Antipsychotic-like effects of a novel phosphodiesterase 10A inhibitor MT-3014 in rats.

Phosphodiesterase (PDE) 10A is an attractive therapeutic target for schizophrenia. Here, we investigated the antipsychotic-like effects of a novel PDE10A inhibitor, 1-({2-(7-fluoro-3-methylquinoxalin-2-yl)-5-[(3R)-3-fluoropyrrolidin-1-yl]pyrazolo[1,5-α]pyrimidin-7-yl}amino)-2-methylpropan-2-ol hydrochloride (MT-3014) in rats. MT-3014 showed a potent and selective inhibitory effect against PDE10A (IC50 = 0.357 nmol/L). Oral administration of MT-3014 (1.0-10 mg/kg) significantly increased the levels of cAMP, cGMP and cAMP response element-binding protein (CREB) phosphorylation in the rat striatum. MT-3014 decreased MK-801 (0.075 mg/kg)-induced hyperactivity (ED50 = 0.30 mg/kg) in a dose-dependent manner, although it decreased spontaneous locomotion in control rats (ED50 = 0.48 mg/kg); its effects were equivalent to those of risperidone. MT-3014 (0.3-3.0 mg/kg and 0.2 mg/kg) attenuated MK-801-induced prepulse inhibition deficits and cognitive deficits in rats, respectively, whereas risperidone attenuated MK-801-induced prepulse inhibition at only a high dose and failed to improve MK-801-induced cognitive deficits. Similar to risperidone (ID50 = 0.63 mg/kg), MT-3014 suppressed the conditioned avoidance response (ID50 = 0.32 mg/kg). Interestingly, MT-3014 did not elicit catalepsy and plasma prolactin increases at high doses. Furthermore, it also did not affect body weight. A positron emission tomography study using [11C]IMA107 showed a plasma concentration-dependent increase in brain PDE10A occupancy after oral administration of MT-3014 within the pharmacological dose range in rats. Brain PDE10A occupancy corresponding to the ID50 value in the conditioned avoidance response was approximately 60%, predicting the target occupancy in patients with schizophrenia. These results suggest that MT-3014 may be a novel antipsychotic drug, which is expected to have additional effects on cognitive impairment, without the prominent side effects associated with current atypical antipsychotics.

Important Role of Endothelial Caveolin-1 in the Protective Role of Endothelium-dependent Hyperpolarization Against Nitric Oxide-Mediated Nitrative Stress in Microcirculation in Mice.

AIMS: Nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) play important roles in maintaining cardiovascular homeostasis. We have previously demonstrated that endothelial NO synthase (eNOS) plays diverse roles depending on vessel size, as a NO generating system in conduit arteries and an EDH-mediated system in resistance arteries, for which caveolin-1 (Cav-1) is involved. However, the physiological role of endothelial Cav-1 in microvessels remains to be elucidated. METHODS AND RESULTS: We newly generated endothelium-specific Cav-1-knockout (eCav-1-KO) mice. eCav-1-KO mice showed loss of endothelial Cav-1/eNOS complex and had cardiac hypertrophy despite normal blood pressure. In eCav-1-KO mice, as compared to wild-type controls, the extent of eNOS phosphorylation at inhibitory Thr495 was significantly reduced in mesenteric arteries and the heart. Isometric tension and Langendorff-perfused heart experiments showed that NO-mediated responses were enhanced, whereas EDH-mediated responses were reduced in coronary microcirculation in eCav-1-KO mice. Immunohistochemistry showed increased level of 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a marker of nitrative stress, in the heart from eCav-1-KO mice. S-guanylation of cardiac H-Ras in eCav-1-KO mice was also significantly increased compared with wild-type controls. CONCLUSIONS: These results suggest that eCav-1 is involved in the protective role of EDH against nitrative stress caused by excessive NO to maintain cardiac microvascular homeostasis.

Important role of endothelium-dependent hyperpolarization in the pulmonary microcirculation in male mice: implications for hypoxia-induced pulmonary hypertension.

Endothelium-dependent hyperpolarization (EDH) plays important roles in the systemic circulation, whereas its role in the pulmonary circulation remains largely unknown. Furthermore, the underlying mechanisms of pulmonary hypertension (PH) also remain to be elucidated. We thus aimed to elucidate the role of EDH in pulmonary circulation in general and in PH in particular. In isolated perfused lung and using male wild-type mice, endothelium-dependent relaxation to bradykinin (BK) was significantly reduced in the presence of Nω-nitro-l-arginine by ~50% compared with those in the presence of indomethacin, and the combination of apamin plus charybdotoxin abolished the residual relaxation, showing the comparable contributions of nitric oxide (NO) and EDH in the pulmonary microcirculation under physiological conditions. Catalase markedly inhibited EDH-mediated relaxation, indicating the predominant contribution of endothelium-derived H2O2. BK-mediated relaxation was significantly reduced at day 1 of hypoxia, whereas it thereafter remained unchanged until day 28. EDH-mediated relaxation was diminished at day 2 of hypoxia, indicating a transition from EDH to NO in BK-mediated relaxation before the development of hypoxia-induced PH. Mechanistically, chronic hypoxia enhanced endothelial NO synthase expression and activity associated with downregulation of caveolin-1. Nitrotyrosine levels were significantly higher in vascular smooth muscle of pulmonary microvessels under chronic hypoxia than under normoxia. A similar transition of the mediators in BK-mediated relaxation was also noted in the Sugen hypoxia mouse model. These results indicate that EDH plays important roles in the pulmonary microcirculation in addition to NO under normoxic conditions and that impaired EDH-mediated relaxation and subsequent nitrosative stress may be potential triggers of the onset of PH. NEW & NOTEWORTHY This study provides novel evidence that both endothelium-dependent hyperpolarization and nitric oxide play important roles in endothelium-dependent relaxation in the pulmonary microcirculation under physiological conditions in mice and that hypoxia first impairs endothelium-dependent hyperpolarization-mediated relaxation, with compensatory upregulation of nitric oxide, before the development of hypoxia-induced pulmonary hypertension.

A major locus involved in the formation of the radial oxygen loss barrier in adventitious roots of teosinte Zea nicaraguensis is located on the short-arm of chromosome 3.

A radial oxygen loss (ROL) barrier in roots of waterlogging-tolerant plants promotes oxygen movement via aerenchyma to the root tip, and impedes soil phytotoxin entry. The molecular mechanism and genetic regulation of ROL barrier formation are largely unknown. Zea nicaraguensis, a waterlogging-tolerant wild relative of maize (Zea mays ssp. mays), forms a tight ROL barrier in its roots when waterlogged. We used Z. nicaraguensis chromosome segment introgression lines (ILs) in maize (inbred line Mi29) to elucidate the chromosomal region involved in regulating root ROL barrier formation. A segment of the short-arm of chromosome 3 of Z. nicaraguensis conferred ROL barrier formation in the genetic background of maize. This chromosome segment also decreased apoplastic solute permeability across the hypodermis/exodermis. However, the IL and maize were similar for suberin staining in the hypodermis/exodermis at 40 mm and further behind the root tip. Z. nicaraguensis contained suberin in the hypodermis/exodermis at 20 mm and lignin at the epidermis. The IL with ROL barrier, however, did not contain lignin in the epidermis. Discovery of the Z. nicaraguensis chromosomal region responsible for root ROL barrier formation has improved knowledge of this trait and is an important step towards improvement of waterlogging tolerance in maize.

In situ detection of laccase activity and immunolocalisation of a compression-wood-specific laccase (CoLac1) in differentiating xylem of Chamaecyparis obtusa.

The secondary cell wall of compression wood tracheids has a highly lignified region (S2L) in its outermost portion. To better understand the mechanism of S2L formation, we focussed on the activity of laccase (a monolignol oxidase) and performed in situ studies of this enzyme in differentiating compression wood. Staining of differentiating compression wood demonstrated that laccase activity began in all cell wall layers before the onset of lignification. We detected no activity of peroxidase (another monolignol oxidase) in any cell wall layer. Thus, laccase likely plays the major role in monolignol oxidisation during compression wood differentiation. Laccase activity was higher in the S2L region than in other secondary wall regions, suggesting that this enzyme was responsible for the high lignin concentration in this region of the cell wall. Immunolabelling demonstrated the expression of a compression-wood-specific laccase (CoLac1) immediately following the onset of secondary wall thickening, this enzyme was localised to the S2L region, whereas much less abundant in the S1 layer or inner S2 layer. Thus, the CoLac1 protein is most likely localised to the outer part of S2 and responsible for the high lignin concentration in the S2L region.

Enhanced MET Translation and Signaling Sustains K-Ras-Driven Proliferation under Anchorage-Independent Growth Conditions.

Oncogenic K-Ras mutation occurs frequently in several types of cancers, including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage-independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage-independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage-independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage-independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global downregulation of mRNA translation during anchorage-independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras-mediated Met expression drives "K-Ras addiction" in anchorage-independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage-independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients.

Plasma fluctuation in estradiol-17ß and bone resorption markers around parturition in dairy cows.

Blood samples were obtained sequentially from 10 dairy cows around the time of parturition to assess plasma fluctuations in estradiol-17ß (E2) levels in association with those of several bone resorption markers. Plasma E2 concentration increased sharply a few days prepartum and decreased quickly after parturition. In terms of bone resorption markers, the plasma level of tartrate-resistant acid phosphatase isoform 5b (TRAP5b) rose significantly, commencing 1 week prepartum, and was maintained at this level to a few days postpartum. The plasma concentration of carboxyterminal collagen cross-links of type-I collagen (CTx) increased significantly after parturition. These observations suggest that osteoclast-mediated bone resorption was activated after parturition when plasma E2 concentrations decreased.

Inhibiting RORγt/Th17 axis for autoimmune disorders.

The recent success reported in late-stage clinical trials for the treatment of psoriasis by antibodies directed against interleukin (IL)-17 or its receptor has validated and strongly supports the development of inhibitors of the IL-17 pathway as a new therapeutic modality in chronic inflammation and autoimmunity. These results also encourage the drug discovery of orally available small molecules that can modulate down the production of IL-17 by Th17 cells (the major IL-17 producers) or the downstream signaling of the IL-17 receptor. Here, we review these strategies with an emphasis on inhibiting the retinoic-acid-related orphan nuclear receptor RORγt, which is the master regulator of Th17 cells and a promising therapeutic target for the treatment of multiple autoimmune disorders.

Changes in xylem tissue and laccase transcript abundance associated with posture recovery in Chamaecyparis obtusa saplings growing on an incline.

Lignin is a major component of plant cell walls and is synthesised through oxidative polymerisation of monolignols. The transcription level of laccase, an enzyme implicated in monolignol polymerisation, is higher in the tissue forming compression wood than in normal wood. Compression wood, which is a special xylem tissue that develops to reorient inclined stems, also has a higher lignin content than normal wood. In the present study, Chamaecyparis obtusa Endl. saplings were grown on an incline and the following variables were tracked for 10 weeks: posture recovery of the saplings; development of xylem tissue on the lower side of inclined stems; and the transcription level of laccase. The posture of saplings approached vertical after 8 weeks, the development of compression wood reached a peak around 6 weeks and laccase transcription was the highest after 4 weeks. These results suggest a sequence of righting mechanisms. Inclination stimulates an increase in the abundance of laccase transcript and this increase encourages the formation of compression wood. The accumulation of compression wood then causes the stem to bend upward.

Structural basis of digoxin that antagonizes RORgamma t receptor activity and suppresses Th17 cell differentiation and interleukin (IL)-17 production.

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4(+) T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.

Beneficial effect of a diet containing heat-killed Lactobacillus paracasei K71 on adult type atopic dermatitis.

The purpose of this study was to investigate the clinical effect of a supplementary diet containing heat-killed lactic acid bacterium Lactobacillus paracasei K71 (LAB diet) on adult patients with atopic dermatitis (AD). A randomized, double-blind, placebo-controlled study was conducted in 34 adult type AD subjects who were treated with conventional topical corticosteroid and tacrolimus. LAB diet or placebo was added over 12 weeks. The primary end-point was the clinical severity of AD which was evaluated by a severity scoring system proposed by the guideline of the Japanese Dermatological Association. The effect was also secondarily evaluated by itch scores of visual analog scales (VAS), quality-of-life (QOL) impairment scores of Skindex 16 and consumption amounts of topical therapeutics. Data on these four assessment variables were collected at baseline and at week 4, 8 and 12. Within the study population, the skin severity scores were significantly decreased from baseline at week 8 (P<0.05) and at week 12 (P<0.01) in the LAB diet group but not in the placebo group. Influence of LAB diet on itch scores or QOL impairment scores was not evident. The consumption of topical therapeutics in the placebo group was 1.9-times greater in total amount compared with the corresponding value in the LAB diet group during the intervention period, although there was no significant difference. No LAB diet- or placebo-related adverse events were observed. We concluded that the LAB diet may have some benefits as a complementary therapy for adult AD patients who are managed with the conventional treatment.

Facilitated acquisition of whole-heart coronary magnetic resonance angiography with visual feedback of respiration status.

Three dimensional (3D) whole-heart (WH) coronary MR angiography (CMRA) requires an extended imaging time, but it may be reduced by providing a subject with visual feedback (VFB). Thirteen healthy volunteers were scanned and quality of 3D WH-CMRA images was compared among three scan conditions: free breathing (FB) with and without VFB (FB + VFB and FB - VFB, respectively) and multiple breath-holds with VFB (MBH + VFB). All but two subjects were able to complete all scans. The average scan times were 10.0 +/- 2.2, 10.0 +/- 2.5, and 8.2 +/- 1.3 min for FB - VFB, FB + VFB, and MBH + VFB, respectively. In the MBH + VFB condition, scan time was significantly reduced by 18% compared with both FB scans. No significant difference in image quality was observed between the FB - VFB and MBH + VFB conditions, but scores were significantly deteriorated at some segments in the FB + VFB condition. The MBH + VFB scan can be performed with a shorter scan time without failure or impairment of image quality.

Whole-heart coronary magnetic resonance angiography with parallel imaging: comparison of acceleration in one-dimension vs. two-dimensions.

PURPOSE: To evaluate visualization of the whole-heart coronary arteries accelerated with parallel imaging (PI) applied in two-dimension (2D) in comparison with one-dimension (1D). MATERIALS AND METHODS: Seventeen healthy subjects were studied with a 1.5-T scanner equipped with a whole body phased array coil system and 16-channel receivers. Using 16 coil elements, whole-heart coronary magnetic resonance angiography (CMRA) was acquired in two conditions of 1D-PI and 2D-PI. The former scan was accelerated in phase direction by factor of 2 and the latter in phase and slice directions by factors of 2.5 and 2, respectively. Visualized length of right coronary artery (RCA), left anterior descending artery (LAD), and left circumflex artery (LCX) was measured. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) was also measured. The CMRA quality was assessed in segment-wise with a five-point scale. RESULTS: The average scan time decreased to 5.3+/-2.2 min in 2D-PI from 11.6+/-3.5 min in 1D-PI, reducing the scan time to 45%. The visualized length, SNR, and CNR in average were smaller for images of 2D-PI compared with those of 1D-PI, however, statistically significant results were observed only in RCA (P<0.05). Score reduction of 2D-PI image quality was limited to 0.34 in average, and only two out of fifteen segments (#2, 6) showed significant score deterioration (P<0.05). CONCLUSIONS: Compared with the relatively limited degree of image degradation, 2D-PI offered a large reduction of the acquisition time, which is of large benefit in clinical situations.

Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells.

Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat brain capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat brain capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat brain capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat brain capillary fraction (per total mRNA) was 35.6-fold greater than that in whole brain, while the level of claudin-12 mRNA was only 13.9% of that in whole brain, suggesting that expression of claudin-12 mRNA is not restricted to brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell-cell boundaries. The permeability surface product of [(14)C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 x 10(-3) and 11.6 x 10(-3) cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in brain capillaries, and plays a key role in the appearance of barrier properties of brain capillary endothelial cells.

Involvement of 3-phosphoinositide-dependent protein kinase-1 in the MEK/MAPK signal transduction pathway.

The phosphatidylinositide-3-OH kinase/3-phospho-inositide-dependent protein kinase-1 (PDK1)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Despite their importance, however, the cross-talk between these two pathways has not been fully understood. Here we report that PDK1 promotes MAPK activation in a MEK-dependent manner. In vitro kinase assay revealed that the direct targets of PDK1 in the MAPK pathway were the upstream MAPK kinases MEK1 and MEK2. The identified PDK1 phosphorylation sites in MEK1 and MEK2 are Ser222 and Ser226, respectively, and are known to be essential for full activation. To date, these sites are thought to be phosphorylated by Raf kinases. However, PDK1 gene silencing using small interference RNA demonstrates that PDK1 is associated with maintaining the steady-state phosphorylated MEK level and cell growth. The small interference RNA-mediated down-regulation of PDK1 attenuated maximum MEK and MAPK activities but could not prolong MAPK signaling duration. Stable and transient expression of constitutively active MEK1 overcame these effects. Our results suggest a novel cross-talk between the phosphatidylinositide-3-OH kinase/PDK1/Akt pathway and the Raf/MEK/MAPK pathway.

Phosphorylation of p27Kip1 at threonine 198 by p90 ribosomal protein S6 kinases promotes its binding to 14-3-3 and cytoplasmic localization.

The cyclin-dependent kinase inhibitor p27Kip1 plays an important role in cell cycle regulation. The cyclin-dependent kinase-inhibitory activity of p27Kip1 is regulated by changes in its concentration and its subcellular localization. Several reports suggest that phosphorylation of p27Kip1 at serine 10, threonine 157, and threonine 187 regulate its localization. We have previously identified that carboxyl-terminal threonine 198 (Thr198) in p27Kip1 is a novel phosphorylation site and that Akt is associated with the phosphorylation at the site (Fujita, N., Sato, S., Katayama, K., and Tsuruo, T. (2002) J. Biol. Chem. 277, 28706-28713). We show herein that activation of the Ras/Raf/mitogen-activated protein kinase kinase (MAPK kinase/MEK) pathway also regulates phosphorylation of p27Kip1 at Thr198. MAPKs were not directly associated with p27Kip1 phosphorylation at Thr198, but the p90 ribosomal protein S6 kinases (RSKs) could bind to and directly phosphorylate p27Kip1 at Thr198 in a Ras/Raf/MEK-dependent manner. RSK-dependent phosphorylation promoted the p27Kip1 binding to 14-3-3 and its cytoplasmic localization. To prove the direct relationship between 14-3-3 binding and cytoplasmic localization, we constructed a p27Kip1-R18 fusion protein in which the R18 peptide was fused to the carboxyl-terminal region of p27Kip1. The R18 peptide is known to interact with 14-3-3 independent of phosphorylation. The p27Kip1-R18 distributed mainly in the cytosol, whereas mutant p27Kip1-R18 (p27Kip1-R18-K2) that had no 14-3-3 binding capability existed mainly in the nucleus. These results indicate that RSKs play a crucial role in cell cycle progression through translocation of p27Kip1, in addition to Akt, to the cytoplasm in a phosphorylation and 14-3-3 binding-dependent manner.

Regulation of kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitro through the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3 in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241.

Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization.

In many human cancers, the cyclin-dependent kinase inhibitor p27(Kip1) is expressed at low or undetectable levels. The decreased p27(Kip1) expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G(1) arrest, in part by up-regulating p27(Kip1). However, the role of Akt-dependent phosphorylation in p27(Kip1) regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27(Kip1). Screening p27(Kip1) phosphorylation sites identified the COOH-terminal Thr(198) residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27(Kip1) through Thr(198) only when it was phosphorylated by Akt. Although Akt also phosphorylated p27(Kip1) at Ser(10) and Thr(187), these two sites were not involved in the binding to 14-3-3 proteins. p27(Kip1) phosphorylated at Thr(198) exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27(Kip1) and cytoplasmic localization.

Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-hydroxystaurosporine).

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the AGC subfamily of protein kinases. In particular, PDK1 plays an important role in the regulation of Akt/PKB survival pathway by phosphorylating Akt on Thr308. Here we show that UCN-01 (7-hydroxystaurosporine), a drug now in clinical trials and with a unique fingerprint pattern, induced dephosphorylation and inactivation of Akt, resulting in the turn-off of the survival signals and the induction of apoptosis. Further analysis revealed that UCN-01-mediated Akt inactivation was caused by inhibiting upstream Akt kinase PDK1 (IC50=33 nM) both in vitro and from cells, but not by suppressing Akt itself or phosphatidylinositide-3-OH kinase. UCN-01-induced PDK1 inhibition was also observed in in vivo murine and human tumor xenografts. Overexpression of active form of Akt diminished the cytotoxic effects of UCN-01, suggesting that UCN-01 may in part exert its cytotoxicity by inhibiting PDK1-Akt survival pathway. Because UCN-01 has already proved to have potent anti-tumor activity in vivo, PDK1-Akt survival pathway is a new, attractive target for cancer chemotherapy.

Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1.

Serine/threonine kinase Akt is thought to mediate many biological actions toward anti-apoptotic responses. Screening of drugs that could interfere with the Akt signaling pathway revealed that Hsp90 inhibitors (e.g. geldanamycin, radicicol, and its analogues) induced Akt dephosphorylation, which resulted in Akt inactivation and apoptosis of the cells. Hsp90 inhibitors did not directly affect Akt kinase activity in vitro. Thus, we examined the effects of Hsp90 inhibitors on upstream Akt kinases, phosphatidylinositide-3-OH kinase (PI3K) and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Hsp90 inhibitors had no effect on PI3K protein expression. In contrast, treatment of the cells with Hsp90 inhibitors decreased the amount of PDK1 without directly inhibiting PDK1 kinase activity. We found that the kinase domain of PDK1 was essential for complex formation with Hsp90 and that Hsp90 inhibitors suppressed PDK1 binding to Hsp90. PDK1 degradation mechanisms revealed that inhibition of PDK1 binding to Hsp90 caused proteasome-dependent degradation of PDK1. Treatment of proteasome inhibitors increased the amount of detergent-insoluble PDK1 in Hsp90 inhibitor-treated cells. Therefore, the association of PDK1 with Hsp90 regulates its stability, solubility, and signaling. Because Akt binding to Hsp90 is also involved in the maintenance of Akt kinase activity, Hsp90 plays an important role in PDK1-Akt survival signaling pathway.