Calcium-dependent Signaling Pathways in T Cells

Engagement of β1 integrin receptors initiates an increase in intracellular calcium concentrations in T cells, potentially affecting calcium-sensitive signaling pathways. The calcium-activated cysteine protease, calpain, regulates a variety of cell functions by calcium-dependent limited proteolysis. To investigate the function of calpain in T cells, we sought to determine the role of this protease in calcium-dependent signaling events. Subsequent to elevations in intracellular calcium concentrations induced by ionomycin or adherence to fibronectin, calpain activity translocated to the cytoskeletal/membrane fraction of T cells. In addition, stimulation of T cells with these agents initiated the proteolytic cleavage of protein tyrosine phosphatase 1B by calpain. Enzymatic cleavage of protein tyrosine phosphatase 1B occurs near the endoplasmic reticulum-targeting sequence and results in the generation of an enzymatically active form of the phosphatase. Furthermore, we show that both the native and the cleaved forms of protein tyrosine phosphatase 1B interact with p130Cas in T cells. This interaction may serve to relocate protein tyrosine phosphatase 1B to sites of focal contact resulting in potential interactions with substrates previously inaccessible to the endoplasmic reticulum-associated phosphatase. Thus, we describe a novel calcium-dependent signaling pathway in T cells that may mediate signals generated by β1 integrin adherence to the extracellular matrix.

Calpain (calcium-activated cysteine protease; EC 3.4.22.17) is ubiquitously expressed in mammalian cells, yet its precise physiologic function in T cells remains to be determined. The majority of calpain is localized in the cytosol of cells (1)(2)(3)(4). However, raising intracellular calcium concentrations ([Ca 2ϩ ] i ) 1 in cells induces calpain translocation to the plasma membrane, the cytoskeleton, and points of attachment between cells and the extracellular matrix (ECM) (5)(6)(7)(8)(9). Furthermore, interactions between integrins and ECM proteins in some cell types, induces the translocation and activation of calpain in response to elevated [Ca 2ϩ ] i (5, 10 -12). Calpain can induce the proteolytic modification of proteins associated with multiple signaling cascades, such as protein kinase C, phospholipase C, pp60 Src , and focal adhesion kinase (13)(14)(15)(16) as well as the limited proteolysis of cytoskeletal proteins including talin, actinbinding protein, and paxillin (17)(18)(19). Thus, calpain is implicated in cytoskeletal reorganization as well as signal transduction pathways in response to elevations in [Ca 2ϩ ] i . This suggests that calpain has a crucial role in integrin-induced alterations in cellular physiology and function. Interaction of integrins with their ligands activates cellular signaling pathways and reorganization of the cytoskeletal system (20 -24). An increase in [Ca 2ϩ ] i represents one second messenger that is quantitatively altered as a consequence of these interactions. This would further suggest the possibility that calpain is activated by the rise in [Ca 2ϩ ] i occurring as a consequence of integrins binding to their ligands. Indeed, there is evidence for this occurring in platelets (10,11). In lymphocytes, ␤1 integrins mediate important interactions with the ECM, yet the biochemical signals transduced to the intracellular biomolecular systems and pathways that modify T cell function are only now beginning to be elucidated (21,24). T cells express ␣4␤1 and ␣5␤1 integrins, which are the principal receptors for the ECM protein fibronectin (FN) (25). Engagement of these receptors with their appropriate ligand results in increased [Ca 2ϩ ] i , suggesting a role for integrin-mediated calcium signaling in T cells (26,27).
In the present study, we have used a calcium ionophore, ionomycin (IONO), to investigate the role of calpain in integrinmediated signaling pathways in T cells. Our studies indicate that calpain undergoes translocation and activation in response to elevations in [Ca 2ϩ ] i resulting in proteolytic cleavage of protein tyrosine phosphatase 1B (PTP-1B). Moreover, we show that translocation and activation of calpain induces proteolytic cleavage of PTP-1B in Jurkat T cells binding the ECM protein FN. In addition, PTP-1B associates with p130 Cas in T cells. Cas may serve as a targeting protein to relocate the proteolytically modified PTP-1B to sites of cell contact resulting in potential interactions with substrates previously inaccessible to the endoplasmic reticulum (ER)-associated form of the PTPase. Thus, we describe a novel signaling pathway in T cells that may be involved in integrin-mediated signaling cascades.
Isolation of T cells-Heparinized venous blood obtained from healthy adult donors was separated on a Ficoll-Hypaque gradient; lymphocytes were harvested, and T cells were isolated by sheep erythrocyte rosetting (28).
Immunoblotting-T cells or Jurkat T cells (2 ϫ 10 6 ), in a final volume of 200 l of RPMI 1640 unless otherwise indicated, were warmed in a 37°C water bath for 5 min prior to addition of stimulants. In some experiments calpeptin (100 M) was added 15 min prior to stimulation. After incubation for the indicated times, cells were pelleted, resuspended in 50 -100 l of Laemmli sample buffer (Bio-Rad), and boiled 5 min. The lysates were microcentrifuged for 3 min and separated on 10% SDS-PAGE unless otherwise indicated. Insoluble fractions were isolated and suspended in Laemmli sample buffer followed by brief sonication, boiling, and separation on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane (0.22 m; Costar, Cambridge, MA), probed with the indicated primary antibody, and followed by horseradish peroxidase-coupled secondary antibody of the appropriate specificity. Immunoreactivity was determined using the ECL chemiluminescence reaction (Amersham Life Science, Inc.).
Calpain Activity Assay-T cells or Jurkat T cells (4 ϫ 10 6 ) in a final volume of 400 l of RPMI 1640 were stimulated as described under Immunoblotting. After stimulation, cells were pelleted and lysed with 100 l of Tris-HCl (pH 7.3) containing 0.1% Triton X-100 on ice for 10 min and microcentrifuged for 10 min at 4°C. The supernatant (detergent soluble fraction) was transferred to a new tube, and the pellet (detergent insoluble fraction) was resuspended in 100 l of lysis buffer. These fractions were transferred to a 96-well plate (round bottom, Costar) and 100 M calpain substrate (N-succinyl-Leu-Tyr-7-amido-4methylcoumarin, Sigma) added to each well. Plates were incubated at 37°C for 30 min and mean fluorescence measured on a Cytofluor plate reader (excitation: 360 nm, emission: 460 nm).
Immune Complex Phosphatase Assay-Jurkat T cells (20 ϫ 10 6 ) in a final volume of 1 ml RPMI 1640 were unstimulated or stimulated with IONO (1 M) for 10 min and lysed in 0.5% Triton X-100 lysis buffer without sodium orthovanadate as described above. Lysates (soluble fraction) were transferred to fresh tubes and the insoluble fraction was resuspended in radioimmune precipitation lysis buffer. PTP-1B immunocomplexes were isolated as described above, washed 4 times in phosphatase assay buffer (Tris-HCl (pH 7.4)), and resuspended in 60 l of phosphatase assay buffer. The resulting samples were subjected to an immune complex phosphatase assay using protein tyrosine phosphatase assay kit #2 (Upstate Biotechnology) per manufacturer's directions.
In Vitro Calpain Proteolysis of PTP-1B-PTP-1B was immunoprecipitated from Jurkat T cells with goat anti-PTP-1B, immunocomplexes were washed three times in calpain assay buffer (200 mM imidazole (pH 7.5), 10 mM cysteine-HCl) and resuspended in the same buffer. Each 150-l reaction contained either 1 unit of calpain I or calpain II (20 mM MOPS (pH 7.0), 0.5 mM NaCl, 1 mM each EDTA and EGTA, and 5 mM 2-mercaptoethanol). Samples were placed at 30°C, and CaCl 2 (1 mM) was added to initiate the reaction. Aliquots (25 l) were removed at the indicated times, and the reaction was terminated by addition of 25 l of Laemmli sample buffer and boiled for 5 min. The proteins were resolved on SDS-PAGE and analyzed by immunoblotting.
Incubation of T cells with Fibronectin-Culture plates (6-well, Costar) were coated overnight at 4°C with 10 g/ml fibronectin or with 100 g/ml poly-L-lysine in phosphate-buffered saline (pH 7.4) per well. Plates were washed three times in phosphate-buffered saline, blocked with 5% non-fat dry milk for 2 h at room temperature, and washed three times in diphosphate-buffered saline. Jurkat T cells (5 ϫ 10 6 ) in a final volume of 1.5 ml RPMI 1640 were added to each well in the presence of PMA (50 ng/ml), and the plates were incubated at 4°C for 30 min. Plates were warmed rapidly in a humidified atmosphere of 5% CO 2 in air at 37°C for 30 min, cells harvested and counted. Samples were adjusted to equal cell numbers and analyzed for calpain activity or by immunoblot analysis as described.

Translocation of Calpain Activity to the Membrane/Cytoskeletal Fraction of T Cells-Experiments
were done to quantitate calpain activity and translocation in T cells stimulated with various agents. T cells were stimulated with IONO and/or PMA, lysed, separated into soluble (cytosolic) and insoluble (membrane/cytoskeletal) fractions and assayed for calpain activity. In resting T cells, calpain activity was readily detectable in the soluble fraction, but very little activity was associated with the insoluble fraction ( Fig. 1), suggesting that this protease is predominantly a cytosolic protein under resting conditions. However, T cells or Jurkat T cells (data not shown) stimulated with IONO alone or with PMA had a decrease in calpain activity in the soluble fraction ( Fig. 1a), but a corresponding increase in calpain activity was associated with the insoluble fraction (Fig. 1b). Stimulation of T cells with IONO resulted in approximately a 5-fold increase in calpain activity associated with the cytoskeletal/membrane fraction. Conversely, stimulation of T cells with PMA alone did not alter calpain activity associated with either cellular fraction nor did PMA synergize with IONO. The increase in calpain activity induced by IONO stimulation of T cells requires extracellular Ca 2ϩ because chelation of extracellular Ca 2ϩ ions by EGTA inhibited IONO induced increase in calpain activity associated with either fraction (Fig. 1a, b). Furthermore, thapsigargin, which depletes intracellular stores of Ca 2ϩ , failed to alter calpain activity in T cells (data not shown). The specificity of the calpain assay was shown by the addition of the calpain inhibitor, calpeptin, to isolated cellular fractions ( Fig. 1) or by preincubation of T cells with calpeptin prior to the addition of stimuli (data not shown).
PTP-1B Is Cleaved by Calpain in Ionomycin-stimulated T Cells-Further experiments were done to determine potential substrates for calpain in stimulated T cells. Jurkat T cells were stimulated with PMA and IONO, IONO alone, or PMA alone, cell lysates prepared and subjected to SDS-PAGE, followed by immunoblot analysis with appropriate monoclonal antibodies. One protein of interest that underwent proteolytic modification in response to PMA and IONO, IONO alone, but not PMA alone, was PTP-1B (Fig. 2a). In nonstimulated Jurkat T cells (Fig. 2, lane 1), PTP-1B migrated as a 50-kDa band corresponding to the full-length form of the phosphatase. However, IONO stimulation of T cells induced the appearance of a modified form of PTP-1B that showed increased electrophoretic mobility and an apparent molecular weight of 42-kDa (Fig. 2a, lane 3). This modification of PTP-1B was inhibited by calpeptin (Fig.  2b, lane 3) or by EGTA implicating the action of calpain (Fig.  2b, lane 4). Furthermore, cleavage of PTP-1B was rapid and dose-dependent with the appearance of the cleaved form of PTP-1B within 1 min after IONO stimulation of Jurkat T cells and T cells (Fig. 3, a and b, lane 2) and at concentrations as low as 50 nM (Fig. 3c, lane 6). These results clearly indicate that conditions that induce calpain activity are also associated with the proteolytic cleavage of PTP-1B in IONO-stimulated T cells.
PTP-1B Is an in Vitro Substrate of Both Calpain I and II-The following experiments were done to determine whether calpain I or calpain II is responsible for the proteolytic cleavage of PTP-1B. PTP-1B was immunoprecipitated from Jurkat T cells and used as a substrate for either calpain I or calpain II. Initially, PTP-1B migrated as a 50-kDa protein corresponding to the full-length form of the phosphatase (Fig. 4, a and b, lane  1). However, after the addition of calcium to initiate calpain I or calpain II proteolytic activity, PTP-1B migrated as a 42-kDa protein (Fig. 4, a and b, lanes 2-4). These in vitro results are identical to the calpain-catalyzed cleavage of PTP-1B observed after stimulation of T cells with IONO. Cleavage of PTP-1B by calpain was also extremely rapid in that no full-length PTP-1B was detectable within 1 min of calpain activation. These results indicate that PTP-1B is a substrate for both calpain I and calpain II.
Proteolytic Cleavage of PTP-1B in T Cells Results in a Soluble Enzymatically Active Form of the Phosphatase-PTP-1B is a ubiquitously expressed 50-kDa nontransmembrane PTP that localizes to the ER via a 35-amino acid C-terminal hydrophobictargeting sequence (29,30). This orients the N-terminal enzymatic domain of PTP-1B toward the cytosol and would restrict the number of substrates to which PTP-1B has access. Proteolytic cleavage of PTP-1B by calpain in IONO-stimulated T cells resulted in a 42-kDa form of the phosphatase. To evaluate at which terminus calpain modifies PTP-1B, Jurkat T cells were stimulated with IONO, lysates were prepared, and immunoblot analysis was performed using antibodies specific for either the N or C terminus of the phosphatase. The 42-kDa cleaved form of PTP-1B was recognized by polyclonal antibodies raised against a peptide corresponding to amino acids 4 -22 mapping at the amino terminus of PTP-1B (Fig. 5a, lane 2), but not by polyclonal antibodies directed against amino acids 387-405 mapping at the carboxyl terminus of the phosphatase (Fig. 5b,  lane 2). These data indicate that the 42-kDa form of PTP-1B was a result of calpain cleaving the phosphatase near the ER-targeting C-terminal sequence.
Next experiments were done to determine the cellular location and enzymatic activity of PTP-1B in IONO-stimulated Jurkat T cells. PTP-1B was immunoprecipitated from the soluble and insoluble fractions obtained from stimulated T cells and subjected to an immune complex phosphatase assay. Aliquots from each sample were removed and subjected to immunoblot analysis. The results revealed that most of the 42-kDa PTP-1B was found in the detergent soluble fraction, as well as a small fraction of the 50-kDa intact form of the phosphatase (Fig. 6, insert). The presence of the intact phosphatase in the soluble fraction is probably the result of disruption of the ER membrane, although it is possible that a fraction of intact PTP-1B localizes to sites other than the ER in T cells. The enzymatic activity of PTP-1B associated with the soluble fraction increased 2-fold in IONO-stimulated cells and corresponded to the increased PTP-1B protein content associated with this fraction resulting from calpain catalyzed cleavage (Fig. 6). Conversely, PTP-1B activity associated with the insoluble fraction was slightly higher in unstimulated cells as compared with IONO-stimulated cells, corresponding to decreased PTP-1B associated with this fraction as a result of calpain modification (Fig. 6). Total phosphatase activity was increased in IONO-stimulated cells as compared with unstimulated cells suggesting the cleaved form of PTP-1B (42-kDa) exhibited increased enzymatic activity as a result of proteolytic cleavage.
These experiments show that calpain cleaves PTP-1B near the C terminus, resulting in a soluble enzymatically active form of the phosphatase. Notably, a small fraction of the 42-kDa form of PTP-1B was found consistently to associate with the insoluble fraction after IONO stimulation of Jurkat T cells or T cells (Fig. 6, insert and data not shown). Association of the 42-kDa form of PTP-1B with the insoluble fraction suggests translocation to the plasma membrane or the cytoskeleton allowing the phosphatase to interact with potential substrates. Thus, proteolytic cleavage of PTP-1B by calpain in response to increased [Ca 2ϩ ] i may modify the substrate specificity of the phosphatase by altering intracellular localization.
Intact 50-kDa and Cleaved 42-kDa Forms of PTP-1B Associate with p130 Cas -Experiments were done to determine whether intact or cleaved forms of PTP-1B associated with p130 Cas . PTP-1B was immunoprecipitated from either unstimulated or IONO-stimulated T cells or Jurkat T cells and precipitates examined for the co-precipitation of p130 Cas (Fig.  7). Anti-PTP-1B immunoprecipitates contained an approximately 110-kDa protein corresponding to the molecular weight of p130 Cas in T cells (31), and it is recognized by anti-p130 Cas antibodies. This band was not observed in control precipitates (Fig. 7, lane 1) and migrates in a manner similar to p130 Cas present in T cell lysates (Fig. 7, lane 6). Furthermore, p130 Cas was co-precipitated with anti-PTP-1B from unstimulated (Fig.  7, lanes 2 and 4) and IONO-stimulated (Fig. 7, lanes 3 and 5) cells, but anti-PTP-1B precipitates from IONO-stimulated cells co-precipitated less p130 Cas than that observed in resting cells. These data suggest that PTP-1B and p130 Cas interact in T cells. However, these experiments do not address whether this association is maintained after the proteolytic modification of PTP-1B by calpain.

FIG. 4. PTP-1B is an in vitro substrate of both calpain I and II.
PTP-1B was immunoprecipitated from Jurkat T cells (20 ϫ 10 6 ), and incubated in the presence of 1 unit of either purified human placental calpain I (a) or calpain II (b) at 30°C in calpain assay buffer (see "Experimental Procedures"). The calpain reaction was initiated by the addition of 1 mM CaCl 2 to each sample. Aliquots (25 l) were removed at indicated intervals, and the reaction was terminated by boiling in equal volumes of 2 ϫ Laemmli sample buffer. Cleavage of PTP-1B was analyzed by immunoblotting. Nitrocellulose membranes were immunoblotted with anti-PTP-1B mAb. However, immunoprecipitates obtained using irrelevant antibody (Fig. 8, lane 1) of the same isotype also contain a band of the same molecular weight. We believe that the reagents used in protein visualization were detecting the immunoglobulin heavy chain used in immunoprecipitating p130 Cas , thus, masking any 50-kDa form of PTP-1B that co-precipitates with p130 Cas . Importantly, anti-p130 Cas immunoprecipitates from IONO-stimulated cells contained an additional protein of 42-kDa that was recognized by anti-PTP-1B antibodies (Fig. 8,  lane 3). This band was not present in isotype-matched irrelevant antibody immunoprecipitates of IONO-stimulated T cells (Fig. 8, lane 1) or in anti-p130 Cas immunoprecipitates of unstimulated T cells. Thus, PTP-1B interacts with p130 Cas in resting T cells, and this interaction can be maintained in response to cleavage of PTP-1B by calpain.
Incubation of T Cells with Fibronectin Induces Calpain-cat-alyzed Cleavage of PTP-1B-Because integrin-mediated signaling events are associated with increased [Ca 2ϩ ] i (26,27), calpain activation (10,11), and cytoskeletal rearrangement (23), we determined if adhesion of T cells to the ECM protein FN would induce calpain translocation and proteolytic cleavage of PTP-1B. Jurkat T cells were stimulated with PMA and adhered to poly-L-lysine or FN coated plates. Cells plated on poly-Llysine displayed very little calpain activity associated with the insoluble fraction (Fig. 9). However, Jurkat T cells adhering to FN displayed a marked increase in calpain activity associated with this fraction. Immunoblot analysis of Jurkat T cell lysates plated on poly-L-lysine contained predominantly the full-length 50-kDa form of PTP-1B (Fig. 10, lane 1). PMA-activated Jurkat T cells adhering to FN triggered the cleavage of PTP-1B (Fig.  10, lane 2), and pretreatment with calpeptin (Fig. 10, lane 3) FIG. 8. The cleaved form (42-kDa) of PTP-1B associates with p130 Cas in IONO-stimulated T cells. The p130 Cas was immunoprecipitated with rabbit anti-p130 Cas (lanes 2 and 3) or an irrelevant mAb of the same isotype (control, lane 1) from Jurkat T cells that were unstimulated (lane 2) or stimulated with IONO for 20 min (lanes 1, 3,  and 4). The immunoprecipitates or Jurkat T cell lysates (lane 4) were analyzed by immunoblotting. The nitrocellulose membranes were immunoblotted with anti-PTP-1B mAb.

FIG. 6. 42-kDa cleaved form of PTP-1B obtained from IONO-stimulated Jurkat T cells is soluble and enzymatically active.
Jurkat T cells (20 ϫ 10 6 ) were unstimulated or stimulated with IONO (1 M) for 10 min at 37°C. Cells were separated into soluble or insoluble fractions, and PTP-1B was immunoprecipitated from each fraction. Equal aliquots were removed from the samples prior to the phosphatase assay and analyzed by immunoblotting (insert) as described in Fig. 3. The immunocomplexes were subjected to an immune complex phosphatase assay as described under "Experimental Procedures." Data are expressed as the average pmols of phosphate released from the synthetic phosphopeptide from triplicate samples. Collectively these data suggest a novel calcium signaling pathway in T cells that is contingent upon calpain activation and the cleavage of PTP-1B and its interaction with p130 Cas .
In T cells, calcium acts as an important second messenger regulating cell shape, motility, and function (32)(33)(34)(35)(36). Although calcium-linked signaling is involved in integrin-mediated signal transduction, the mechanism(s) is obscure (20,26). Our results begin to define this mechanism by linking increases in T cell [Ca 2ϩ ] i occurring after IONO stimulation or adherence to FN with the activation of calpain and its subsequent translocation to the cytoskeletal/membrane fraction. Furthermore, stimulation of T cells with these substances causes calpaininduced proteolytic cleavage of PTP-1B, resulting in a 42-kDa enzymatically active form of the phosphatase as a result of C-terminal truncation. Interestingly, PTP-1B can be cleaved by calpain I and calpain II, both of which are present in T cells (37). PTP-1B normally associates with the ER membrane via a C terminus-targeting sequence, whereas the catalytic domain is located near the N terminus (29,30). Cleavage of PTP-1B by calpain results in the loss of the ER-targeting sequence and potential relocalization of the 42-kDa phosphatase to sites of focal contact. Our observations support this by showing that activated T cells bound to immobilized FN activate calpain resulting in the proteolytic cleavage of PTP-1B. This is consistent with the fact that calpain can associate with membranes, cytoskeleton, as well as sites of focal contact after appropriate stimulation of cells (5)(6)(7)(8)(9). In support of this is the finding that calpain cleaves PTP-1B in response to platelet aggregation that is dependent on the ␣IIb␤3 integrin (38). However, to our knowledge, this is the first report of proteolytic modification of PTP-1B by calpain in nucleated cells and further suggests the presence of a novel integrin-mediated calcium signaling pathway in T cells.
Although PTP-1B can regulate cell cycle progression (39), as well as insulin receptor and epidermal growth factor receptor signaling pathways (40,41), the function and potential substrates of this protein tyrosine phosphatase remain elusive. PTP-1B does not contain either Src homology 2 or Src homology 3 domains that often mediate protein-protein interactions involved in multiple signaling pathways. However, PTP-1B does contain two polyproline-rich segments at amino acids 301-315 and 386 -397 that have the consensus sequences (PXXPX(R/K)) for class II Src homology 3-binding proteins (42). Liu et al. (42) recently reported that the class II Src homology 3 binding domain of p130 Cas binds the more N-terminal polyproline-rich region of PTP-1B in vitro and in the v-crk transformed fibroblast cell line 3Y1. Our results show not only an association between PTP-1B and p130 Cas in resting T cells and Jurkat T cells, but that p130 Cas associates with both the 50-kDa native form and the 42-kDa calpain-cleaved form of PTP-1B. However, IONO stimulation of T cells markedly decreases the amount of p130 Cas that co-precipitates with PTP-1B. These data indicate FIG. 9. Adherence to fibronectin increases calpain activity associated with the membrane/cytoskeletal fraction in PMA-stimulated Jurkat T cells. Jurkat T cells (20 ϫ 10 6 ) were incubated on poly-L-lysine-or FN-coated culture plates in the presence of PMA (50 ng/ml) at 37°C for 30 min. Cells were harvested from each well, and cell numbers were equalized prior to isolation of the insoluble (membrane/cytoskeletal) fraction. Calpain activity was determined as described under "Experimental Procedures." The results are expressed as the average mean fluorescence Ϯ S.E. of triplicate samples from a representative experiment. that the PTP-1B⅐p130 Cas complex dissociates after cleavage of PTP-1B by calpain. This may occur subsequent to relocalization of the complex to sites of focal contact, resulting in potential interactions of PTP-1B with substrates previously inaccessible to the ER bound phosphatase.
The p130 Cas protein contains several consensus sequences known to mediate protein-protein interactions, including a Src homology 3 domain, a polyproline-rich region, and multiple potential Src homology 2 binding domains. The unique structure of this docking protein suggests it participates in integrinmediated signal transduction by acting as a platform for the assembly of multimeric protein complexes (43). In support of this, p130 Cas undergoes tyrosine phosphorylation in response to ␤1 integrin stimulation and also localizes to sites of focal contact between cells and the ECM (44 -46). Whether the PTP-1B⅐p130 Cas complex is involved is unclear; however, the association of PTP-1B and p130 Cas suggests a potential regulatory role in the formation of multimeric complexes by regulating the phosphorylation levels of p130 Cas .
Integrin-mediated T cell adhesion to ECM proteins is temporally regulated (25). Integrins expressed on resting T cells exist in a low avidity state; after activation of the cells a high avidity state is induced by "inside-out" signaling (47,48). Thus, integrins are targets for functional regulation, permitting cellular adhesiveness to be modulated in response to extracellular signals. Phosphorylation of proteins is involved in transmembrane-signaling events and cytoskeletal reorganization. In this respect, protein tyrosine phosphatases have been implicated in modulating the integrity of focal contacts (49,50) and the avidity state of lymphocyte integrin molecules (51). Furthermore, integrin-mediated calcium mobilization is involved in ␤1 integrin activation and regulation (52,53).
The work presented here supports an "outside-in" signaling pathway in integrin-mediated calcium signaling in T cells. The activation of calpain in these cells is dependent on the increase in [Ca 2ϩ ] i . The translocation of calpain activity and the proteolytic cleavage of PTP-1B by calpain occurs after T cell adherence to the ECM protein FN as well as stimulation of these cells with IONO. Cleavage of PTP-1B by calpain causes the relocalization of the 42-kDa form of the phosphatase from the ER to sites of focal contact via its association with p130 Cas . This would allow PTP-1B to interact with potential substrates previously inaccessible to the ER-localized phosphatase, and mediate "inside-out" signaling events involved in the regulation of ␤1 integrin function, cytoskeletal assembly, and ultimately cell behavior. Not only may p130 Cas be an in vivo substrate for PTP-1B, but it also may serve as a targeting protein to relocate the calpain cleaved form of PTP-1B.