Inositol polyphosphate 4-phosphatase is inactivated by calpain-mediated proteolysis in stimulated human platelets.

Inositol polyphosphate 4-phosphatase (4-phosphatase), an enzyme that catalyzes the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate, was shown to be a substrate for the calcium-dependent protease calpain in vitro and in stimulated human platelets. Stimulation of platelets with the calcium ionophore, A23187, resulted in complete proteolysis of 4-phosphatase and a 75% reduction in enzyme activity. Thrombin stimulation of platelets resulted in partial proteolysis of 4-phosphatase and a 41% reduction in enzyme activity (n = 8, range of 36-51%). In addition, preincubation with the calpain inhibitor, calpeptin, suppressed the accumulation of phosphatidylinositol 3,4-bisphosphate in thrombin-stimulated platelets by 36% (n = 2, range = 35-37%). These data suggest that the calpain-mediated inhibition of 4-phosphatase is involved in the phosphatidylinositol 3,4-bisphosphate accumulation in thrombin-stimulated platelets.

An enzyme recently implicated in the degradation of PtdIns(3,4)P 2 is inositol polyphosphate 4-phosphatase (4-phosphatase). The 4-phosphatase was originally characterized as a Mg 2ϩ -independent enzyme that catalyzed the hydrolysis of the 4-position phosphate of Ins(3,4)P 2 and inositol 1,3,4-triphosphate (11). Recently, 4-phosphatase has been shown to preferentially hydrolyze the analogous lipid, PtdIns(3,4)P 2 , with a first order rate constant 2 orders of magnitude greater than that obtained using the soluble substrates (12). Antiserum raised against a C-terminal peptide of 4-phosphatase immunoprecipitates Ͼ95% of the PtdIns(3,4)P 2 phosphatase activity from rat brain supernatant (13). Human and rat brain 4-phosphatase cDNA have been cloned and predict proteins that are highly conserved with 97% amino acid identity (13). Sequence analysis of 4-phosphatase indicated the presence of PEST sequences; proline, glutamate/aspartate, serine/threonine rich motifs, that are common features of proteins that are substrates for the calcium-dependent thiol protease, calpain (14,15). Several proteins important for signal transduction have been shown to be regulated by calpain-mediated proteolysis, including protein phosphotyrosine phosphatase 1B (16), phospholipase C-␤ 3 (17), and integrin ␤ 3 (18).
In this study, we demonstrate that 4-phosphatase is a substrate for calpain and is inactivated by calpain-mediated proteolysis in vitro and in stimulated human platelets. In addition, we show that calpain inhibition suppresses the accumulation of PtdIns(3,4)P 2 in thrombin-stimulated human platelets. These results suggest a role for 4-phosphatase in the regulation of intracellular PtdIns(3,4)P 2 levels.
In Vitro Calpain Treatment of Recombinant 4-Phosphatase-Six histidine-tagged recombinant human 4-phosphatase was expressed in Escherichia coli using the 6HisTrc vector (Clontech), and recombinant protein was purified on a nickel-nitrilotriacetic acid-agarose column (Qiagen). Recombinant six-histidine-tagged 4-phosphatase (100 ng/ml) was incubated with various amounts of type I porcine calpain in 20 mM Hepes (pH 7.5), 100 mM NaCl, 2 mM EDTA, 3 mM CaCl 2 , and 1 mM dithiothreitol for 10 min at 37°C. Reactions were stopped by the addition of 10 mM EDTA and 1 g of calpastatin/ml.
Platelet Preparation and Stimulation-Washed platelets were prepared from plasma obtained from healthy donors as described previously (19) with the modifications that the platelet-rich plasma was incubated at 37°C for 20 min with 1 mM acetylsalicylic acid and 10 M prostaglandin E 1 prior to centrifugation. Platelets (10 9 /ml) were stimulated by 1 M A23187 or 1 unit of thrombin/ml in 15 mM Tris (pH 7.4), 140 mM NaCl, 5.5 mM glucose, and 2.5 mM CaCl 2 (platelet aggregation buffer) unless otherwise indicated. Platelets were stirred at 37°C using a aggregometer (Payton).
Preparation of Platelet Lysates and 4-Phosphatase Activity Assay-Platelets lysates were prepared by the addition of 0.5 ml of 2% Triton X-100, 20 mM Hepes (pH 7.5), 10 mM EDTA, 10 g of leupeptin/ml, and * This work was supported by Grants HL 16634 and HL 55672 from the National Institutes of Health and by the American Society of Hematology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The abbreviations used are: PtdIns 3-kinase, phosphatidylinositol 3-kinase; PtdIns(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; 4-phosphatase, inositol-polyphosphate 4-phosphatase; TBS, Tris-buffered saline; HPLC, high performance liquid chromatography.
4-Phosphatase Immunoblotting-Platelet suspensions were boiled for 4 min in SDS loading buffer containing 50 mM Tris (pH 6.8), 2% SDS, 10% ␤-mercaptoethanol 0.05% bromphenol blue, 10 mM EDTA, and 10 g of leupeptin/ml. Samples were separated by SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were blocked with 5% powdered milk, 0.05% Tween 20 in Tris-buffered saline (TBS) for 1 h at room temperature and then incubated with 1:2000 dilution of rabbit antiserum directed against the 4-phosphatase C-terminal peptide in TBS containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature. Membranes were washed with TBS-T and then incubated with 1:4000 diluted anti-rabbit IgG-Horseradish peroxidase (Amersham Corp.) in TBS-T for 1 h at room temperature. Membranes were then washed with TBS-T and 4-phosphatase was detected by Supersignal™ chemiluminescence reagents (Pierce) and Biomax™ Film (Eastman Kodak Co.). 4 Labeling of Platelets, Phospholipid Extraction, and Deacylation-Platelets were labeled by incubating 10 9 platelets/ml suspended in platelet aggregation buffer without CaCl 2 containing 1 mCi 32 P0 4 /ml for 1.5 h at 37°C. Platelets were then centrifuged at 4000 ϫ g for 1 min and resuspended at 10 9 platelets/ml in platelet aggregation buffer. Platelets were stimulated as indicated, and the phospholipids were extracted and deacylated as described previously (19,20).

PO
HPLC Analysis of Glycerophosphorylinositols-Glycerophosphorylinositols were separated on a Partisil SAX column using flow rate of 1 ml/min and a gradient of 0 -1 M ammonium phosphate (pH 3.8) consisting of linear gradient from O to 25% solvent B over 60 min followed by a linear gradient to 100% over 50 min (pump A: water, pump B: 1 M ammonium phosphate). 32 P0 4 -labeled glycerophosphorylinositols derivatives were detected using an A-100 radioactive flow detector (Radiomatic).
Miscellaneous Techniques-Protein concentration was determined using the Bio-Rad protein assay reagent. Lipid phosphate was determined using the method of Ames and Dubin (21).

RESULTS AND DISCUSSION
The presence of PEST sequences in the predicted amino acid sequence of 4-phosphatase suggested that this enzyme might be a substrate for the calcium-dependent protease, calpain (13). As shown in Fig. 1A, immunoblot analysis of recombinant 4-phosphatase treated with calpain in vitro using anti-4-phosphatase C-terminal peptide antiserum indicates that the enzyme is a substrate for calpain. A proteolytic fragment of 104 kDa is detected using this antiserum, indicating that a calpain cleavage site exists near the 4-phosphatase N terminus. However, this 104-kDa fragment is proteolyzed further to fragments that are not detected by this antiserum, indicating the presence of at least one additional cleavage site near the Cterminal epitope. This proteolysis results in a 75% decrease in the activity of recombinant 4-phosphatase (Fig. 1B). The 25% residual 4-phosphatase activity is resistant to calpain treatment with 500 ng/ml (data not shown).
To determine whether 4-phosphatase is a substrate for calpain in vivo, human platelets were stimulated with calcium ionophore A23187, an agonist known to activate platelet calpain in the presence of extracellular calcium (16,17). As shown in Fig. 2A, immunoblot analysis of lysates prepared from unstirred platelets stimulated with 1 M A23187 using anti-4phosphatase C-terminal peptide antiserum indicates that the full-length 105-kDa 4-phosphatase is rapidly (half-life of 2 min) and completely proteolyzed in the presence extracellular calcium with the generation of a 39-kDa immunoblotting proteolytic fragment (lanes 1-5). In addition, the presence of EDTA (lane 6) or preincubation of platelets with the cell-permeant calpain inhibitor, calpeptin (lane 7), blocks A23187-stimulated proteolysis of 4-phosphatase ( Fig. 2A). This proteolysis of 4-phosphatase correlates with a 75% decrease in the observed enzyme activity in platelet lysates which was prevented by the presence of EDTA or preincubation with calpeptin (Fig. 2B). The remaining 25% of platelet 4-phosphatase activity was not inactivated by 30-min ionophore stimulation (data not shown). The amount of enzyme activity that is resistant to calpainmediated proteolysis in platelets is similar to that observed when 4-phosphatase is treated with calpain in vitro.
Stimulation of platelets with the physiological agonist, thrombin, is also known to activate platelet calpain via a mechanism that requires both extracellular calcium (17) and platelet aggregation (22). As shown in Fig. 3A, immunoblot analysis of lysates prepared from stirred platelets stimulated for 5 min with 1 unit/ml thrombin indicates that the 4-phosphatase is partially proteolyzed in the presence of extracellular calcium resulting in a 39-kDa immunoblotting proteolytic fragment similar to that observed in ionophore-stimulated platelets (lane 2). This proteolysis was blocked by preincubation with calpeptin (lane 3), the presence of RGDS, a tetrapeptide that prevents platelet aggregation (lane 4), or the presence of EDTA (lane 5). Thrombin stimulation resulted in an average decrease of 41% (n ϭ 8, range of 36 -51%) in 4-phosphatase activity observed in platelet lysates, and this activity decrease was blocked by the agents that prevent calpain-mediated proteolysis (Fig. 3B).
To evaluate the possible role of calpain-mediated inactiva-tion of 4-phosphatase on the levels of PtdIns(3,4)P 2 in thrombin-stimulated platelets, the effect of calpain inhibition on the accumulation of PtdIns(3,4)P 2 was measured. As shown in Fig.  4, preincubation of platelets with calpeptin prior to thrombin stimulation suppressed the accumulation of PtdIns(3,4)P 2 by an average of 36% (n ϭ 2, range of 35-37%). A similar suppression of PtdIns(3,4)P 2 levels of approximately 40% has been reported previously for platelets stimulated with thrombin in the absence of extracellular calcium (23) or in the presence of RGDS (24,25), two factors known to prevent calpain activation. PtdIns(3,4)P 2 accumulation in thrombin-stimulated platelets is biphasic with a rapid (within 20 s) calcium-independent phase and a slow (after 90 s) calcium-dependent phase (23). It has been proposed that the biphasic rise in PtdIns(3,4)P 2 levels is a result of a rapid activation of PtdIns 3-kinase followed by a slower calcium/aggregation-dependent inactivation of a PtdIns(3,4)P 2 phosphatase that produces the further sustained rise in PtdIns(3,4)P 2 (23,25). The data reported here are consistent with this model and suggest a mechanism involving the inactivation of 4-phosphatase by calpain-mediated proteolysis to produce the calcium/aggregation-dependent accumulation of PtdIns(3,4)P 2 in thrombin-stimulated platelets.