Osamu Hazeki

Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase

Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.

Heterodimeric Phosphoinositide 3-Kinase Consisting of p85 and p110β Is Synergistically Activated by the βγ Subunits of G Proteins and Phosphotyrosyl Peptide

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the βγ subunits of G protein (Gβγ) from rat liver. The Gβγ-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110β and p85 (or their related subunits). The stimulation by Gβγ was inhibited by the GDP-bound α subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of Gβγ was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110β/p85α expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110α and p85 in the same rat liver, together with a recombinant p110α/p85α, was not activated by Gβγ, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110β/p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.

Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3-kinase rather than receptor-operated Ca2+ influx.

Superoxide anion and arachidonic acid were produced in guinea pig neutrophils in response to a chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Both responses were markedly, but the former response to a phorbol ester was not at all, inhibited when the cellular cAMP level was raised by prostaglandin E1 combined with a cAMP phosphodiesterase inhibitor. Increasing cAMP was also inhibitory to fMLP-induced activation of phosphatidylinositol (PI) 3-kinase and Ca2+ influx without any effect on the cation mobilization from intracellular stores. The fMLP-induced respiratory burst was abolished when PI 3-kinase was inhibited by wortmannin or LY294002, but was not affected when Ca2+ influx was inhibited. On the contrary, fMLP released arachidonic acid from the cells treated with the PI 3-kinase inhibitors as well as from nontreated cells, but it did not so when cellular Ca2+ uptake was prevented. The chemotactic peptide activated PI 3-kinase even in cells in which the receptor-mediated intracellular Ca2+ mobilization and respiratory burst were both abolished by exposure of the cells to a permeable Ca2+-chelating agent. Thus, stimulation of fMLP receptors gave rise to dual effects, activation of PI 3-kinase and intracellular Ca2+ mobilization; both effects were necessary for the fMLP-induced respiratory burst. Increasing cellular cAMP inhibited the respiratory burst and arachidonic acid release as a result of the inhibitions of PI 3-kinase and Ca2+ influx, respectively, in fMLP-treated neutrophils.

TYROSINE PHOSPHORYLATION OF THE C-CBL PROTO-ONCOGENE PRODUCT MEDIATED BY CELL SURFACE ANTIGEN CD38 IN HL-60 CELLS

The human cell surface antigen CD38 is a 46-kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We demonstrated previously that the extracellular domain of CD38 has NAD glycohydrolase (NADase) activity and that the ecto-form NADase activity induced in HL-60 cells during cell differentiation by retinoic acid is due to CD38. In the present study, we investigated the intracellular signaling mediated by CD38 in retinoic acid-differentiated HL-60 cells with an anti-CD38 monoclonal antibody. The addition of anti-CD38 monoclonal antibody to the cells induced rapid tyrosine phosphorylation of the cellular proteins with molecular weights of 120,000, 87,000, and 77,000. An increase in tyrosine kinase activity in the anti-phosphotyrosine immunoprecipitates of the cells was also observed after the addition of anti-CD38 monoclonal antibody. Moreover, one of the prominent tyrosine-phosphorylated proteins stimulated by the anti-CD38 monoclonal antibody was identified as the c-cbl proto-oncogene product, p120. These results indicated that tyrosine phosphorylation of cellular proteins, including p120, is possibly involved in transmembrane signaling mediated by CD38.

Opposite Effects of Wortmannin and 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one Hydrochloride on Toll-Like Receptor-Mediated Nitric Oxide Production: Negative Regulation of Nuclear Factor-κB by Phosphoinositide 3-Kinase

A number of previous studies have suggested the involvement of phosphoinositide 3-kinase (PI3K) in Toll-like receptor (TLR) signaling. However, there have also been a number of conflicting reports. The PI3K inhibitor wortmannin greatly enhanced TLR-mediated inducible nitric-oxide synthase (iNOS) expression and cytokine production in the mouse macrophage cell line Raw264.7. The effect of wortmannin was common to TLR2, -3, -4, and -9 and was accompanied by activation of nuclear factor-κB and up-regulation of cytokine mRNA production. We were surprised to find that another PI3K inhibitor, LY294002, strongly suppressed the production of iNOS and cytokines. This effect of 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) was based on its inhibitory effect on mRNA synthesis. Expression of dominant-negative mutants of PI3K in macrophages augmented the lipopolysaccharideinduced expression of iNOS. Introduction of a pH1 vector producing short hairpin RNA that targets a catalytic subunit of PI3K (p110β) also enhanced the TLR-mediated responses. Thus, the augmentation of TLR signals by wortmannin was mediated through the inhibition of PI3K, whereas the effect of LY294002 was not explained by its effect on PI3K. These discrepancies in the effects of pharmacological inhibitors in TLR-signaling may have caused confusion regarding the role of PI3K in innate immunity.

Specific association of phosphatidylinositol 3-kinase with the protooncogene product Cbl in Fcγ receptor signaling

A tyrosine‐phosphorylated protein with a molecular mass of 115 kDa was reported to be tightly associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3‐kinase, when the enzyme was essentially activated upon ligand engagement of Fcγ receptors (FcγR) leading to engulfment of IgG‐coated erythrocytes by phagocytes [Ninomiya et al. (1994) J. Biol. Chem. 269, 22732‐22737]. Here, the 115‐kDa protein is identified as the product of human c‐cbl protooncogene. Cross‐linking of FcγRII on the surface of THP‐1 cells triggered (a) prominent tyrosine phosphorylation of Cbl, (b) activation of PI 3‐kinase that was immunoprecipitated with the anti‐Cbl or the anti‐phosphotyrosine antibody, and (c) specific association of Cbl with p85, Thus, Cbl functions in phagocytes as a result of its association with PI 3‐kinase in response to FcγR ligation.

Activation of PI 3-kinase by G protein βγ subunits

Abstract We have reported that fMLP-induced activation of pertussis toxin-sensitive GTP-binding proteins in THP-1 cells potentiates the insulin-induced accumulation of PtdIns(3,4,5)P 3 , a product of phosphoinositide 3-kinase (T. Okada et al., Biochem. J. 317 , 475–480, 1996). The synergism in PtdIns(3,4,5)P 3 accumulation was observed in Chinese hamster ovary cells expressing both insulin and fMLP receptors. In rat adipocytes, which represent the physiological target cells of insulin, receptor-mediated activation of GTP-binding protein by adenosine and prostaglandin E 2 potentiated the insulin-induced PtdIns(3,4,5)P 3 accumulation. In cell-free systems, the activity of the p85/p110β subtype of phosphoinositide 3-kinase was, while that of p85/p110α was not, stimulated by the βγ subunits of the GTP-binding proteins. We propose here a hypothesis that the p85/p110β subtype is under the control of both the insulin receptors and the GTP-binding protein-coupled receptors in intact cell systems.

A Naphthoquinone Derivative, Shikonin, Has Insulin-Like Actions by Inhibiting Both Phosphatase and Tensin Homolog Deleted on Chromosome 10 and Tyrosine Phosphatases

The 1,4-naphthoquinone derivative, shikonin, has been shown to increase glucose uptake by adipocytes and myocytes with minor effects on protein tyrosine phosphorylation in the cells (Biochem Biophys Res Commun 292:642-651, 2002). The present study was performed to examine the mechanism of this action of shikonin. Shikonin inhibited the phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3) phosphatase activity of recombinant phosphatase and tensin homolog deleted on chromosome 10 (PTEN) with an IC50 value of 2.7 μM. Shikonin induced marked accumulation of PtdIns-3,4,5-P3 and activation of protein kinase B (PKB) in Chinese hamster ovary cells expressing insulin receptors. In addition to its effect on PTEN, shikonin was found to inhibit several protein phosphatases in cell-free systems. Its effect on tyrosine phosphorylation in intact cells was far weaker than that of pervanadate, a widely used tyrosine phosphatase inhibitor, despite the observation that the effect of shikonin on PKB was more potent than that of pervanadate. These results suggested that the inhibition of PTEN provides a clue to its potent insulin-like actions. We also found that naphthoquinones, including 1,2-naphthoquinone, inhibit PTEN in the cell-free system, which suggested that the effect on PTEN (and thus the effect on phosphatidylinositol 3-kinase signaling) should be taken into account when examining the pharmacological actions of naphthoquinone derivatives.

Association of phosphatidylinositol 3‐kinase with the photo‐oncogene product Cbl upon CD38 ligation by a specific monoclonal antibody in THP‐1 cells

We reported that ecto‐NAD+ glycohydrolase activity induced upon differentiation of HL‐60 cells with retinoic acid is localized on the extracellular domain of CD38 and that CD38 ligation by a specific monoclonal antibody, HB‐7, is followed by rapid tyrosine phosphorylation of cellular proteins including a proto‐oncogene product, Cbl. In the present study, we investigated intracellular signaling linked to the HB‐7‐induced Cbl phosphorylation in dibutyryl cAMP‐treated THP‐1 cells. The 85‐kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3‐kinase was immunoprecipitated with anti‐Cbl antibody in a manner dependent on the tyrosine phosphorylation of Cbl. PI 3‐kinase activity was also observed in the immunoprecipitated fractions containing tyrosine‐phosphorylated Cbl. The phosphorylated form of Cbl, which had been separated from the CD38‐stimulated cells, was capable of directly binding to a recombinant p85 fused to glutathione S‐transferase. Thus, the direct association of tyrosine‐phosphorylated Cbl with PI 3‐kinase, possibly leading to the kinase activation, appeared to be involved in intracellular signaling caused by the CD38 ligation.

Involvement of the βγ subunits of inhibitory GTP-binding protein in chemoattractant receptor-mediated potentiation of cyclic AMP formation in guinea pig neutrophils

Abstract Cellular cyclic AMP formation in response to prostaglandin (PG) E1 was markedly potentiated by the chemoattractant formyl-Met-Leu-Phe (fMLP) in guinea pig neutrophils. This potentiation by fMLP was abolished by prior treatment of the cells with pertussis toxin, but not by the prevention of an fMLP-induced intracellular Ca z+ increase in the cells, indicating the direct involvement of the inhibitory GTP-binding protein (G1), but not Ca2+, in the fMLP-induced potentiation of cyclic AMP formation. Cyclic AMP formation in the neutrophils was also unique in response to forskolin; the diterpene inhibited cyclic AMP formation stimulated by PGE1 plus fMLP at low concentrations, but it slightly stimulated the basal and fMLP-induced cyclic AMP formation at high concentrations. Such a forskolin-induced inhibition was also observed in the adenylyl cyclase of the cell membranes and detergent extract therefrom only when the cyclase was activated by GTP or its nonhydrolyzable analogue (GTPγS). The forskolin-inhibitable activity could be affinity-purified from the GTPγS-treated cell membranes with a forskolin-agarose column. The cyclase appeared to be purified as a complex with the GTPγS-bound α subunit of the stimulatory GTP-binding protein (Gsα), but not with the (βγ subunits, as judged from immunoblot analysis with specific antisera. The GTPγS-bound Gsα-stimulated cyclase activity was further enhanced by (βγ, and this enhancement was again inhibited by forskolin. These results suggest that the GTP-bound Gsα produced by PGE1 receptor stimulation and the (βγ subunits released from G1 by fMLP receptor stimulation were acting synergistically in the cyclic AMP formation of intact neutrophils.