immunoprecipitates were subjected on SDS-polyacrylamide gels containing MBP, and MBP kinases assays were performed in the gel.



There is much evidence that MAP kinases may be involved in cell growth and differentiation by phosphorylating and thereby activating nuclear transcriptional factors(20, 21, 22, 24, 25) . Meanwhile, there are many compelling examples of gene expression induced by adhesive interactions with ECM(6, 7, 8, 9) . Thus, MAP kinase may play a pivotal role in adhesion-dependent gene activation through the integrin-mediated signaling pathways. The fact that MAP kinase is activated by both growth factor- and integrin-mediated signals suggests that these two signaling pathways converge at this or upstream points. In this regard, it is interesting to note that pp125 which was the molecule discovered to undergo tyrosine phosphorylation after integrin-mediated signals, has now been found to also undergo tyrosine phosphorylation after stimulation with several growth factors(31) . However, resolving the nature and degree of interactions between integrin- and growth factor-mediated signaling pathways, and their relative contributions, must await a more complete definition and understanding of integrin-mediated signal transduction pathways.

Schaller et al.(19) have recently identified Tyr-397 as the major autophosphorylation site on pp125 both in vivo and in vitro. Of special interest is that tyrosine phosphorylation of this site results in the stable binding of pp125 to the SH2 domain of Src family tyrosine kinases such as pp60 and pp59(18, 19) . They proposed that the association of pp60 and pp59 with pp125 may lead to their enzymatic activation(19) . If this proposed model actually operates in the cell adhesion process, Src family kinases might be a strong candidate in the beta1 integrin signal transduction pathway leading to the MAP kinase activation. So far, there has been no direct evidence that Src kinases are activated by cell adhesion. In this regard, further characterization of pp130 whose tyrosine phosphorylation is enhanced by cell adhesion (Fig. 1) would be important, since pp60 has been shown to be tightly associated with a phosphotyrosyl protein with an apparent molecular weight of 130 kDa(18, 32) . We are currently investigating these possibilities. Such efforts will shed more light on the mechanism of the integrin-mediated signal transduction.


FOOTNOTES

*
This work was supported by Grants-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, and by Japan Rheumatism Foundation Grant for 1993 (to Y. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 03-3815-5411; Fax: 03-5684-3987.

(^1)
The abbreviations used are: ECM, extracellular matrix protein; MAP, mitogen-activated protein; HSF, human skin fibroblast; FN, fibronectin; MBP, myelin basic protein; PLL, poly-L-lysine; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; MHC, major histocompatibility complex.


ACKNOWLEDGEMENTS

We thank Dr. David Rothstein (Yale University, New Haven, CT) for helpful discussions.


REFERENCES

  1. Hemler, M. E. (1990) Annu. Rev. Immunol. 8, 365-400 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hynes, R. O. (1992) Cell 69, 11-25 [Medline] [Order article via Infotrieve]
  3. Juliano, R. L., and Haskill, S. (1993) J. Cell Biol. 120, 577-585 [Medline] [Order article via Infotrieve]
  4. Nojima, Y., Humphries, M. J., Mould, A. P., Komoriya, A., Yamada, K. M., Schlossman, S. F., and Morimoto, C. (1990) J. Exp. Med. 172, 1185-1192 [Abstract]
  5. Shimizu, Y., Van Seventer, G. A., Horgan, K. J., and Shaw, S. (1990) J. Immunol. 145, 59-67 [Abstract/Free Full Text]
  6. Haskill S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr. (1991) Cell 65, 1281-1289 [Medline] [Order article via Infotrieve]
  7. Schmidhauser, C., Bissell, M. J., Myers, C. A., and Casperson, G. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9118-9122 [Abstract]
  8. Dhawan, J., and Farmer, S. R. (1990) J. Biol. Chem. 265, 9015-9021 [Abstract/Free Full Text]
  9. Werb, Z., Tremble, P. M., Behrendtsen, O., Crowley, E., and Damsky, C. H. (1989) J. Cell Biol. 109, 877-889 [Abstract]
  10. Ingber, D. E., Prusty, D., Fragioni, J. V., Cragoe, E. J., Lechene, C., and Schwartz, M. A. (1990) J. Cell Biol. 110, 1803-1811 [Abstract]
  11. Kornberg, L. J., Earp, H. S., Turner, C. E., Prockop, C., and Juliano, R. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8392-8396 [Abstract]
  12. Guan, J.-L., Trevithick, J. E., and Hynes, R. O. (1991) Cell Regul. 2, 951-964 [Medline] [Order article via Infotrieve]
  13. Nojima, Y., Rothstein, D. M., Sugita, K., Schlossman, S. F., and Morimoto, C. (1992) J. Exp. Med. 175, 1045-1053 [Abstract]
  14. Schaller, M. D., Borgman, C. A., Cobb, B. S., Vines, R. R., Reynolds, A.-B., and Parsons, J. T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5192-5196 [Abstract]
  15. Guan, J.-L., and Scalloway, D. (1992) Nature 358, 690-692 [CrossRef][Medline] [Order article via Infotrieve]
  16. Kornberg, L., Earp, H. S., Parsons, J. T., Schaller, M., and Juliano, R. L. (1992) J. Biol. Chem. 267, 23439-23442 [Abstract/Free Full Text]
  17. Akiyama, S. K., Yamada, S. S., Yamada, K. M., and LaFlamme, S. E. (1994) J. Biol. Chem. 269, 15961-15964 [Abstract/Free Full Text]
  18. Cobb, B. S., Schaller, M. D., Leu, T.-H., and Parsons, J. T. (1994) Mol. Cell. Biol. 14, 147-155 [Abstract]
  19. Schaller, M. D., Hildebrand, J. D., Shannon, J. D., Fox, J. W., Vines, R. R., and Parsons, J. T. (1994) Mol. Cell. Biol. 14, 1680-1688 [Abstract]
  20. Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., and Yancopoulos, G. D. (1991) Cell 65, 663-675 [Medline] [Order article via Infotrieve]
  21. Thomas, G. (1992) Cell 68, 3-6 [Medline] [Order article via Infotrieve]
  22. Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892 [Abstract]
  23. Nakielny, S. Cohen, P., Wu, J., and Sturgill, T. W. (1992) EMBO J. 11, 2123-2129 [Abstract]
  24. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
  25. Pulverer, B. J., Kyriakis, J. M., Avruch, J., Nikolakaki, E., and Woodgett, J. R. (1991) Nature 353, 670-674 [CrossRef][Medline] [Order article via Infotrieve]
  26. Tobe, K., Kadowaki, T., Tamemoto, H., Ueki, K., Hara, K., Koshio, O., Momomura, K., Gotoh, Y., Nishida, E., Akanuma, Y., Yazaki, Y., and Kasuga, M. (1991) J. Biol. Chem. 266, 24793-24803 [Abstract/Free Full Text]
  27. Burridge, K., Turner, C. E., and Romer, L. H. (1992) J. Cell Biol. 119, 893-903 [Abstract]
  28. Bockholt, S. M., and Burridge, K. (1993) J. Biol. Chem. 268, 14565-14567 [Abstract/Free Full Text]
  29. Haimovich, B., Lipfert, L., Brugge, J. S., and Shattil, S. J. (1993) J. Biol. Chem. 268, 15868-15877 [Abstract/Free Full Text]
  30. Burridge, K., Fath, K., Kelly, T., Nuckolls, G., and Turner, C. (1987) Annu. Rev. Cell Biol. 4, 487-525 [CrossRef]
  31. Zachary, I., Sinnett-Smith, J., and Rozengurt, E. (1992) J. Biol. Chem. 267, 19031-19034 [Abstract/Free Full Text]
  32. Kanner, S. B., Reynolds, A. B., Wang, H. C., Vines, R. R., and Parsons, J. T. (1991) EMBO J. 10, 1689-1698 [Abstract]

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