(Received for publication, February 19, 1997)
From the Division of Hematology, Departments of Internal Medicine and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110
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.
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-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)P2 in thrombin-stimulated human platelets. These results suggest a role for 4-phosphatase in the regulation of intracellular PtdIns(3,4)P2 levels.
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-PhosphataseSix 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 StimulationWashed 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 AssayPlatelets 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 ImmunoblottingPlatelet 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 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.).
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 GlycerophosphorylinositolsGlycerophosphorylinositols 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 TechniquesProtein concentration was determined using the Bio-Rad protein assay reagent. Lipid phosphate was determined using the method of Ames and Dubin (21).
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).
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.
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).
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.
We thank Cecil Buchanan for his assistance with the phosphate assays and Dr. Monita Wilson and Xiaoling Zhang for their helpful discussions.