COMMUNICATION:
Inositol Polyphosphate 4-Phosphatase Is Inactivated by Calpain-mediated Proteolysis in Stimulated Human Platelets*

(Received for publication, February 19, 1997)

F. Anderson Norris , Robert C. Atkins and Philip W. Majerus

From the Division of Hematology, Departments of Internal Medicine and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

The activation of phosphatidylinositol 3-kinases (PtdIns 3-kinase)1 is an essential element of receptor-mediated signal transduction that results in the rapid accumulation of two potential lipid second messengers, phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) (1-3). Although the intracellular targets of these lipids are unknown, both have been shown to activate calcium-independent isozymes of protein kinase C in vitro (4, 5). Moreover, the phosphorylation of a major protein kinase C substrate, pleckstrin, in thrombin-stimulated human platelets correlates temporally with rising levels of PtdIns(3,4)P2 (6) and the addition of exogenous PtdIns(3,4)P2 or PtdIns(3,4,5)P3 to permeabilized human platelets results in the pleckstrin phosphorylation (6, 7). Furthermore, growth factor-mediated activation of the serine/threonine protein kinase PKB/Akt requires the activation of PtdIns 3-kinase (8, 9), and the pleckstrin homology domain of PKB/Akt, a domain implicated in the binding of phosphoinositides, is required for its activation in vivo (9). Recently, PKB/Akt has been shown to be specifically activated by PtdIns(3,4)P2 in vitro (10). These data suggest that PtdIns(3,4)P2 and PtdIns(3,4,5)P3 function as activators of protein kinases.

An enzyme recently implicated in the degradation of PtdIns(3,4)P2 is inositol polyphosphate 4-phosphatase (4-phosphatase). The 4-phosphatase was originally characterized as a Mg2+-independent enzyme that catalyzed the hydrolysis of the 4-position phosphate of Ins(3,4)P2 and inositol 1,3,4-triphosphate (11). Recently, 4-phosphatase has been shown to preferentially hydrolyze the analogous lipid, PtdIns(3,4)P2, 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 >95% of the PtdIns(3,4)P2 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-beta 3 (17), and integrin beta 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)P2 in thrombin-stimulated human platelets. These results suggest a role for 4-phosphatase in the regulation of intracellular PtdIns(3,4)P2 levels.


EXPERIMENTAL PROCEDURES

Materials

Bovine thrombin, A23187, Triton X-100, dithiothreitol, prostaglandin E1, and acetylsalicylic acid were purchased from Sigma. Pefabloc® SC and leupeptin were purchased from Boehringer Mannheim. Porcine erythrocyte calpain type I and human recombinant calpastatin were purchased from Calbiochem. Calpeptin was purchased from LC Laboratories. [32P]H3PO4 was purchased from ICN Biomedicals. Ins(3,4)P2 and [3H]Ins(3,4)P2 were prepared as described previously (11).

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 CaCl2, and 1 mM dithiothreitol for 10 min at 37 °C. Reactions were stopped by the addition of 10 mM EDTA and 1 µg 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 µM prostaglandin E1 prior to centrifugation. Platelets (109/ml) were stimulated by 1 µM 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 CaCl2 (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 µg of leupeptin/ml, and 1 mM Pefabloc® to a 0.5-ml suspension of platelets (109/ml). Platelet lysates were assayed for 4-phosphatase activity using [3H]Ins(3,4)P2 as substrate as described previously (11).

4-Phosphatase Immunoblotting

Platelet suspensions were boiled for 4 min in SDS loading buffer containing 50 mM Tris (pH 6.8), 2% SDS, 10% beta -mercaptoethanol 0.05% bromphenol blue, 10 mM EDTA, and 10 µg 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 SupersignalTM chemiluminescence reagents (Pierce) and BiomaxTM Film (Eastman Kodak Co.).

32PO4 Labeling of Platelets, Phospholipid Extraction, and Deacylation

Platelets were labeled by incubating 109 platelets/ml suspended in platelet aggregation buffer without CaCl2 containing 1 mCi 32P04/ml for 1.5 h at 37 °C. Platelets were then centrifuged at 4000 × g for 1 min and resuspended at 109 platelets/ml in platelet aggregation buffer. Platelets were stimulated as indicated, and the phospholipids were extracted and deacylated as described previously (19, 20).

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). 32P04-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 C-terminal 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).


Fig. 1. Immunoblot analysis and enzyme activity of recombinant inositol-polyphosphate 4-phosphatase-treated with calpain in vitro. 100 ng/ml of six His-tagged human 4-phosphatase was treated for 10 min at 37 °C with the amounts of type II calpain indicated. Proteolysis was detected by immunoblot analysis using a C-terminal peptide antiserum (A), and the effect of proteolysis on enzyme activity was determined using Ins(3,4)P2 as substrate (B).
[View Larger Version of this Image (41K GIF file)]


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 µM A23187 using anti-4-phosphatase 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 calpain-mediated proteolysis in platelets is similar to that observed when 4-phosphatase is treated with calpain in vitro.


Fig. 2. Immunoblot analysis and enzyme activity of inositol-polyphosphate 4-phosphatase from calcium ionophore-stimulated human platelets. The anti-4-phosphatase immunoblot (A) and enzyme activity using Ins(3,4)P2 as substrate (B) was determined for unstirred human platelets stimulated with 1 µM A23187 for 0 min (lane 1), 1 min (lane 2), 2 min (lane 3), 5 min (lane 4), 10 min (lane 5), 10 min following pretreatment with 30 µM calpeptin (lane 6), and 10 min in the presence of 2 mM EDTA (lane 7). The 4-phosphatase specific activity of lysates from unstimulated platelets was 6 × 10-4 µmol/min/mg of protein.
[View Larger Version of this Image (72K GIF file)]


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 = 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).


Fig. 3. Immunoblot analysis and enzyme activity of inositol-polyphosphate 4-phosphatase from thrombin-stimulated human platelets. The anti-4-phosphatase immunoblot (A) and 4-phosphatase activity using Ins(3,4)P2 as substrate (B) were determined for stirred human platelet that were unstimulated (lane 1), stimulated with 1 unit/ml thrombin (lane 2), stimulated with 1 unit/ml thrombin after pretreatment with 30 µM calpeptin (lane 3), in the presence of 0.5 mM RGDS (lane 4) or 2 mM EDTA (lane 5). For the immunoblot in A, the 39-kDa fragment was detected with an exposure five times longer than that used to detect the 105-kDa 4-phosphatase. The error bars in B indicate the range of enzyme activity values. The 4-phosphatase specific activity of lysates from unstimulated platelets was 6 × 10-4 µmol/min/mg of protein.
[View Larger Version of this Image (58K GIF file)]


To evaluate the possible role of calpain-mediated inactivation of 4-phosphatase on the levels of PtdIns(3,4)P2 in thrombin-stimulated platelets, the effect of calpain inhibition on the accumulation of PtdIns(3,4)P2 was measured. As shown in Fig. 4, preincubation of platelets with calpeptin prior to thrombin stimulation suppressed the accumulation of PtdIns(3,4)P2 by an average of 36% (n = 2, range of 35-37%). A similar suppression of PtdIns(3,4)P2 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)P2 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)P2 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)P2 phosphatase that produces the further sustained rise in PtdIns(3,4)P2 (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)P2 in thrombin-stimulated platelets.


Fig. 4. The effect of calpain inhibitor on the accumulation of PtdIns(3,4)P2 in thrombin-stimulated human platelets. PtdIns(3,4)P2 (cpm/nmol total lipid phosphate) was measured by HPLC of deacylated lipid for [32P]PO4-labeled human platelets stimulated with 1 unit of thrombin/ml for 5 min without pretreatment (sample 1) and with pretreatment with 30 µM calpeptin (sample 2). The error bars indicate the range of values.
[View Larger Version of this Image (55K GIF file)]



FOOTNOTES

*   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. The 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)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; 4-phosphatase, inositol-polyphosphate 4-phosphatase; TBS, Tris-buffered saline; HPLC, high performance liquid chromatography.

ACKNOWLEDGEMENTS

We thank Cecil Buchanan for his assistance with the phosphate assays and Dr. Monita Wilson and Xiaoling Zhang for their helpful discussions.


REFERENCES

  1. Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175 [Medline] [Order article via Infotrieve]
  2. Traynor-Kaplan, A. E., Thompson, B. L., Harris, A. L., Taylor, P., Omann, G. V., and Sklar, L. A. (1989) J. Biol. Chem. 264, 15668-15673 [Abstract/Free Full Text]
  3. Jackson, T. R., Stephens, L. R., and Hawkins, P. T. (1992) J. Biol. Chem. 267, 16627-16636 [Abstract/Free Full Text]
  4. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13-16 [Abstract/Free Full Text]
  5. Toker, A., Meyer, M., Reddy, K. K., Falck, J. R., Aneja, R., Aneja, S., Parra, A., Burns, D. J., Ballas, L. M., and Cantley, L. C. (1994) J. Biol. Chem. 269, 32358-32367 [Abstract/Free Full Text]
  6. Toker, A., Bachelot, C., Chen, C.-S., Falck, J. R., Hartwig, J. H., Cantley, L. C., and Kovacsovics, T. J. (1995) J. Biol. Chem. 270, 29525-29531 [Abstract/Free Full Text]
  7. Zhang, J., Falck, J. R., Reddy, K. K., Abrams, C. S., Zhao, W., and Rittenhouse, S. E. (1995) J. Biol. Chem. 270, 22807-22810 [Abstract/Free Full Text]
  8. Burgering, B. M. T., and Coffer, P. J. (1995) Nature 376, 599-602 [CrossRef][Medline] [Order article via Infotrieve]
  9. Franke, T. F., Yang, S.-I., Chan, T. O., Datta, K., Kazlauskas, A., Morrison, D. K., Kaplan, D. R., and Tsichlin, P. N. (1995) Cell 81, 727-736 [Medline] [Order article via Infotrieve]
  10. Franke, T. F., Kaplan, D. R., Cantley, L. C., and Toker, A. (1997) Science 275, 665-668 [Abstract/Free Full Text]
  11. Bansal, V. S., Caldwell, K. K., and Majerus, P. W. (1990) J. Biol. Chem. 265, 1806-1811 [Abstract/Free Full Text]
  12. Norris, F. A., and Majerus, P. W. (1994) J. Biol. Chem. 269, 8716-8720 [Abstract/Free Full Text]
  13. Norris, F. A., Auethavekiat, V., and Majerus, P. W. (1995) J. Biol. Chem. 270, 16128-16133 [Abstract/Free Full Text]
  14. Rogers, S., Wells, R., and Rehsteiner, M. (1986) Science 234, 364-368 [Medline] [Order article via Infotrieve]
  15. Wang, K. K. W., Villalobo, A., and Roufogalis, B. D. (1989) Biochem. J. 262, 693-706 [Medline] [Order article via Infotrieve]
  16. Frangioni, J. V., Oda, A., Smith, M., Salzman, E. W., and Neel, B. G. (1993) EMBO J. 12, 4843-4856 [Abstract]
  17. Banno, Y., Nakashima, S., Hachiya, T., and Nozawa, Y. (1995) J. Biol. Chem. 270, 4318-4324 [Abstract/Free Full Text]
  18. Du, X., Saido, T. C., Tsubuki, S., Indig, F. E., Williams, M. J., and Ginsberg, M. H. (1995) J. Biol. Chem. 270, 26146-26151 [Abstract/Free Full Text]
  19. Baenziger, N. L., and Majerus, P. W. (1974) Methods Enzymol. 31, 149-155 [Medline] [Order article via Infotrieve]
  20. Lips, D. L., and Majerus, P. W. (1989) J. Biol. Chem. 264, 19911-19915 [Abstract/Free Full Text]
  21. Ames, B. N., and Dubin, D. T. (1960) J. Biol. Chem. 235, 769-775 [Medline] [Order article via Infotrieve]
  22. Fox, J. E. B., Taylor, R. G., Taffarel, M., Boyles, J. K., and Goll, D. E. (1993) J. Cell Biol. 120, 1501-1507 [Abstract]
  23. Sorisky, A., King, W. G., and Rittenhouse, S. E. (1992) Biochem. J. 286, 581-584 [Medline] [Order article via Infotrieve]
  24. Sultan, C., Plantavid, M., Bachelot, C., Grondin, P., Breton, M., Mauco, G., Lévy-Toledano, S., Caen, J. P., and Chap, H. (1991) J. Biol. Chem. 266, 23554-23557 [Abstract/Free Full Text]
  25. Rittenhouse, S. E. (1996) Blood 88, 4401-4414 [Free Full Text]

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