©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Regulation of Protein-tyrosine Phosphatases by Integrin through Cytoskeletal Reorganization and Tyrosine Phosphorylation in Human Platelets (*)

Yasuharu Ezumi, Hiroshi Takayama(§), and Minoru Okuma

From the (1) First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, 54 Shogoin-Kawaramachi, Sakyo-ku, Kyoto 606, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The major platelet integrin (glycoprotein IIb-IIIa) has been implicated in the regulation of tyrosine phosphorylation and dephosphorylation in activated platelets. To investigate the mechanisms of the -dependent tyrosine dephosphorylation, normal platelets or thrombasthenic platelets lacking were stimulated with thrombin and fractionated into Triton X-100-soluble or -insoluble subcellular matrices. We then examined the kinetics of the tyrosine-phosphorylated proteins and distribution of protein-tyrosine phosphatases in these fractions and whole cell lysates. First, -dependent tyrosine dephosphorylation was recovered mainly in the cytoskeleton with similar kinetics to the whole cell lysate. Second, protein-tyrosine phosphatase (PTP) 1B and its cleaved 42-kDa form were associated with the cytoskeleton in an aggregation-dependent manner, whereas association of PTP1C with the cytoskeleton was regulated differentially both by thrombin stimulation and by -mediated aggregation. Several calpain inhibitors did not affect either tyrosine phosphorylation and dephosphorylation or relocation of PTP1B, but they did inhibit cleavage of PTP1B. Cytochalasin D blocked relocation of both PTP1B and PTP1C but not PTP1B cleavage. SH-PTP2 was distributed in the other fractions than the cytoskeleton and showed no relocation on thrombin stimulation. Finally, the cytoskeleton-associated PTP1C became tyrosine-phosphorylated in an -mediated aggregation-dependent manner. Thus, integrin was involved differentially in the regulation of PTP1B and PTP1C.


INTRODUCTION

Platelets are nonproliferative, terminally differentiated cells, which offer an attractive model system to study the various biochemical events leading to structural and functional alterations in activated cells. When platelets are exposed to stimuli such as thrombin and collagen, platelets become activated, undergo a dramatic shape change, adhere to each other, and aggregate. In these processes, the major platelet integrin (also called glycoprotein IIb-IIIa), which is a heterodimeric adhesion receptor (1) , plays a critical role. Integrin can recognize Arg-Gly-Asp-containing adhesive ligands including fibrinogen, fibronectin, von Willebrand factor, and vitronectin (2, 3) . Among these adhesive molecules, the binding of fibrinogen to is the major mechanism for platelet aggregation. Besides functioning as an adhesive receptor, integrin is involved in signal transduction from the extracellular matrix to the cytoplasm (4) .

Protein-tyrosine phosphorylation has been implicated in most biochemical events that involve transmembrane signaling and cytoskeletal reorganization. Agonist-stimulated platelets show rapid changes in tyrosine phosphorylation of multiple proteins (5, 6) , some of which are dependent on platelet aggregation mediated through the binding of immobilized fibrinogen to under stirring condition (7, 8) . The net phosphorylation of tyrosine residues on substrate proteins is regulated by activities of both protein-tyrosine kinases (PTKs)() and protein-tyrosine phosphatases (PTPases). Platelets contain numerous cytosolic PTKs including five members of the Src family (pp60, pp60, pp60, pp61, and pp54/58 ) (9, 10, 11) , pp125(12) , and pp72(13) . pp60, which is the most abundant PTK in platelets (9) , shows an increase in tyrosine kinase activity with thrombin-induced activation (14) and translocates to the Triton X-100-insoluble, cytoskeleton-rich fraction dependent on platelet aggregation (15, 16) . pp125, which is located in focal adhesion, is phosphorylated on tyrosine and activated in an aggregation-dependent manner (12) .

However, little information exists about the physiological role or regulation of PTPases. Like the PTK family, PTPases are grouped into two forms: non-transmembrane cytoplasmic PTPases and receptor-type transmembrane PTPases (17) . Non-transmembrane PTPases consist of a single catalytic domain and N- or C-terminal extensions, which are important for enzymatic regulation and intracellular localization. For example, PTP1B (18) and T-cell PTPase (19) are localized to the endoplasmic reticulum or the particulate fraction via C-terminal targeting sequences, whereas other non-transmembrane PTPases, PTP1C (20) (also known as SH-PTP1C, HCP, SHP, or PTP6N; Refs. 21-24) and SH-PTP2 (25) (also called PTP2C, SH-PTP3, Syp, or PTP1D; Refs. 26-29), contain two Src homology 2 (SH2) domains. PTP1C is expressed predominantly in hematopoietic cells (22) , whereas PTP1B and SH-PTP2 are expressed ubiquitously (18, 25) . Until now, two forms of non-transmembrane PTPases have been documented in platelets (30, 31) , whereas receptor-type transmembrane PTPases, such as CD45, have not been found in platelets (32) .

We previously reported that is involved in the tyrosine-specific dephosphorylation of certain proteins, suggesting for the first time the engagement of in the activation of some PTPases present in platelets (33) . Here we report that cytoskeletal reorganization and protein-tyrosine phosphorylation are also involved in the -dependent regulation of PTPases in platelets.


EXPERIMENTAL PROCEDURES

Materials

Anti-phosphotyrosine monoclonal antibodies 4G10 and PY20 were purchased from Upstate Biotechnology, Inc. (UBI, Lake Placid, NY) and ICN Biomedicals, Inc. (Costa Mesa, CA), respectively. Affinity-purified rabbit polyclonal antibodies against PTP1B, PTP1C, and SH-PTP2 were from UBI. Monoclonal antibody 327 is specific for pp60 and was obtained from Oncogene Science, Inc. (Uniondale, NY). Prostaglandin E was kindly provided by Ono Pharmaceutical Co. (Osaka, Japan). Cytochalasin D and aprotinin were obtained from Sigma. Leupeptin was from Peptide Institute, Inc. (Minoh, Japan). Calpeptin was from LC Laboratories (Woburn, MA). EST and MDL were generous gifts of Dr. M. Tamai of Taisho Pharmaceutical Co. (Saitama, Japan) and Dr. E. H. W. Bohme of Marion Merrell Dow Research Institute (Cincinnati, OH), respectively. All other reagents were obtained as described previously (33) .

Preparation and Activation of Platelets

After informed consent was obtained, venous blood was collected from healthy adult donors or from patients with Glanzmann's thrombasthenia. Anti-coagulation of blood and preparation of washed platelets were performed as described previously (33) . Washed platelets (0.5-1.0 10 cells/ml) were activated by 1 unit/ml thrombin with or without stirring at 1,000 rpm in an aggregometer at 37 °C for appropriate intervals. In some experiments, washed platelets were preincubated for 10 min at 37 °C with the following inhibitors prior to activation with thrombin: 1 mM RGDS, 20 µM cytochalasin D, 500 µM EST, 250 µM calpeptin, or 250 µM MDL.

Fractionation of Platelets

Subcellular fractionation of platelets was carried out using a modification of the method described by Fox et al.(34) . After washed platelets (5 10 cells/ml) were activated with thrombin, reactions were stopped by lysing the cells with 0.5 volume of 3 lysis buffer containing 3% Triton X-100, 15 mM EGTA, 15 mM EDTA, 30 mM benzamidine, 3 mM phenylmethylsulfonyl fluoride (PMSF), 3 mM NaVO, 60 µg/ml leupeptin, 60 µg/ml aprotinin, 30 µM cytochalasin D, and 50 mM Tris-HCl, pH 7.4. In some experiments, cytochalasin D was omitted from the lysis buffer. This and all subsequent steps were performed at 4 °C. The cytoskeleton was sedimented immediately by centrifugation of the lysate at 10,000 g for 5 min. The membrane skeleton was isolated from the 10,000 g supernatant by centrifugation at 100,000 g for 3 h in a 1.5-ml microcentrifuge tube (Eppendorf, Hamburg, Germany) with a Beckman TL-100 using a TLA-100.3 rotor (Beckman Instruments, Inc., Palo Alto, CA). The cytoskeleton and the membrane skeleton then were washed three times with 1 lysis buffer containing 1% Triton X-100, 5 mM EGTA, 5 mM EDTA, 10 mM benzamidine, 1 mM PMSF, 1 mM NaVO, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 50 mM Tris-HCl, pH 7.4, and solubilized in 1 SDS sample buffer (2% SDS, 5% glycerol, 5% -mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8). The Triton X-100-soluble fraction from the 100,000 g supernatant was diluted with 0.5 volume of 3 concentrated SDS sample buffer. When the samples of the whole lysate of platelets were needed, thrombin-activated platelets were solubilized directly by addition of 0.5 volume of 3 concentrated SDS sample buffer. Following solubilization in SDS sample buffer, the samples were promptly boiled for 5 min.

Immunoblotting

The samples (proteins from 1 10 platelets/lane) were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (35) . Separated proteins were transferred electrophoretically to a nitrocellulose membrane (Bio-Rad) with a semidry blotter. The membranes were blocked for 1 h at room temperature or overnight at 4 °C with 3% nonfat dried milk in TPBS (0.1% Tween 20, 137 mM NaCl, 2.7 mM KCl, 8 mM NaHPO, 1.5 mM KHPO) and washed three times in TPBS. The membranes then were incubated for 2 h with primary antibodies against proteins of interest in TPBS containing 1% bovine serum albumin. The primary antibodies used were as follows: a mixture of anti-phosphotyrosine monoclonal antibodies 4G10 and PY20 (1 µg/ml each); anti-pp60 monoclonal antibody 327 (1 µg/ml); polyclonal antibodies against PTP1B (0.5 µg/ml), PTP1C (1 µg/ml), or SH-PTP2 (1 µg/ml). The membranes were washed four times in TPBS and incubated for 1 h either with horseradish peroxidase-conjugated goat anti-mouse (1 µg/ml) or anti-rabbit (0.5 µg/ml) IgG in TPBS containing 1% bovine serum albumin. Immunoreactivity was determined using the ECL chemiluminescence reaction (Amersham International plc, Little Chalfont, United Kingdom). In some experiments, the membranes once probed were stripped of bound antibodies by incubation in buffer containing 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7, for 30 min at 60 °C and then reprobed with other antibodies as described above.

Immunoprecipitation

Washed platelets (1 10 cells/ml) were stimulated with thrombin for appropriate periods and lysed for 1 h in 0.5 volume of 3 radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 15 mM EGTA, 3% Triton X-100, 3% sodium deoxycholate, 0.3% SDS, 3 mM PMSF, 3 mM NaVO, 60 µg/ml leupeptin, 60 µg/ml aprotinin, and 50 mM Tris-HCl, pH 7.4). This and all subsequent steps were carried out at 4 °C. The lysates were clarified by centrifugation at 16,000 g for 20 min, precleared with 50 µl of protein A-Sepharose CL-4B (50% slurry) (Pharmacia LKB Biotechnology, Inc., Uppsala, Sweden) by sedimentation at 16,000 g for 3 min, and then incubated for 2 h with anti-PTP1B antibody (2 µg/ml), anti-PTP1C antibody (4 µg/ml), anti-SH-PTP2 antibody (10 µg/ml), or rabbit anti-mouse IgG (10 µg/ml) as a control. Immune complexes were incubated for 1 h with 50 µl of protein A-Sepharose CL-4B (50% slurry). Immunoprecipitates were sedimented by brief centrifugation and washed four times in 1 RIPA buffer (150 mM NaCl, 5 mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM NaVO, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 50 mM Tris-HCl, pH 7.4). For immunoprecipitation of proteins from the subcellular fractions, the cytoskeleton and the membrane skeleton of thrombin-activated platelets were solubilized in RIPA buffer following fractionation of cells. Immunoprecipitated PTP1C was obtained from each solubilized fraction in RIPA buffer. Immunoprecipitated proteins (from 4 10 platelets/lane) were eluted from protein A-Sepharose beads in 1 SDS sample buffer, boiled for 5 min, resolved on SDS-PAGE, and analyzed by immunoblotting as described above.

RESULTS

Protein-tyrosine Phosphorylation and Dephosphorylation in the Subcellular Fractions of Thrombin-activated Platelets

To investigate the involvement of integrin in protein-tyrosine phosphorylation and dephosphorylation, we first examined tyrosine-phosphorylated proteins by immunoblot assay in whole lysates of platelets from normal donors and from patients with Glanzmann's thrombasthenia. As shown in Fig. 1(A and B), thrombasthenic platelets lacking showed different patterns from normal platelets in tyrosine dephosphorylation as well as in tyrosine phosphorylation on stimulation with thrombin. In normal platelets, tyrosine-phosphorylated protein bands with molecular masses of 130, 120, and 115 kDa peaked at 10 s, 30 s, and 2 min, respectively, then quickly declined, and disappeared. However, in thrombasthenic platelets, the 130- and 120-kDa protein bands persisted and were not dephosphorylated until 5 min after stimulation, whereas the 115-kDa protein band did not appear. Two clusters of protein bands with molecular masses of 68-78 and 53-60 kDa also showed tyrosine-specific dephosphorylation in normal platelets but did not in thrombasthenic platelets. Immunoblot analysis with anti-pp60 antibody revealed that the most prominent band in the 53-60-kDa bundle was pp60 (data not shown). The 102-kDa tyrosine-phosphorylated protein band appeared at 2 min and persisted until 5 min following thrombin stimulation in normal platelets, but did not appear in thrombasthenic platelets. The 95-kDa band peaked at 30 s and was persistent until 5 min either in normal or thrombasthenic platelets. These results indicated that was involved in the tyrosine phosphorylation of the 115- and 102-kDa protein bands and the tyrosine dephosphorylation of the 130-, 120-, 68-78-, and 53-60-kDa protein bands. Thrombasthenic platelets also differed from normal platelets in kinetic patterns of other doublet bands of 36/37 and 33/34 kDa in tyrosine phosphorylation and dephosphorylation. Following stimulation with thrombin, these doublets peaked at 10 s, quickly declined, and disappeared in normal platelets, whereas these bands in thrombasthenic platelets peaked at 30 s and declined slowly.


Figure 1: Time course of thrombin-induced protein-tyrosine phosphorylation in whole lysates and subcellular fractions of normal and thrombasthenic platelets. Washed platelets from normal donors (A, C, E, and G) or integrin -deficient subjects with Glanzmann's thrombasthenia (B, D, F, and H) were stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions. The platelets were lysed directly in SDS sample buffer to obtain whole cell lysates (A and B) or in 1% Triton X-100-containing lysis buffer with cytochalasin D, and fractionated into the cytoskeleton (1% Triton X-100-insoluble, 10,000 g pellets) (C and D), the membrane skeleton (1% Triton X-100-insoluble, 100,000 g pellets) (E and F), and the 1% Triton X-100-soluble fraction (G and H) as described under ``Experimental Procedures.'' The same experiment as shown in panelC was also performed using the lysis buffer without cytochalasin D (C` in parentheses). Proteins from the same number of platelets were subjected to 10% SDS-PAGE and probed by immunoblotting with anti-phosphotyrosine antibodies. Molecular masses of major tyrosine-phosphorylated protein bands and molecular mass markers are indicated in kDa on the left and right of the panels, respectively.



Because previous reports have suggested that many tyrosine-phosphorylated proteins are components of Triton X-100-insoluble, cytoskeleton-rich fractions of activated platelets, we explored whether the Triton X-100-soluble or -insoluble subcellular fraction was the site of the tyrosine dephosphorylation observed in the whole lysate of thrombin-activated platelets. This investigation led us to the finding that the conventionally used lysis buffer (34) should be modified by including cytochalasin D to observe the protein-tyrosine dephosphorylation patterns in the subcellular fractions with similar kinetics to those in the whole cell lysate. When we used the lysis buffer without cytochalasin D, protein-tyrosine phosphorylation observed in the cytoskeleton of normal platelets was persistent and quite different from that in the whole cell lysate (Fig. 1C`, parentheses). On the contrary, using the modified lysis buffer containing cytochalasin D as described under ``Experimental Procedures,'' nearly identical patterns of tyrosine phosphorylation and dephosphorylation of proteins observed in the whole cell lysate were noted in the cytoskeletons of normal and thrombasthenic platelets (Fig. 1, C-H), except for several protein bands. One exception to these observations was pp60. pp60 was redistributed from the Triton X-100-soluble fraction and the Triton X-100-insoluble membrane skeleton into the cytoskeleton in aggregated normal platelets but not in thrombasthenic platelets, as has been reported by several laboratories (14, 15, 16) . Other exceptions were the 36/37- and 33/34-kDa tyrosine-phosphorylated protein bands. We could not obtain reproducible results for the cytoskeletal localization of these protein bands. However, we detected only the 36/37-kDa bands in the cytoskeleton of normal platelets. When normal platelets were activated with thrombin in the presence of RGDS, an inhibitor of -fibrinogen binding, or without stirring to prevent platelet aggregation, we obtained the same patterns of tyrosine phosphorylation and dephosphorylation as observed in thrombasthenic platelets (data not shown).

Cytochalasin D but Not Calpain Inhibitors Affects the Patterns of Tyrosine Phosphorylation

Next, we examined whether an inhibitor of actin polymerization, cytochalasin D, influenced the state of tyrosine-phosphorylated proteins in the whole cell lysate and in the cytoskeleton. As shown in Fig. 2, pretreatment of normal platelets with 20 µM cytochalasin D did not inhibit thrombin-induced aggregation (data not shown), but markedly reduced the thrombin-induced tyrosine-phosphorylated protein bands both in the whole cell lysate and in the cytoskeleton. This suggested that tyrosine phosphorylation of the cytoskeletal components was largely dependent on actin polymerization. Furthermore, cytochalasin D blocked the relocation of pp60 to the cytoskeleton in aggregated normal platelets (Fig. 2, C, E, and G), confirming the findings by Oda et al.(16) .


Figure 2: Effects of an actin polymerization inhibitor, cytochalasin D, and a calpain inhibitor, EST, on thrombin-induced tyrosine phosphorylation in whole lysates and subcellular fractions of normal platelets. Washed platelets from normal donors were pretreated with 20 µM cytochalasin D (A, C, E, and G) or 500 µM EST (B, D, F, and H) for 10 min and stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions. The platelets were lysed directly in SDS sample buffer to obtain whole cell lysates (A and B) or in 1% Triton X-100-containing lysis buffer with cytochalasin D, and fractionated into the cytoskeleton (C and D), the membrane skeleton (E and F), and the 1% Triton X-100-soluble fraction (G and H) as described under ``Experimental Procedures.'' Proteins from the same number of platelets were subjected to 10% SDS-PAGE and probed by immunoblotting with anti-phosphotyrosine antibodies. Molecular masses of major tyrosine-phosphorylated protein bands and molecular mass markers are indicated in kDa on the left and right of the panels, respectively.



Recently, Frangioni et al.(30) have reported that, in activated platelets, a calcium-dependent neutral protease, calpain, cleaves PTP 1B in an aggregation-dependent fashion, and that the cleaved 42-kDa form of PTP1B relocates from the membrane to the cytosol and shows 2-fold higher enzymatic activity than the intact form. Hence we studied the effects of calpain inhibitors EST (36), MDL (37) , and calpeptin (38) on the -regulated tyrosine dephosphorylation in platelets. However, as shown in Fig. 2 , EST affected neither aggregation (data not shown) nor the patterns of tyrosine phosphorylation and dephosphorylation in the whole cell lysate and the subcellular fractions, particularly the cytoskeleton. At the same time, we confirmed that the cleavage of PTP1B occurred in thrombin-activated normal platelets as aggregation proceeded but not in thrombin-activated thrombasthenic platelets which showed no aggregation; EST completely inhibited this aggregation-dependent cleavage of PTP1B (Fig. 3, a, b, and d). The other calpain inhibitors, MDL and calpeptin, did not affect either aggregation or tyrosine phosphorylation and dephosphorylation. However, calpeptin inhibited the cleavage of PTP1B less than EST or MDL (data not shown). By contrast, cytochalasin D did not prevent cleavage of PTP1B (Fig. 3c).


Figure 3: Aggregation-dependent cleavage of PTP1B in thrombin-activated platelets is inhibited completely by EST but not by cytochalasin D. Untreated normal (a) or thrombasthenic platelets (b) were stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions. Normal platelets pretreated with 20 µM cytochalasin D (c) or 500 µM EST (d) were stimulated with thrombin. Platelets were lysed in SDS sample buffer. Proteins from the same number of platelets were subjected to 10% SDS-PAGE and probed by immunoblotting with anti-PTP1B antibody. The positions of PTP1B (arrow) and the cleaved form of PTP1B (arrowhead) are indicated on the right of the blots.



Association of PTP1B and Its Cleaved 42-kDa Form with the Cytoskeleton

In order to show the presence of PTPases in the cytoskeleton, we studied subcellular distributions of PTPases, PTP1B, PTP1C, and SH-PTP2 by immunoblot analysis. As shown in Fig. 4A, PTP1B translocated from the detergent-soluble fraction to the cytoskeleton in thrombin-activated normal platelets under stirring conditions. Interestingly, not only PTP1B itself but also its cleaved 42-kDa form were associated with the cytoskeleton in a time-dependent manner as platelets aggregated (Fig. 4A, a). In thrombasthenic platelets stimulated with thrombin, PTP1B did not translocate to the cytoskeleton (Fig. 4B, a). We confirmed that PTP1B did not translocate to the cytoskeleton if normal platelets were activated with thrombin in the presence of RGDS or under unstirring conditions to prevent aggregation (data not shown). Pretreatment of normal platelets with cytochalasin D prior to thrombin stimulation blocked the relocation of PTP1B to the cytoskeleton, whereas a calpain inhibitor, EST, which prevented the cleavage of PTP1B, did not interfere with the relocation of PTP1B (Fig. 4B, b and c). These results indicated that PTP1B association with the cytoskeleton was dependent on an -mediated aggregation as well as actin polymerization.


Figure 4: PTP1B translocates into the cytoskeleton in thrombin-activated platelets in an aggregation- and actin polymerization-dependent manner. A, normal platelets were stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions, lysed in 1% Triton X-100-containing lysis buffer with cytochalasin D, and fractionated into the cytoskeleton (CSK) (a), the membrane skeleton (MSK) (b), and the detergent-soluble fraction (SOL) (c) as described under ``Experimental Procedures.'' B, thrombasthenic (a) and normal platelets, which were pretreated with 20 µM cytochalasin D (b) or 500 µM EST (c) for 10 min, were stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions and lysed. Cytoskeletons were subjected to 10% SDS-PAGE and probed by immunoblotting with anti-PTP1B antibody. The positions of PTP1B (arrow) and the cleaved form of PTP1B (arrowhead) are indicated on the right of the blots.



Redistribution of PTP1C in the Subcellular Fractions

As shown in Fig. 5, PTP1C was present in the detergent-soluble fraction and the membrane skeleton of unstimulated platelets. When normal platelets were activated with thrombin under stirring conditions, PTP1C relocated to the cytoskeleton (Fig. 5A). PTP1C was redistributed to the cytoskeleton also in thrombin-activated thrombasthenic platelets (Fig. 5B, a). However, the intensity of the PTP1C band was weaker in the cytoskeleton of thrombasthenic platelets than in aggregated normal platelets (Fig. 5A and B, panels a). When normal platelets were activated with thrombin in the presence of RGDS or without stirring, we obtained the same results as in thrombasthenic platelets (data not shown). Pretreatment of normal platelets with cytochalasin D prior to thrombin stimulation attenuated the association of PTP1C with the cytoskeleton and diminished the intensity of the PTP1C band in a time-dependent manner as platelet aggregation proceeded (Fig. 5B, b). These findings may be derived from detachment of the associated PTP1C from the cytoskeleton. Another PTPase containing SH2 domains, SH-PTP2, was distributed in the membrane skeleton and in the detergent-soluble fraction but not in the cytoskeleton of unstimulated platelets. Stimulation with thrombin did not change the subcellular distribution of SH-PTP2 (data not shown).


Figure 5: Redistribution of PTP1C between the subcellular fractions in thrombin-activated platelets. A, normal platelets were stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions, lysed in 1% Triton X-100-containing lysis buffer with cytochalasin D, and fractionated into the cytoskeleton (CSK) (a), the membrane skeleton (MSK) (b), and the detergent-soluble fraction (SOL) (c) as described under ``Experimental Procedures.'' B, thrombasthenic (a) or normal platelets pretreated with 20 µM cytochalasin D (b) were stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions and lysed. Cytoskeletons were subjected to 10% SDS-PAGE and probed by immunoblotting with anti-PTP1C antibody. Arrows indicate the position of PTP1C.



Phosphorylation of PTP1C on Tyrosine Residues in an Aggregation-dependent Manner

Tyrosine phosphorylation of proteins, especially proteins containing SH2 domains, is an important mechanism to regulate activities or interactions with other molecules (39) . We examined tyrosine phosphorylation of immunoprecipitated PTPases from activated platelets with thrombin by immunoblotting. We first found that stimulation of platelets with thrombin under stirring conditions caused tyrosine phosphorylation of PTP1C in a time-dependent fashion (Fig. 6A, a). Furthermore, the tyrosine phosphorylation of PTP1C occurred only when thrombin-activated platelets were stirred simultaneously to induce aggregation (Fig. 6B, a). Blockage of aggregation of thrombin-activated platelets by not stirring or by addition of RGDS prevented tyrosine phosphorylation of PTP1C. In addition, pretreatment of platelets with cytochalasin D did not inhibit platelet aggregation but did abolish tyrosine phosphorylation of PTP1C (Fig. 6B, a). Immunoblot analysis with anti-PTP1C antibody showed that the amount of immunoprecipitated PTP1C was nearly unchanged in these experiments (Fig. 6, A and B, panelsb). We could not detect tyrosine phosphorylation of PTP1B and SH-PTP2 in unstimulated platelets or in thrombin-stimulated ones (data not shown). These data indicated that tyrosine phosphorylation of PTP1C was dependent on -mediated aggregation as well as actin polymerization. Furthermore, tyrosine-phosphorylated PTP1C was seen in the cytoskeleton but not in the membrane skeleton or the detergent-soluble fraction (Fig. 6C, lanes 1-3). However, the intensity of tyrosine-phosphorylated PTP1C band in the cytoskeleton was much weaker than that in the whole cell lysate, whereas a considerable amount of PTP1C was immunoprecipitated from each fraction (Fig. 6C, lanes 4 and 5).


Figure 6: Thrombin induces tyrosine phosphorylation of PTP1C associated with the cytoskeleton, which is dependent on aggregation and actin polymerization. A, normal platelets were stimulated with 1 unit/ml thrombin for the indicated times under stirring conditions, lysed in RIPA buffer, and immunoprecipitated with anti-PTP1C antibody (-PTP1C) or rabbit anti-mouse IgG (RAM) as a control. B, normal platelets were untreated (lanes 1-3) or pretreated with 1 mM RGDS (lane4) or 20 µM cytochalasin D (lane5) for 10 min prior to stimulation. Unstimulated platelets (lane1) or those stimulated with 1 unit/ml thrombin for 2 min under stirring (lanes 2, 4, and 5) or unstirring conditions (lane3), were lysed inRIPA buffer, and immunoprecipitated with anti-PTP1C antibody. C, normal platelets were stimulated with 1 unit/ml thrombin for 2 min under stirring conditions, lysed in 1% Triton X-100-containing lysis buffer with cytochalasin D, and fractionated into the cytoskeleton (CSK) (lanes 1 and 4), the membrane skeleton (MSK) (lanes2 and 5), and the detergent-soluble fraction (SOL) (lanes3 and 6). The proteins from each fraction were immunoprecipitated with anti-PTP1C antibody. The immunoprecipitates were subjected to 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed by immunoblotting with anti-phosphotyrosine antibodies (A, a; B, a; and C, lanes 1-3). The same membranes were reprobed with anti-PTP1C antibody (A, b; B, b; and C, lanes 4-6). Arrows mark the position of PTP1C.



We also studied whether tyrosine-phosphorylated PTP1C increased its enzymatic activity compared to unphosphorylated PTP1C by an immune complex PTPase assay using para-nitrophenyl phosphate; we did not detect any significant difference in PTPase activity (data not shown). However, this negative result agrees with the suggestions by Lorenz et al.(40) that autodephosphorylation of PTP1C takes place in vitro and would make it difficult to prove any difference in PTP1C activity due to tyrosine phosphorylation. In fact, autodephosphorylation may explain why tyrosine-phosphorylated PTP1C was largely decreased as described above during preparation of the cytoskeleton fraction.

DISCUSSION

Until recently, the physiological roles or modes of regulation of PTPases in platelets have received much less attention than PTKs, although platelets have been suggested to possess several PTPases (30, 31) . Our previous work has shown for the first time that integrin is involved in protein-tyrosine dephosphorylation in activated platelets (33) . It has been suggested that many of the proteins phosphorylated on tyrosine in activated platelets are components of the cytoskeleton (34) . If so, one would speculate that the -regulated protein-tyrosine dephosphorylation would be observed on the tyrosine-phosphorylated proteins of the cytoskeleton in activated platelets. However, this notion has not been successfully proven as yet, although there are a few reports showing protein-tyrosine phosphorylation but not dephosphorylation of cytoskeletons in activated platelets (14, 16, 34) .

When we first studied protein-tyrosine phosphorylation of subcellular fractions after platelets were activated and lysed in the lysis buffer that had been conventionally used in previous studies (14, 16, 34) , we experienced difficulty in recovering the same tyrosine dephosphorylation patterns in subcellular fractions as were observed in the whole cell lysate. Tyrosine-phosphorylated proteins were recovered mostly in the cytoskeleton, but there was little tyrosine dephosphorylation observed on these proteins. This suggested to us that some PTKs were still active in the platelet lysate for unknown reasons, while PTPases were inactivated by orthovanadate included in the lysis buffer. Therefore, we tried to modify the conventionally used lysis buffer by adding various inhibitors in order to preserve tyrosine dephosphorylation following platelet lysis. Although inhibitors of PTKs, such as genistein or tyrphostin, were expected to be suitable for this purpose, they were found to be insufficient to prevent protein-tyrosine phosphorylation in the platelet lysate in our preliminary experiments.

The addition of cytochalasin D to the lysis buffer enabled us to show the reproducible patterns of tyrosine dephosphorylation in the cytoskeleton with similar kinetics to those observed in the whole lysate. This suggests that actin polymerization is still persistent, even after platelets are lysed, and that newly polymerized cytoskeleton keeps associated PTKs active and maintains protein-tyrosine phosphorylation. It is possible that ATP required for such phosphorylation could be supplied from the platelet dense granules which contain abundant ATP (41) after platelets are lysed. Thus, we demonstrated for the first time the presence of tyrosine dephosphorylation in the cytoskeleton which was dependent on -mediated aggregation, as was observed in the whole lysate.

Frangioni et al.(30) have recently shown that is engaged in inducing PTP1B cleavage by a Ca-dependent neutral protease calpain at a site upstream from its C-terminal targeting sequence, resulting in subcellular relocation of the PTP1B catalytic domain from the membrane to the cytosol and a 2-fold increase in its enzymatic activity. They proposed that the PTP1B cleavage, the subcellular relocation into the cytosol, and the enzymatic activation account for the blunted increase in protein-tyrosine phosphorylation seen with aggregation. However, their proposed mechanism can not explain the regulation of the aggregation-dependent dephosphorylation of tyrosine-phosphorylated proteins observed in this study for the following reasons. First, complete blockage of PTP1B cleavage by several calpain inhibitors including EST did not affect the patterns of tyrosine phosphorylation and dephosphorylation in any subcellular fractions as well as in the whole cell lysate. Second, the tyrosine-phosphorylated proteins which were observed to be dephosphorylated in the whole cell lysate were not components of the cytosol but rather the cytoskeleton. In contrast to our study, the central part of their findings was obtained from experiments using washed platelets stimulated with 1 µM A23187 under unstirring conditions such that platelets did not aggregate. Interestingly, A23187 induces protein-tyrosine phosphorylation and its subsequent dephosphorylation of platelets under unstirring conditions, as originally reported by Takayama et al.(42) . This indicates that A23187-induced dephosphorylation of tyrosine-phosphorylated proteins is mediated by different mechanisms from the aggregation-dependent ones that are required for the physiologic agonists, such as thrombin-induced tyrosine dephosphorylation.

The occurrence of dephosphorylation of tyrosine-phosphorylated proteins in the cytoskeleton suggests that the responsible PTPases must be associated with the cytoskeleton to act on their substrates. Among the PTPases investigated in this study, we found that PTP1B and PTP1C were associated with the cytoskeleton in activated platelets, but SH-PTP2 was not. Interestingly, not only intact PTP1B but also its cleaved 42-kDa form were present in the cytoskeleton of aggregated platelets. It appears that PTP1B itself could be responsible for more PTPase activity than its cleaved form for dephosphorylation of tyrosine-phosphorylated proteins, since the inhibition of PTP1B cleavage by calpain inhibitors did not affect tyrosine dephosphorylation of those proteins. However, there is still the possibility that the cleaved 42-kDa form associated with the cytoskeleton could elicit PTPase activity on tyrosine-phosphorylated proteins that were not detected in this study. Furthermore, it is tempting to speculate that PTP1B cleavage by calpain may occur after PTP1B relocated into the cytoskeleton, since the cleaved 42-kDa form was found to be present only in the cytoskeleton but not in the other fractions of aggregated platelets. The intact PTP1B localizes to the endoplasmic reticulum membrane via its C-terminal sequence (18) . Lee and Chen (43) suggested the involvement of the cytoskeleton in motility of the endoplasmic reticulum in interphase cells. The demonstration of PTP1B association with the cytoskeleton in the present work may also suggest the dynamic association of the endoplasmic reticulum with the cytoskeleton in platelets.

Li et al.(31) have recently reported that PTP1C is associated with the cytoskeleton in an aggregation-dependent manner. In contrast to their report, we found that PTP1C became associated with the cytoskeleton in activated platelets regardless of the occurrence of aggregation, since PTP1C was found to relocate into the cytoskeleton in activated thrombasthenic platelets. However, in view of the aggregation-induced enhancement of PTP1C association with the cytoskeleton, the apparent discrepancy between our findings and those of Li et al. could be due to the difference in sensitivity for detection of associated PTP1C. Therefore, it is reasonable to conclude that the relocation of PTP1C into the cytoskeleton is regulated by two steps: first by the stimulation of thrombin and second by the -mediated aggregation. Such two-step regulated association with the cytoskeleton is also the case with pp60 (see Footnote 2) and pp72(44) . Cytochalasin D did not completely inhibit the relocation of PTP1C into the cytoskeleton but did that of PTP1B, indicating that actin polymerization is prerequisite to the PTP1B relocation but not necessarily to the PTP1C relocation. Since we have recently found that thrombin induces rapid phosphorylation of PTP1C on serine residues,() such phosphorylation may be necessary for the PTP1C relocation.

In addition to the relocation of PTPases, one interesting observation was that PTP1C became phosphorylated on tyrosine as platelet aggregation proceeded. It has been reported that PTP1C becomes tyrosine-phosphorylated in the BAC1.2F5 macrophage cell line in response to colony-stimulating factor 1 (45, 46) , in the megakaryoblastic leukemia cell line Mo7e in response to stem cell factor (46) , and in a T-cell hybridoma line or normal murine thymocytes in response to CD4 or CD8 cross-linking (40) . Similarly to these previous reports, we failed to define the biochemical consequences of PTP1C tyrosine phosphorylation in vivo which may regulate its enzymatic activity, possibly because of autodephosphorylation. However, it should be noted that PTP1C was associated with the cytoskeleton but was not tyrosine-phosphorylated in thrombin-activated platelets without aggregation; PTP1C tyrosine phosphorylation was dependent on -mediated aggregation as well as actin polymerization. Furthermore, we showed that tyrosine phosphorylation of PTP1C was detected only in the cytoskeleton and not in other fractions. Therefore, the functional role of PTP1C tyrosine phosphorylation might not be related to its relocation into the cytoskeleton but rather to the regulation of its enzymatic activity.

Another important new aspect of PTP1C is that -dependent tyrosine phosphorylation and dephosphorylation may be coupled, in part, through PTP1C phosphorylated on tyrosine. Since Ferrell and Martin (7) first reported that was involved in the regulation of tyrosine phosphorylation of a specific set of proteins, pp125 has been identified as one of these proteins (12) . However, it seems unlikely that PTP1C is a substrate of pp125 that is activated by tyrosine phosphorylation, since we observed that tyrosine phosphorylation of PTP1C occurs before pp125. PTP1C appears to be tyrosine-phosphorylated by unidentified PTKs whose activity is regulated by -mediated aggregation.

A model summarizing our findings and the possible mechanisms of -dependent tyrosine dephosphorylation is presented in Fig. 7. Further studies are necessary to identify the tyrosine-phosphorylated proteins whose dephosphorylations are regulated by -mediated mechanisms and their linkage with PTPases as well as PTKs. These efforts will lead to more complete understandings of the functional roles of PTPases.


Figure 7: A model for the aggregation-dependent tyrosine dephosphorylation in the cytoskeleton of thrombin-activated platelets. A, prior to aggregation, stimulation of platelets with thrombin leads to activation of some PTKs, substrates of which reside or relocate mainly in the cytoskeleton, translocation of PTP1C to the cytoskeleton, and activation of . Then fibrinogen is bound to . B, under stirring conditions, activated platelets aggregate through -fibrinogen binding. Aggregation induces cytoskeletal reorganization, calpain-catalyzed cleavage of PTP1B, further relocation of PTP1C to the cytoskeleton, and activation of some PTKs that phosphorylate proteins including PTP1C. In the cytoskeleton, two forms of PTP1B and tyrosine-phosphorylated PTP1C possibly dephosphorylate their substrates, which were phosphorylated on tyrosine residues prior to aggregation. ThR, thrombin receptor; PTKs, protein-tyrosine kinases; Tyr, tyrosine; PY, phosphotyrosine residues.




FOOTNOTES

*
This work was supported by a grant-in-aid for general scientific research from the Ministry of Education, Science and Culture, Japan. This study was presented at the 1994 Annual Meeting of the American Society of Hematology, Nashville, TN and published in abstract form (Ezumi, Y., Takayama, H., Ichinohe, T., Hirai, K., Tomo, K., and Okuma, M. (1994) Blood 84 , 321a). 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. Tel.: 81-75-751-3151; Fax: 81-75-751-3201.

The abbreviations used are: PTK, protein-tyrosine kinase; PTPase, protein-tyrosine phosphatase; SH2, Src homology 2; RGDS, Arg-Gly-Asp-Ser; IgG, immunoglobulin G; PMSF, phenylmethylsulfonyl fluoride; RIPA, radioimmunoprecipitation assay; PAGE, polyacrylamide gel electrophoresis; EST, ethyl (+)-(2S, 3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)]-2-oxiranecarboxylate; MDL, carbamic acid, [1-[[(1-formyl-2-phenylmethyl)amino]carbonyl]-2-methylpropyl]-, phenylmethyl ester.

Y. Ezumi, H. Takayama, and M. Okuma, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Dr. M. Tamai for providing the EST, to Dr. E. H. W. Bohme for providing the MDL, and to Shoko Okamoto for secretarial assistance.


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