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Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan (K.N., K.Y., T.S., E.I., M.T., H.M., K.H., O.H.); and Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan (T.M.)
Received for publication April 16, 2006.
Accepted for publication June 27, 2006.
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Abstract |
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). The SH2 domains of the regulatory subunit of PI 3-kinase bind to the phosphorylated tyrosine residues in the consensus sequence, YXXM. This binding activates PI 3-kinase and results in the accumulation of PtdIns-3,4,5-P3 and PtdIns-3,4-P2. The machinery downstream of PI 3-kinase includes a serine/threonine kinase protein kinase B (PKB, also known as Akt), the activity of which is up-regulated by the products of PI 3-kinase (Alessi and Downes, 1998). Overexpression of a constitutively active form of PKB has been reported to lead to increased glucose uptake and GLUT4 translocation in 3T3L1 adipocytes (Kohn et al., 1996).
The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is an enzyme that dephosphorylates the D3 position of PtdIns-3,4,5-P3 and PtdIns-3,4-P2 (Maehama and Dixon, 1998; Cantley and Neel, 1999). Overexpression of the lipid phosphatase reduces the insulininduced accumulation of PtdIns-3,4,5-P3 in 293 cells (Maehama and Dixon, 1998). Infection with adenovirus expressing PTEN in 3T3L1 adipocytes attenuates both the insulin-induced glucose uptake and the Akt activation (Nakashima et al., 2000). Microinjection of an anti-PTEN antibody potentiates the insulin-induced events (Ono et al., 2001). Thus, PTEN is regarded as a negative regulator of insulin signaling.
Shikon is the dried root of the plant Lithospermum erythrorhizon (Siebold et Zucc.), the extract of which is used in traditional Chinese medicine for the treatment of macular eruptions, measles, sore throat, carbuncles, and burns (Papageorgiou et al., 1999; Chen et al., 2002). With regard to the molecular basis of its actions, shikonin, a naphthoquinone pigment isolated from the root (or its derivatives), have been reported to possess a variety of pharmacological activities, including anti-inflammatory and antitumor activities (Sankawa et al., 1977; Tanaka et al., 1986; Hisa et al., 1998; Hashimoto et al., 2002). Kamei et al. (2002) found another action of shikonin while screening for small molecules with insulin-like actions. The study showed that shikonin effectively increases the glucose uptake of 3T3L1 adipocytes and rat cardiomyocytes. This effect of shikonin is not accompanied by activation of insulin receptor-tyrosine kinase.
Several studies have been performed to clarify the site of action of shikonin in the signal transduction system of cells. In neutrophils, a shikonin derivative, acetylshikonin, has been reported to reduce N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced protein tyrosine phosphorylation by 90% (Wang et al., 1997). This compound inhibits PtdIns-4,5-P2 breakdown and Ins-1,4,5-P3 formation in the cells by inhibiting phospholipase C (Wang and Kuo, 1997). Shikonin has also been reported to inhibit the production of tumor necrosis factor-
by inhibiting the expression of its mRNA (Staniforth et al., 2004). Although these mechanisms may represent the basis of some actions of shikonin, they do not explain its insulin-like action. In the present study, we examined the effects of shikonin on insulin signaling using Chinese hamster ovary cells expressing insulin receptors (CHO-IR cells) as a model system. The results suggested that the inhibitory effect on PTEN provides a clue to its potent insulin-like actions.
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Materials and Methods |
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-32P]ATP were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Monoclonal antibodies against pThr308 and pSer473 of PKB and polyclonal antibodies against PTEN and phospho-PTEN (Ser308) were from Cell Signaling Technology (Beverly, MA). Polyclonal anti-PKB antibody, polyclonal anti-IR
antibody, and monoclonal anti-phosphotyrosine antibody (PY99) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse IgG agarose was from American Qualex Antibodies (San Clemente, CA). pGEX-6P-1, PreScission protease, and PD-10 column were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). pQE-30 expression vector was from QIAGEN (Hilden, Germany). Pervanadate was prepared by mixing sodium orthovanadate and hydrogen peroxide at equimolar concentrations. Naphthoquinone compounds were dissolved in dimethyl sulfoxide before use, and all experiments were performed in the presence of <0.5% dimethyl sulfoxide. Cell Lines. CHO-IR cells were kind gifts from Dr. Yosuke Ebina (Ehime University, Matsuyama, Japan) and were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin.
Recombinant Proteins. cDNAs encoding PTP1B, SHP-1, Cdc25A (GenBank accession numbers NM_002827 [GenBank] , NM_002831 [GenBank] , and NM_001789 [GenBank] , respectively) were obtained by polymerase chain reaction with appropriate cDNA libraries and primers possessing additional nucleotide sequences convenient for subcloning. The cDNA constructs were subcloned into the expression vector pGEX-6P-1 or pQE-30. Glutathione transferase-fused or 6xHis-tagged proteins were expressed in Escherichia coli and adsorbed onto glutathione-Sepharose 4B or high-capacity nickel chelate affinity matrix resin, respectively, in accordance with the manufacturer's instructions. The glutathione beads were incubated with precision protease, and the cleaved proteins were stored at -80°C in Tris-buffered saline containing 1 M dithiothreitol. Histidine-tagged proteins were eluted from the nickel-CAM beads with 250 mM imidazole. The eluate was changed to Tris-buffered saline containing 1 M dithiothreitol using a PD-10 column before storage at -80°C.
Immunoprecipitation and Immunoblotting. The CHO-IR cells were starved of serum and the medium was replaced with incubation buffer consisting of 130 mM NaCl, 4.7 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES-NaOH, pH 7.4, and 0.1% (w/v) bovine serum albumin. After incubation with the indicated compounds at 37°C, the buffer was removed, and the reactions were stopped by adding 1 ml of ice-cold phosphate-buffered saline containing 10 mM NaF and 1 mM sodium orthovanadate. The cells were then lysed in lysis buffer consisting of 25 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 30 mM NaF, 200 µM phenylmethylsulfonyl fluoride, 20 µM (4-amidinophenyl)-methansulfonyl fluoride, 2 µM leupeptin, 2 µM pepstain, and 1% Nonidet P-40. After centrifugation (15,000 rpm for 10 min), aliquots of the supernatant were mixed with SDS-polyacrylamide gel electrophoresis sample buffer and boiled for 5 min. The peptides were separated by SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes (Millipore, Bedford, MA). After blocking, the membranes were incubated with the indicated antibody, washed, and then incubated with horseradish peroxidase-conjugated second antibody. The second antibody was located using an enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences). For immunoprecipitation, the cell lysates were incubated with anti-phosphotyrosine or anti-myc antibody for 1.5 h at 4°C and then mixed with anti-mouse IgG agarose. The immune complex was washed three times with lysis buffer and boiled for 5 min in sample buffer. The peptides were separated and analyzed by immunoblotting as described above.
Protein Kinase B Activity. The CHO-IR cells were cultured on six-well plates and transfected with myc-tagged PKB in the expression vector, pCMV5, as described previously (Kubo et al., 2005). The transfected cells were starved of serum before treatment with insulin or shikonin. The cells were solubilized in 400 µl of lysis buffer and then mixed with anti-myc antibody. The immune complex was washed twice and incubated at 30°C for 15 min in 20 µ l of 50 mM Tris-HCl, pH 7.4, 0.5 mM EGTA, 1 mM dithiothreitol, 10 mM MgCl2, 30 µM Crosstide, and 100 µM [-32P]ATP (0.25 µCi/ml). Aliquots of the incubation mixture were spotted onto P81 anion exchange paper (Whatman, Brentford, UK) to absorb the peptide substrate. The paper was washed vigorously with 1% phosphoric acid, and then the associated radioactivity was determined.
Accumulation of PtdIns-3,4,5-P3. CHO-IR cells, cultured on six-well plates, were starved of serum and labeled with 32Pi (50 µCi/ml) for 1 h. The medium was replaced with incubation buffer, and the cells were stimulated with insulin or shikonin at 37°C. The buffer was removed before the cells were scraped off in 0.6 ml of 8% HClO4. The suspension was mixed with 2.25 ml of chloroform/methanol [1:2 (v/v)] and the extracted lipids were separated onto oxalate-impregnated thin-layer chromatography plates as described previously (Takasuga et al., 1999).
PI 3-Kinase Activity. CHO-IR cells, cultured on six-well plates, were starved of serum and treated with insulin or shikonin. The cells were then solubilized in buffer consisting of 25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 30 mM NaF, 200 µM phenylmethylsulfonyl fluoride, 20 µM (4-amidinophenyl)-methansulfonyl fluoride, 2 µM leupeptin, 2 µM pepstain, and 1% Nonidet P-40. After centrifugation at 15,000 for 20 min, the supernatant was subjected to immunoprecipitation with anti-pTyr antibody. PI 3-kinase activity in the immune complex was determined using phosphatidylinositol as the substrate.
Lipid Phosphatase Activity. PTEN (50 ng) was preincubated at 37°C for 5 min in 10 µl of buffer consisting of 0.1 M Tris-HCl, pH 8.0, 10 mM dithiothreitol, 0.25% octyl glucoside, and 0.1 µg/µl bovine serum albumin, in the presence or absence of the indicated compounds. The phosphatase reaction was started by addition of 0.1 mM diC16-PtdIns-3,4,5-P3 and was stopped after 5 min by the addition of 80 µl of Malachite green reagent, as described previously (Maehama et al., 2000; Schmid et al., 2004). After the color was developed for 15 min, absorbance at 620 nm was measured. The amount of phosphate released was quantified using standard solutions of inorganic phosphate.
pIR Phosphatase Activity. Phosphorylated IR was semipurified from insulin-treated CHO-IR cells using wheat germ agglutinin (WGA)-agarose (Vector Laboratories, Burlingame, CA). His-PTP1B (200 ng) was incubated at 37°C for 10 min in the presence or absence of shikonin in a buffer consisting of 50 mM Tris-HCl, pH 7.6, 100 mM NaCl, and 1 mM dithiothreitol. The tyrosine phosphatase reaction was started by the addition of pIR-bound WGA-agarose beads. After 10 min, the reaction was stopped by the addition of chilled buffer consisting of 50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 0.1 mM sodium orthovanadate, and 1% Nonidet P-40. The beads were washed with the same buffer and boiled in SDS sample buffer for 5 min. The tyrosine phosphorylation of IR was analyzed by immunoblotting.
p-Nitrophenyl Phosphate Phosphatase Activity. His-PTP1B (20 ng), His-SHP1 (500 ng), or Cdc25A (15 µg) was incubated at 37°C for 10 min in 135 µl of reaction buffer containing various concentrations of the inhibitors. The reaction buffer for the activities of PTP1B and SHP1 consisted of 100 mM sodium acetate, pH 5.5, 100 mM NaCl, and 1 mM dithiothreitol, whereas the activity of Cdc25A was assayed in buffer consisting of 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM dithiothreitol. The phosphatase reaction was started by the addition of 15 µl of p-nitrophenyl phosphate (pNPP) solution and stopped after 10 min by the addition of 150 µ l of 1 N NaOH. The concentrations of pNPP approximated the Km values of each phosphatase: PTP1B, 1 mM; SHP1, 26 mM; and Cdc25A, 45 mM. The amount of p-nitrophenol produced was determined from the absorbance at 405 nm. The IC50 value was determined by interpolation of the percentage inhibition versus inhibitor concentration plot.
For examination of the time dependence of the action of shikonin, His-PTP1B (100 ng) was first incubated for various times with 20 µM shikonin in the presence or absence of 5 mM phenylphosphate in Tris-buffered saline containing 0.01% bovine serum albumin and 1 M dithiothreitol. The mixture was diluted 100-fold with a reaction buffer consisting of 100 mM sodium acetate, pH 5.5, 100 mM NaCl, 1 mM dithiothreitol, and 5 mM pNPP. The phosphatase reaction was allowed to proceed for 10 min and was stopped by the addition of 0.5 N NaOH. The samples incubated in the absence of phenylphosphate were diluted with reaction buffer containing 50 µM phenylphosphate. A control experiment was performed by adding shikonin at the start of the phosphatase reaction.
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Results |
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; Alessi and Downes, 1998), we first examined whether shikonin has any effect on PKB in CHO-IR cells. Treatment of the cells with shikonin caused phosphorylation of PKB on both the Ser473 residue in the C-terminal hydrophobic motif and the Thr308 residue in the activation loop (Fig. 1A). The level of phosphorylation reached a maximum 10 min after the addition of shikonin, and the effect of 30 µM shikonin was as potent as 0.1 µM insulin (Fig. 1B). An inhibitor of PI 3-kinase, wortmannin, completely abolished the shikonin-induced phosphorylation and that induced by insulin. Another inhibitor, LY294002, also inhibited the effects of both insulin and shikonin (data not shown). The presence of highly expressed insulin receptors is not required for the shikonin action because the increased phosphorylation of PKB can be observed in the parent CHO cells (data not shown).
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PKB is phosphorylated and activated when the products of PI 3-kinase bind to the PH domain of the enzyme (Alessi and Downes, 1998). Thus, we next examined whether shikonin had any effect on PtdIns-3,4,5-P3 accumulation in the cells. Shikonin induced marked accumulation of [32P]PtdIns-3,4,5-P3 in 32Pi-labeled cells (Fig. 2). The effects of both shikonin and insulin were abolished by wortmannin and LY294002.
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-subunit of the insulin receptor (IR). Shikonin caused tyrosine phosphorylation of a peptide with a mobility similar to that of IR. The effect of shikonin on IR was confirmed when the lysate was first mixed with antipTyr antibody, and the immune complex was analyzed with anti-IR antibody (Fig. 3C).
One possible explanation for the increased tyrosine-phosphorylation of IR was that shikonin directly influenced the autophosphorylation of IR. However, shikonin showed no effect on the activity of insulin receptor-tyrosine kinase, which was purified from both insulin-treated and -untreated CHO-IR cells using WGA-agarose beads (data not shown). An alternative explanation is that the effect of shikonin was mediated by inhibiting the dephosphorylation of IR. Therefore, we examined the effect of shikonin on PTP1B, a tyrosine phosphatase known to dephosphorylate IR. As shown in Fig. 4A, shikonin inhibited the ability of recombinant PTP1B to dephosphorylate the IR prepared from insulin-treated CHO-IR cells. When the PTP1B activity was determined using 1 mM pNPP as the substrate, shikonin inhibited the phosphatase in a dose-dependent manner with an IC50 value of 17 µM (Fig. 4B). Shikonin inhibited the pNPP phosphatase activities of another tyrosine phosphatase, SHP-1, and a dual specificity phosphatase, Cdc25A (Table 1).
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In the experiment shown in Fig. 4C, PTP1B was first incubated with 20 µM shikonin for various times, and the mixture was diluted by 100-fold before measurement of the pNPP phosphatase activity. Prior incubation with shikonin markedly decreased PTP1B activity in the diluted solution. The degree of inhibition was decreased when shikonin treatment was performed in the presence of a poor substrate, phenylphosphate (open symbols). Thus, the action of shikonin is considered to be irreversible and involves binding to the active center of the enzyme, as was the case in inhibition of human prostatic acid phosphatase by 4-difluoromethylphenyl phosphate (Wang et al., 1994).
The above results showed that shikonin possesses the potential ability to activate PI 3-kinase by inactivating tyrosine phosphatases in intact cells. However, it is unlikely that this action is the sole mechanism by which shikonin induces marked accumulation of PtdIns-3,4,5-P3 in the cells. The above results showed that the effects of shikonin on activation of PI 3-kinase (Fig. 3A) and phosphorylation of IR (Fig. 3C) were far weaker than those of insulin. On the other hand, the effects on accumulation of PtdIns-3,4,5-P3 (Fig. 2) and activation of PKB (Fig. 1) were comparable with, or even more effective than the effects of insulin. One possible explanation for this discrepancy is that shikonin prevents the degradation of PtdIns-3,4,5-P3. Thus, we next examined its effect on PTEN, a PtdIns-3,4,5-P3 phosphatase. As shown in Fig. 5A, the expression and phosphorylation status of PTEN was unaffected by the shikonin treatment. In the experiment shown in Fig. 5B, recombinant PTEN was incubated with PtdIns-3,4,5-P3, and the liberation of phosphate was determined by Malachite green assay. Shikonin inhibited the lipid phosphatase activity in a dose-dependent manner with an IC50 value of 2.7 µM. This observation suggested that the effects of shikonin on intact cells are based on not only inhibition of protein tyrosine phosphatases but also on inhibition of PTEN.
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and PKB in CHO-IR cells (Fig. 5C). It is interesting to note that the effect of shikonin on PKB was as prominent as that of pervanadate, although its effect on IR was far weaker (Fig. 5C). This discrepancy may be explained by the fact that vanadate, unlike shikonin, did not inhibit PTEN at concentrations as high as 100 µM (Table 1).
Many naphthoquinone derivatives have been reported to cause diverse effects on intact cells through their effects on protein tyrosine phosphatases (Ham et al., 1998; Ni et al., 1998; Urbanek et al., 2001). Because shikonin is a derivative of 1,4-naphthoquinone, it is intriguing to consider that several effects of the naphthoquinone derivatives are based on their effect on PTEN. Thus, we examined the effects of two simple naphthoquinone compounds, 1,2-naphthoquinone and 5,8-dihydroxy-1,4-naphthoquinone, on PTEN activity. Their effects on PTP1B, SHP-1, and Cdc25A were also examined. As shown in Fig. 6, these compounds inhibited both PTEN and tyrosine phosphatases, although the effective concentrations were different.
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). However, its mechanism of action has not been clarified. The present study suggested that the insulin-like actions of shikonin are due to modulation of PI 3-kinase-dependent signaling. Shikonin induced accumulation of PtdIns-3,4,5-P3, a product of PI 3-kinase, in intact cells (Fig. 2). The activation of the signaling pathway was confirmed by phosphorylation and activation of PKB (Fig. 1). As possible mechanisms of action, shikonin was found to inhibit both the IR phosphatase activity of PTP1B (Fig. 4) and the PtdIns-3,4,5-P3 phosphatase activity of PTEN (Fig. 5) in cell-free systems.
Insulin signaling is initiated by activation of the tyrosine kinase activity of IR and the increased tyrosine phosphorylation of signaling proteins in target cells. Tyrosine phosphatases, including PTP1B, TCPTP, and PTP, have been reported to dephosphorylate and inactivate IR (Moller et al., 1995; Bandyopadhyay et al., 1997; Salmeen et al., 2000; Wälchli et al., 2000; Lacasa et al., 2005). Mice lacking the PTP1B gene show enhanced sensitivity to insulin (Elchebly et al., 1999; Klaman et al., 2000). Insulin-induced phosphorylation of IR and activation of PKB/Akt were enhanced in immortalized TCPTP(-/-) murine embryo fibroblasts (Galic et al., 2003). The results of the present study showed that shikonin, like other naphthoquinone compounds, inhibits tyrosine phosphatases in cell-free systems (Table 1). This inhibition may be one mechanism responsible for its insulin-like action. However, the effect of shikonin on phosphorylation of IR in intact cells was far weaker than those of insulin and pervanadate, despite the observation that the effect on PKB was as potent as or even more potent than that of pervanadate (Fig. 5). These observations suggest that the increased tyrosine phosphorylation is not the sole mechanism of action of shikonin. In agreement with this speculation, low concentrations of shikonin were reported to augment glucose uptake even when tyrosine phosphorylation has been otherwise increased by pervanadate (Kamei et al., 2002).
An intriguing observation in the present study was the inhibition of PTEN by shikonin (Fig. 5). PTEN is a lipid phosphatase that converts the products of PI 3-kinase to their inactive D-3 dephosphorylated forms. The effect on PTEN explained why shikonin caused marked accumulation of PtdIns-3,4,5-P3 with minor effects on PI 3-kinase activity or IR phosphorylation in intact cells (Fig. 3). PTEN possesses a signature motif, HCXXGXXR, present in the active sites of protein tyrosine phosphatases. The results of crystal structure analysis demonstrated similarity between the active pockets of PTEN and tyrosine phosphatases (Lee et al., 1999). Thus, shikonin may inhibit the enzymes by binding to their active pockets. It has been reported that inhibition of Cdc25 by a naphthoquinone derivative involves an interaction with residues within the active site of the enzyme (Kerns et al., 1995; Ham et al., 1997). The results of the present study showed that shikonin inhibits PTP1B in an irreversible manner, and this inhibition is protected by the substrate of the enzyme (Fig. 4C). Thus, shikonin is considered to inhibit PTEN in a similar manner.
We showed that shikonin is an inhibitor of both PTEN and protein tyrosine phosphatases. Its effect on PTEN provides insight into its potent insulin-like actions as discussed above. PTEN is a tumor suppressor, the mutation or deletion of which is observed in approximately 50% of glioblastoma, endometrial carcinoma, prostate carcinoma, and melanoma cases (Cantley and Neel, 1999). It is also known that DJ1, a genetic negative regulator of PTEN, is associated with breast and lung cancers (Cully et al., 2006). In the present study, we showed that naphthoquinone derivatives, including 1,2-naphthoquinone, inhibit PTEN at concentrations similar to that of shikonin. Many naphthoquinone derivatives have been reported to cause diverse effects in intact cells through the inhibition of protein tyrosine phosphatases (Ham et al., 1998; Ni et al., 1998; Urbanek et al., 2001). Their effects on PTEN and thus PI 3-kinase signaling should be taken into account when the pharmacological actions of the naphthoquinone derivatives are examined.
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Footnotes |
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, the subunit of insulin receptor; pNPP, p-nitrophenyl phosphate; PKB, protein kinase B; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; WGA, wheat germ agglutinin; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride. Address correspondence to: Dr. Osamu Hazeki, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan. E-mail: hazeki{at}hiroshima-u.ac.jp
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) or absence (
) of 5 mM phenylphosphate. The mixture was diluted 100-fold before the determination of pNPP phosphatase activity.
), His-PTP1B (
) was incubated at 30°C for 10 min with various concentrations of 1,2-naphthoquinone (A) or 5,8-dihydroxyl-1,4-naphthoquinone (B). The activities of tyrosine phosphatases were determined with pNPP as the substrate. The concentrations of pNPP approximated the Km values of each phosphatase: PTP1B, 1 mM; SHP1, 26 mM; and Cdc25A, 45 mM. The PTEN activity was determined with 0.1 mM diC16-PtdIns-3,4,5-P3 as the substrate.