Regulation of Integrin-mediated p130Cas Tyrosine Phosphorylation in Human B Cells
A ROLE FOR p59Fyn AND SHP2*

(Received for publication, February 7, 1997, and in revised form, April 3, 1997)

Serge N. Manié Dagger §, Anne Astier Dagger §, Nilou Haghayeghi Dagger §, Timothy Canty Dagger §, Brian J. Druker , Hisamaru Hirai par and Arnold S. Freedman Dagger §**

From the Dagger  Department of Medicine, Harvard Medical School and the § Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, the  Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon 97201, and the par  Third Department of Internal Medicine, University of Tokyo, Hongo, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Engagement of beta 1 integrins in terminally differentiated human B cell lines, such as ARH-77, leads to prominent tyrosine phosphorylation of the p130 Crk-associated substrate (Cas). Cas regulates the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes, which are potentially involved in the propagation of downstream signals. We demonstrate here that immunoprecipitated Cas from beta 1 integrin-stimulated ARH-77 cells was associated with tyrosine kinase and phosphatase activities and that integrin ligation led to the recruitment of at least p59Fyn tyrosine kinase and SHP2 tyrosine phosphatase in Cas immune complexes. Cotransfection studies in COS-7 cells further indicated that Fyn/Cas physical interaction and Fyn-mediated Cas phosphorylation required amino acids 638-889 in the C-terminal region of Cas. This sequence contains both c-Src SH2 and SH3 domain-binding motifs. In vitro binding studies using glutathione S-transferase fusion proteins derived from the SH2 or SH3 domains of Fyn suggested that both Fyn domains can participate in Fyn/Cas interaction. These data implicate Fyn and SHP2 as potential modulators of Cas signaling complexes in B cells.


INTRODUCTION

Integrins are alpha /beta heterodimeric adhesion receptors that are involved in cell/cell and cell/matrix interactions (1, 2). With regard to B lymphocytes, integrins are involved in cell localization within specific microenvironments (3, 4) and in regulating cell survival (5-8). One of the intracellular signaling events initiated by integrins is the activation of a cascade of tyrosine phosphorylation events (9). In many cell types, including B lymphocytes, there is prominent tyrosine phosphorylation of proteins of 105-130 kDa. Several of these substrates have been identified, including p125FAK (FAK (focal adhesion kinase)) (10-14); RAFTK (related adhesion focal tyrosine kinase; also known as PYK2 and CAKbeta ) (15-18); p120c-cbl, the cellular homologue of the oncogene v-cbl (19); p130Cas (Cas (Crk-associated substrate)) (20-24); and the Cas-like molecule p105HEF1 (human enhancer of filamentation 1), also known as Cas-L for lymphocyte-type Cas protein (24, 25).

Cas was originally identified as one of the major tyrosine-phosphorylated proteins in v-crk- or v-src-transformed cells (26, 27). Cas belongs to a new family of structurally related proteins that are thought to act as "docking molecules," i.e. regulating the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes. This family includes three members so far: Cas, HEF1/Cas-L (25, 28), and Efs/Sin (embryonal Fyn-associated substrate/Src-interacting or signal-integrating protein) (29, 30). They all contain an SH3 domain in the N-terminal region; a cluster of SH2 domain-binding motifs that have been named the "substrate domain" (27); and, with the exception of HEF1/Cas-L, several potential binding motifs for SH3 domains.

Following beta 1 integrin cross-linking, Cas phosphorylation was most prominent in B cell lines representative of a more differentiated state, such as the multiple myeloma cell lines ARH-77, IM-9, and RPMI 8226, and was minimally detectable in normal mature B cells (24). In contrast, HEF1 was consistently tyrosine-phosphorylated in all immature, mature, and terminally differentiated B cell lines as well as in normal B cells following both beta 1 integrin and B cell antigen receptor ligation. Therefore, the phosphorylation of these two related molecules appears to be differentially regulated in B cells.

Proteins interacting with Cas include FAK and PTP-1B, which bind to the Cas SH3 domain (31-33); Crk family members, which interact with tyrosine-phosphorylated Cas through SH2 domain-binding motifs (23, 34-36); c-Src, which interacts with tyrosine-phosphorylated Cas through SH2 and SH3 domain-binding motifs in the Cas C-terminal region (37); and the protein-tyrosine phosphatase PTP-PEST (38). Cas also associates with the focal adhesion proteins paxillin and tensin (39). Binding of Crk family members to tyrosine-phosphorylated Cas illustrates the assembly of signaling complexes since the SH3 domain of Crk proteins can bind in turn to a number of proteins, including two guanine nucleotide exchange factors, Sos and C3G, which regulate Ras and Rap1 activation, respectively (40-44). The Cas-Crk-Sos or Cas-Crk-C3G signaling complexes are potentially involved in the propagation of downstream signals.

Studies performed in fibroblasts have shown that c-Src is primarily responsible for integrin-mediated Cas phosphorylation and that FAK might recruit Src family kinases to phosphorylate Cas (34, 35). To date, the identification of Cas-associated Src kinases in nontransfected cells is not well documented. In this study, we present evidence that in the human multiple myeloma cell line ARH-77, p59Fyn tyrosine kinase (Fyn) and SHP2 tyrosine phosphatase are recruited to the Cas complex following integrin ligation. This study provides insight into the control of integrin-mediated tyrosine phosphorylation of Cas in B cells.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

ARH-77 cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum. Antibodies used in this study were directed against the following: CD29/beta 1 integrin (mAb1 K20, provided by Prof. Alain Bernard, U146 INSERM, Nice, France), phosphotyrosine (mAb 4G10), p59Fyn or p130Cas (Cas) (rabbit polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA), GST (GST mAb, Santa Cruz Biotechnology, Santa Cruz, CA), SHP2 (PTP-1D mAb, Transduction Laboratories, Lexington, KY), hemagglutinin (HA mAb, Boehringer Mannheim), and affinity-purified rabbit anti-mouse Ig (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). GST-Fyn fusion proteins were provided by Drs. Joan Brugge and Martyn Botfield (Ariad Pharmaceuticals, Cambridge, MA).

Activation of Cells

ARH-77 cells were stimulated with anti-integrin antibodies plus rabbit anti-mouse Ig as described previously (14). Cells were then lysed in 0.5% Nonidet P-40 buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 mM NaF, and 1 mM sodium vanadate.

Cell Transfection

Fyn cDNA inserted into the pSRalpha 2 expression vector was provided by Dr. Antonio Da Silva (Dana-Farber Cancer Institute) (45). Rat Cas cDNA and deletion mutants of Cas cDNA were cloned into a modified version of the pcDL-SRalpha 296 expression plasmid, termed pSP65-SRalpha .2-HAtag-Hygro, containing a hygromycin B phosphotransferase gene and an HA epitope tag sequence in frame with the Cas cDNAs (46, 47). Briefly, 5'-fragments of the cDNAs with an in-frame XbaI site were generated using polymerase chain reaction and cloned together with the corresponding 3'-fragment into the restriction sites XbaI and EcoRI of pSP65-SRalpha .2-HAtag-Hygro. The expression plasmids were used to transiently transfect COS-7 cells by the DEAE-dextran/Me2SO method as described (48).

Precipitations and in Vitro Kinase Assay

For immunoprecipitation studies, cell lysates were precleared with protein G-Sepharose beads (Pharmacia, Uppsala) and then preincubated with specific antibody for 1 h at 4 °C, followed by the addition of protein G-Sepharose beads for 1 h at 4 °C. For precipitations with GST fusion proteins, lysates were incubated for 2 h at 4 °C with 25 µg of fusion proteins bound to glutathione beads (Pharmacia). Precipitated proteins were washed four times with lysis buffer and subjected to kinase assay or eluted by boiling in sample buffer (2% SDS, 10% glycerol, 0.1 M Tris, pH 6.8, 0.02% bromphenol blue). For sequential immunoprecipitation, washed beads were boiled for 5 min in the presence of 2% SDS, and the supernatants were reprecipitated with antibodies in lysis buffer containing a 0.1% final SDS concentration. In vitro kinase assays were performed by washing Cas immunoprecipitates once in kinase buffer (10 mM Hepes, pH 7.3, containing 50 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, and 100 µM sodium vanadate) and incubating the pellet in kinase buffer containing 0.1 mM ATP (Sigma) for 10 min at room temperature. Proteins were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to Immobilon-PTM membranes (Millipore Corp., Bedford, MA). Membranes were blocked using 5% nonfat dried milk in TBS-T (20 mM Tris, pH 7.6, 130 mM NaCl, 0.1% Tween 20) and incubated for 1 h with specific antibodies in TBS-T. Immunoreactive bands were visualized using secondary horseradish peroxidase-conjugated antibodies (Promega, Madison, WI) and chemiluminescence (ECL, Amersham International, Buckinghamshire, United Kingdom).


RESULTS

In Vivo Association between p59Fyn and Cas following beta 1 Integrin Ligation in the ARH-77 B Cell Line

To examine Cas-associated tyrosine kinase(s), the B cell line ARH-77 was stimulated with the anti-beta 1 integrin mAb K20 followed by rabbit anti-mouse Ig. Cell lysates were then immunoprecipitated with anti-Cas antibody, and subjected (+) or not (-) to an in vitro kinase assay for 10 min (Fig. 1A, upper panels). Anti-phosphotyrosine Western blot analysis showed that Cas immune complexes contained a transient tyrosine kinase(s) activity (maximally detected at 5 and 15 min), which resulted in increased in vitro tyrosine phosphorylation of Cas plus an additional main band ranging from 55 to 60 kDa. The same membrane was reprobed with anti-Cas antibody to show that equivalent amounts of immunoprecipitated Cas were loaded in each lane (Fig. 1A, middle panels). The in vitro phosphorylated 55-60-kDa band contained a sharp 59-kDa protein, which was suggestive of the presence of p59Fyn kinase. To investigate this possibility, the membrane was reprobed with anti-p59Fyn antibody. As shown in Fig. 1A, (lower panels), anti-Fyn antibody reacted with a faint band detectable only in the 5 and 15 min lanes of stimulation. To further confirm the identity of this 59-kDa protein, anti-Cas or control (Ct) immunoprecipitates from 15-min beta 1 integrin-stimulated ARH-77 cells were subjected to in vitro kinase assays (Ip 1), and half of the samples were then reimmunoprecipitated with anti-Fyn antibody (Ip 2). As shown in Fig. 1B (lower panel), Fyn could be reimmunoprecipitated from Cas immune complexes, but not from control immunoprecipitates. Fyn was tyrosine-phosphorylated (Fig. 1B, upper panel) and comigrated with the sharp pp59. These results indicate that Fyn kinase associates in an integrin-regulated manner with Cas and strongly suggest that it participates in Cas phosphorylation.


Fig. 1. In vivo association between p59Fyn and Cas following beta 1 integrin ligation in the ARH-77 B cell line. A, the human B cell line ARH-77 was stimulated with anti-beta 1 integrin antibodies plus rabbit anti-mouse Ig and then lysed as a function of time. Cell lysates were immunoprecipitated (IP) with anti-Cas antibody and subjected (+) or not (-) to an in vitro kinase assay (IVKA). Samples were then immunoblotted with anti-phosphotyrosine antibody (P-Tyr; upper panels), stripped, and reblotted with anti-Cas antibody (middle panels) or antiserum to Fyn (lower panels) as indicated. B, lysates of 15-min anti-beta 1 integrin-stimulated ARH-77 cells were first immunoprecipitated (Ip 1) with anti-Cas antibody (Cas) or an irrelevant control antibody (Ct) and subjected to an in vitro kinase assay. A second immunoprecipitation (Ip 2) with anti-Fyn antibodies (Fyn) was then performed on eluates of the first immunoprecipitations. All eluates were then analyzed by Western blotting with anti-Tyr(P) antibody (upper panels) and after stripping, with anti-Cas (middle panel) or anti-Fyn (lower panel) antibodies as indicated. The positions of Cas and Fyn proteins and immunoglobulin (bracket) as well as molecular mass markers (in kDa) are shown. Blots were imaged by chemiluminescence.
[View Larger Version of this Image (74K GIF file)]

Tyrosine Phosphorylation of Cas by Coexpression with Fyn Kinase in COS-7 Cells Requires the Src-binding Sites in the C-terminal Region of Cas

The C terminus of Cas contains a proline-rich sequence (RPLPSPP) and a YDYV motif, which have been shown to bind to Src SH3 and SH2 domains, respectively (37). Since we have previously reported that a GST fusion protein containing the RPLPSPP sequence bound to Fyn (24), we further examined the requirement of this region of Cas for Fyn/Cas interaction. cDNAs encoding HA-tagged wild-type Cas or deletion mutants of Cas were transiently expressed in COS-7 cells in the presence or absence of cDNA encoding Fyn. The mutants of Cas included the following: Cas Delta SD, in which the sequence from amino acids 213 to 514, which contains the substrate domain with 15 out of 27 potential sites of tyrosine phosphorylation within Cas, was deleted; and Cas Delta SB, in which the sequence from amino acids 638 to 889, which contains the Src SH2 and SH3 domain-binding motifs, was deleted (37) (Fig. 2). To discriminate transfected Cas proteins from endogenous Cas proteins, lysates of transfected COS-7 cells were immunoprecipitated with anti-HA tag antibody. Anti-phosphotyrosine immunoblotting of the anti-HA tag immunoprecipitations indicated that the presence of cotransfected Fyn led to Cas phosphorylation (Fig. 3A). The Cas Delta SD mutant, although lacking some of the potential sites of tyrosine phosphorylation, showed increased tyrosine phosphorylation in the presence of Fyn. In contrast, the Cas Delta SB mutant cotransfected with Fyn demonstrated reduced tyrosine phosphorylation when compared with Cas or Cas Delta SD. Therefore, the SB domain of Cas was necessary for Fyn-mediated Cas phosphorylation. Comparable levels of expression of Cas, Cas mutants (Fig. 3B), and Fyn (Fig. 3D) were detected regardless of the cotransfection conditions. More important, Fyn kinase was co-immunoprecipitated with Cas and Cas Delta SD, but not with Cas Delta SB (Fig. 3C), indicating that the SB region was required for a physical interaction between Fyn and Cas.


Fig. 2. Schematic diagram representing the various p130Cas constructs used. Shown are intact Cas; Cas Delta SD, in which the substrate domain containing 15 YXXP motifs was deleted; and Delta SB, in which the C-terminal domain containing the Src SH3-binding motif (RPLPSPP) and the Src SH2-binding motif (YDYV) was deleted.
[View Larger Version of this Image (16K GIF file)]


Fig. 3. Tyrosine phosphorylation of Cas by coexpression with Fyn kinase in COS-7 cells requires the Src-binding site of Cas. COS-7 cells were transiently transfected with control plasmid (Mock) or with plasmid encoding HA-tagged wild-type Cas (Cas WT), HA-tagged Cas with the Src-binding site deleted (Delta SB), or HA-tagged Cas with the substrate domain deleted (Delta SD). A second set of cells were also cotransfected with a plasmid encoding Fyn (+ Fyn). Transfected Cas was immunoprecipitated (Ip) using anti-HA antibodies and analyzed by Western blotting with anti-Tyr(P) antibody (P-Tyr) (A). The positions of Cas, Cas Delta SB, Cas Delta SD, and Fyn are indicated. The membrane was then stripped and reblotted with anti-Cas (Cas; B) or anti-Fyn (Fyn; C) antibodies as indicated. Cotransfected cell lysates were also immunoprecipitated with anti-Fyn antibodies to control for the amount of Fyn expression within the cells (D).
[View Larger Version of this Image (36K GIF file)]

In Vitro Interaction of Cas with Fyn SH2 and SH3 Domains

To further study the interaction between Cas and Fyn, we performed binding experiments with truncated GST-Fyn fusion proteins corresponding to the SH2 or SH3 domain of Fyn. Fusion proteins were incubated with lysates of unstimulated (Fig. 4A, 0 lanes) or beta 1 integrin-stimulated (beta 1 lanes) ARH-77 cells, and the presence of Cas was analyzed by immunoblotting. To control for the integrin-mediated Cas tyrosine phosphorylation, anti-phosphotyrosine immunoprecipitations from the same samples were also analyzed. Fig. 4A shows that Cas did not bind to GST protein alone. In contrast, Cas that was derived from stimulated cells was able to bind to the GST-Fyn SH2 domain, whereas Cas from both unstimulated and stimulated cells bound to the GST-Fyn SH3 domain. In this experiment, Cas was seen to migrate as 105- and 130-kDa bands as described previously (24), with the main increased phosphorylation in the 130-kDa form. Hyperphosphorylated Cas precipitated with anti-phosphotyrosine antibody or GST-Fyn SH2 protein resolved with a slower migration (27). The membrane was reprobed with anti-GST antibody to show that comparable amounts of GST fusion proteins were used to precipitate Cas (Fig. 4B). These results indicate that the Fyn SH3 domain binds to Cas in vitro and that integrin-stimulated phosphorylation of Cas creates a binding site for the Fyn SH2 domain.


Fig. 4. Interaction of Cas in vitro with Fyn SH2 and SH3 domains. ARH-77 cells were unstimulated (0 lanes) or stimulated for 15 min with cross-linked anti-beta 1 integrin antibody (beta 1 lanes), and cell lysates were either immunoprecipitated with anti-Tyr(P) antibody (P-Tyr) or precipitated with the following GST fusion proteins as indicated: GST alone, the GST-Fyn SH2 domain, and the GST-Fyn SH3 domain. Eluates were immunoblotted with anti-Cas antibody (Cas; A), stripped, and reblotted with anti-GST antibodies (GST; B).
[View Larger Version of this Image (46K GIF file)]

In Vivo Association of SHP2 Tyrosine Phosphatase with Cas following Integrin Ligation

To investigate the presence of a Cas-associated tyrosine phosphatase, Cas immunoprecipitates from beta 1 integrin-stimulated ARH-77 cells were subjected to an in vitro kinase assay with or without the tyrosine phosphatase inhibitor sodium vanadate (Fig. 5A). The tyrosine phosphorylation of Cas was markedly increased with the addition of sodium vanadate in the kinase assay. These results indicate that a tyrosine phosphatase activity is associated with Cas immunoprecipitates. Because the tyrosine phosphatase SHP2 has been shown recently to associate through its SH2 domains with the Cas-related Cas-L (HEF1) protein (25), we tested whether SHP2 also associated with Cas. Unstimulated or beta 1 integrin-stimulated ARH-77 cells were immunoprecipitated with anti-Cas (Cas) or control (Ct) antibodies and immunoblotted with anti-SHP2 antibody (Fig. 5B). Compared with unstimulated cells and control immunoprecipitates, a 72-kDa band reactive with anti-SHP2 antibody was clearly increased in Cas complexes isolated from beta 1 integrin-stimulated cells. ARH-77 cells were then beta 1 integrin-stimulated for 0, 2, 5, and 15 min, and cell lysates were immunoprecipitated with anti-Cas antibody (Fig. 5C). Cas quantification was comparable at 2, 5, and 15 min and showed increased tyrosine phosphorylation, which correlated with Cas-SHP2 complex formation, which was increased at the 5- and 15-min time points. These results suggest that similar to HEF1/Cas-L, SHP2 associates with Cas in an integrin-regulated manner. Therefore, SHP2 is likely to be associated with the phosphatase activity observed in vitro and might play a role in the in vivo control of Cas dephosphorylation.


Fig. 5. In vivo association of SHP2 tyrosine phosphatase with Cas following integrin ligation. A, ARH-77 cells were stimulated for 15 min with anti-beta 1 integrin antibody plus rabbit anti-mouse Ig, and cell lysates were immunoprecipitated (Ip) with anti-Cas antibody (Cas). Cas immunoprecipitates were then subjected to an in vitro kinase assay in the absence (-) or presence (+) of Na3VO4 and analyzed by Western blotting with anti-Tyr(P) antibody (P-Tyr). The same membrane were stripped and reblotted with anti-Cas antibody as indicated. B, 15-min beta 1 integrin-stimulated ARH-77 cells (beta 1 lanes) or unstimulated cells (0 lane) were immunoprecipitated with anti-Cas antibody or an irrelevant control antibody (Ct) and analyzed by Western blotting with anti-Tyr(P) antibody (left panel). The membrane was then stripped and reblotted with anti-Cas or anti-SHP2 antibodies (right panels) as indicated. The position of SHP2 is indicated by an arrow. C, ARH-77 cells were stimulated as indicated with anti-beta 1 integrin antibody and lysed as a function of time. Anti-Cas immunoprecipitates were analyzed with anti-Tyr(P) antibody (left panel). The membrane was then stripped and reblotted with anti-Cas or anti-SHP2 antibodies (right panels) as indicated. The position of SHP2 is indicated by an arrow.
[View Larger Version of this Image (46K GIF file)]


DISCUSSION

Integrins are involved in the regulation of proliferation, differentiation, and cell survival in a variety of cell types, events that are dependent upon tyrosine phosphorylation (49). Cas has been identified to be a major tyrosine-phosphorylated substrate following integrin ligation (20-24). In this study, we found that precipitations of Cas immune complexes from integrin-stimulated cells contained both tyrosine kinase and tyrosine phosphatase activities, which modulated the in vitro phosphorylation of Cas. Furthermore, Fyn tyrosine kinase and SHP2 tyrosine phosphatase were recruited in Cas complexes, suggesting that they participate in modulating Cas phosphorylation.

The C-terminal proline-rich region of Cas can associate in vitro with several Src kinases, including p59Fyn, p59/62Hck, and p53/56Lyn (24). In this study, we found that in ARH-77 cells, Fyn was the most obvious kinase detectable. The focal adhesion kinases FAK and RAFTK can associate with Cas (18, 31, 32, 36). In B cells, RAFTK and, to a lesser extent, FAK are both tyrosine-phosphorylated following integrin ligation (14, 18). Similar to Src kinases, the kinase activity of RAFTK and FAK correlates with an increase in autophosphorylation activity in certain cell types (15, 50). However, we previously reported that Cas associated with RAFTK is mainly nonphosphorylated on tyrosine residues (18). In addition, we did not observe tyrosine-phosphorylated bands corresponding in size to RAFTK and FAK, 120 and 125 kDa, respectively. This results suggest that in nontransfected cells and under the conditions of these assays, these two kinases may not be sufficient for Cas phosphorylation. Further support of this is the observation that Cas phosphorylation is reduced in fibroblasts lacking Src kinases, but remains unaffected in fibroblasts lacking FAK (34, 35).

In a cotransfection assay in COS-7 cells, we found that the Fyn/Cas physical interaction and subsequent Fyn-mediated Cas phosphorylation required amino acids 638-889 in the C-terminal region of Cas. This sequence contains a polyproline stretch (RPLPSPP) and a YDYV motif to which both Fyn SH3 and SH2 domains could bind in vivo. In vitro studies using GST fusion proteins derived from the SH2 and SH3 domains of Fyn indicated that both domains could participate in this Fyn/Cas interaction. However, the in vivo association of Fyn and Cas was transient and correlated with Cas tyrosine phosphorylation, implying a regulation of this interaction. Whether the Fyn SH3 domain is primarily involved in Cas binding or is subsequent to Fyn SH2 binding to reinforce the interaction between the two proteins is not known. A precedent for the predominant binding of the SH2 domain over the SH3 domain has been described for the interaction of p120Cbl with phosphoinositol 3'-kinase (51).

The Cas-like protein Sin leads to activation of Src kinase activity on binding of Sin to the c-Src SH3 domain (30). Similarly, it is possible that Fyn binding to Cas will stimulate the kinase activity of Fyn, which in turn will lead to the effective phosphorylation of Cas and allow the recruitment of SH2 domain-containing molecules such as the adapter proteins of the Crk family (23, 24).2 In support of this is the finding that Fyn-phosphorylated Cas in COS cells interacts with Crk (data not shown). The assembly of this complex of proteins might further enable recruited proteins to interact with and potentially be phosphorylated by Fyn.

Since the phosphorylation of Cas is likely to be an important event in the propagation and amplification of downstream signals of integrin ligation, a mechanism of down-regulating that process is critical. A role for SHP2 tyrosine phosphatase in modulating the phosphorylation of Cas in B cells came from the finding that SHP2 was recruited in Cas complexes following integrin ligation. The Cas-SHP2 association correlated with the extent of Cas tyrosine phosphorylation. In view of the recent finding that SHP2 associates with HEF1/Cas-L through its SH2 domains (25), it is possible that a similar mechanism occurs for Cas-SHP2 assembly. This might result in activation of SHP2 activity since engagement of its SH2 domains following binding to platelet-derived growth factor receptor beta  stimulates its tyrosine phosphatase activity (52). Two other protein-tyrosine phosphatases, namely PTP-1B and PTP-PEST, can also associate with Cas (33, 38) and might participate in the Cas-associated phosphatase activity.

The precise mechanism by which integrin cross-linking leads to the activation of the kinase(s) and phosphatase(s) is presently unclear. Integrin-mediated phosphorylated Cas localizes to focal adhesions, whereas nonphosphorylated Cas remains in the cytosol (22, 27). Such Cas localization may be driven by its constitutive association through its SH3 domain with FAK, due to the FAK C-terminal focal adhesion targeting domain (50). Therefore, redistributed Cas during integrin stimulation might localize Cas to a region of the cell where Cas will be accessible to tyrosine kinases. Support for this hypothesis is that optimal Cas phosphorylation requires an intact cytoskeleton since inhibitors of cytoskeletal assembly also inhibit integrin-mediated Cas tyrosine phosphorylations (20, 24). A potential sequence of events leading to Cas phosphorylation in ARH-77 cells could be that integrin-mediated cytoskeletal reorganization allows the co-compartmentalization of Cas and Fyn. Activated Fyn, possibly by its binding to Cas, phosphorylates Cas, leading to the formation of a signaling complex including Crk family members and SHP2. SHP2, which becomes activated following SH2-mediated binding to phosphorylated Cas, could then participate in downstream signaling events and/or attenuate the activity of the complex by dephosphorylating Cas.

Cas and the Cas-like protein HEF1 are both expressed in normal B cells and most B cell lines (24). However, Cas is the predominant substrate that is phosphorylated in terminally differentiated B cell lines following beta 1 integrin ligation, whereas HEF1 is the major phosphorylated substrate in normal tonsillar B cells and other B cell lines. It is unknown why Cas or HEF1 is the favored substrate in certain cells, although this may be related to the compartmentalization and activation of specific kinases during integrin-induced cytoskeleton reorganization.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants CA55207 and CA66996, American Cancer Society Grant DHP-145, and a fellowship from the Lymphoma Foundation of America (to S. N. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
**   To whom correspondence and reprint requests should be addressed: Div. of Hematologic Malignancies, Dana-Farber Cancer Inst., 44 Binney St., Boston, MA 02115. Tel.: 617-632-3441; Fax: 617-632-5167; E-mail: arney_freedman{at}dfci.harvard.edu.
1   The abbreviations used are: mAb, monoclonal antibody; GST, glutathione S-transferase; HA, hemagglutinin.
2   Astier, A., Manié, S. N., Law, S. F., Canty, T., Hagheyeghi, N., Druker, B. J., Salgia, R., Golemis, E. A., and Freedman, A. S. (1997) Leukemia Lymphoma, in press.

ACKNOWLEDGEMENTS

We thank Prof. Alain Bernard for K20 antibody, Drs. Joan Brugge and Martyn Botfield for Fyn constructs, and Dr. Antonio Da Silva for providing Fyn cDNA. We greatly appreciate Dr. Andreas Beck for help in preparing Cas constructs. We also thank Drs. Andreas Beck, Antonio Da Silva, Bernard Mari, Susan Law, and Erica Golemis for helpful discussions. We thank Janet Walsh for assistance in preparing the manuscript.


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