Trajectory of criterion symptoms of major depression under newly started antidepressant treatment: sleep disturbances and anergia linger on while suicidal ideas and psychomotor symptoms disappear early.

OBJECTIVE: In modern psychiatry, depression is diagnosed with the diagnostic criteria; however, the trajectory of each of the criterion symptoms is unknown. This study aims to examine this. METHODS: We made repeated assessments of the nine diagnostic criterion symptoms with the Patient Health Questionnaire-9 (PHQ-9) among 2011 participants of a 25-week pragmatic randomised controlled trial of sertraline and/or mirtazapine for hitherto untreated major depressive episodes. The changes from baseline were estimated with the mixed-effects model with repeated measures. The time to disappearance of each symptom was modeled using the Kaplan-Meier survival analysis. RESULTS: The total score on PHQ-9 was 18.5 (SD = 3.9, n = 2011) at baseline, which decreased to 15.3 (5.2, n = 2011) at week 1, to 11.5 (5.9, n = 1953) at week 3, to 7.8 (6.0, n = 1927) at week 9, and to 6.0 (5.9, n = 1910) at week 25. Suicidal ideas, psychomotor symptoms decreased rapidly, while anergia and sleep disturbance also decreased but only slowly. The survival analyses confirmed the primary analyses. CONCLUSIONS: Upon initiation of antidepressant treatment, patients with newly treated major depressive episodes can expect their suicidal ideas and psychomotor symptoms to disappear first but sleep disturbances and anergia to linger on.

Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV.

Serum levels of IL-6 and IL-1ß can predict the efficacy of gemcitabine in patients with advanced pancreatic cancer.

BACKGROUND: With this study, we sought to characterise the impact of pro-inflammatory cytokines on the outcomes of gemcitabine monotherapy (GEM) in patients with pancreatic cancer (PC). METHODS: Treatment-naive patients with advanced PC and no obvious infections were eligible for enrolment. All of the patients were scheduled to undergo systemic chemotherapy. Serum pro-inflammatory cytokines were measured using an electro-chemiluminescence assay method before chemotherapy. High cytokine levels were defined as values greater than the median. Clinical data were collected prospectively. RESULTS: Sixty patients who received GEM were included in the analysis. High IL-6 and IL-1ß levels were poor prognostic factors for overall survival in a multivariate analysis (P=0.011 and P=0.048, respectively). Patients with both a high IL-6 level and a high IL-1ß level exhibited shortened overall and progression-free survival, a reduction in the tumour control rate, and a high dose intensity of GEM compared with patients with low levels of both IL-6 and IL-1ß. CONCLUSION: The serum levels of IL-6 and IL-1ß predict the efficacy of GEM in patients with advanced PC.

Age-adjusted relative suicide risk by marital and employment status over the past 25 years in Japan.

BACKGROUND: There have been no longitudinal studies in Japan examining national-level data for suicide risk by marital and employment status. We examined the age-adjusted relative suicide risk (RR) by marital and employment status from national data acquired for all suicides in Japan occurring in the past 25 years. METHODS: All deaths identified as suicides according to ICD-9 and ICD-10 were extracted from vital statistics data of Japan for the years 1980, 1985, 1990, 1995, 2000 and 2005. Population statistics for Japanese residents aged ≥15 years were obtained from the census. RESULTS: Suicide rates for almost all categories analyzed decreased in both genders between 1985 and 1990 and increased between 1995 and 2000, especially among men. Unemployed and divorced men had a consistently higher RR in each year analyzed. Unemployed and divorced women had a higher risk than those in other categories, especially in 2000 and 2005. In women, particularly in 1980, 1985 and 1990, those who were unemployed and never married had a similar RR to those who were unemployed and divorced. CONCLUSIONS: Unemployed and divorced people were at a high risk of suicide over the past 25 years, particularly in 2000 and 2005. Our findings suggest that the effects of divorce and unemployment on suicide risk are synergistic.

Skimmed, sterilized, and concentrated bovine late colostrum promotes both prevention and recovery from intestinal tissue damage in mice.

Bovine colostrum is a rich source of tissue repair and growth factors, and inhibits gastrointestinal injury induced by the side effects of nonsteroidal antiinflammatory drugs (NSAID), such as indomethacin. Nonsteroidal antiinflammatory drugs are drugs with analgesic and antipyretic effects, but in higher doses they have inflammatory effects. The pathogenesis of small intestinal damage caused by NSAID is unclear. The present study was performed to investigate the antiinflammatory effects of skimmed, sterilized, and concentrated bovine late colostrum on intestinal injury induced by side effects of NSAID, and then to identify the active ingredient in the colostrum for intestinal tissue. In Japan, the sale of bovine colostrum within 5 d after parturition is prohibited by law. Therefore, we focused on bovine late colostrum obtained from healthy lactating cows 6 to 7 d after parturition. Proliferation of small intestine epithelial cells was stimulated in mice fed the colostrum for 1 wk. With regard to indomethacin-induced enteropathy, both prefeeding and postfeeding with colostrum facilitated growth of the intestinal villi, indicating preventive and healing effects. Furthermore, to identify the active ingredient in the colostrum responsible for this effect, the casein and whey fractions were prepared from the colostrum and fed to normal mice. Only the colostrum casein fraction stimulated intestinal villus elongation, whereas the whey fraction and mature milk casein showed no such effect. Taken together, these observations indicate that the skimmed, sterilized, and concentrated bovine late colostrum, especially the casein fraction, could be used to treat the injurious effects of NSAID in the intestine and could be effective for treatment of other ulcerative conditions in the bowel, suggesting that the colostrum has therapeutic potential for intestinal inflammation.

Multiple-dose therapy with bovine colostrum confers significant protection against diarrhea in a mouse model of human rotavirus-induced gastrointestinal disease.

Rotavirus is the most important etiologic agent of severe gastroenteritis. Previously, we reported that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal unimmunized cows at 6 to 7d after parturition effectively prevented against human rotavirus (HRV)-induced severe gastroenteritis in vivo, when administered as a single dose 60 min before viral inoculation. In the present study, we examined the efficacy of multiple administrations of SCBLC at smaller dosages after viral inoculation in vivo. We demonstrate that multiple administrations within 24h after virus inoculation resulted in earlier recovery from diarrheal symptoms, in an administration frequency-dependent manner. Furthermore, we investigated whether isolated IgG anti-HRV activity in SCBLC was equivalent to that of IgG isolated from bovine mature milk as measured by in vitro activity assays. We found that IgG-containing fractions from SCBLC and mature milk exhibited approximately the same level of anti-HRV activity. We concluded that the SCBLC contains a high level of IgG against HRV-induced severe gastroenteritis, which will be possible to use in protective effects in immunocompromised hosts, such as children and the elderly. Multiple doses of SCBLC during the early stages of infection or lower dosage of SCBLC given as a single dose both resulted in relief of diarrheal symptoms.

Ezetimibe improves postprandial hyperlipidaemia in patients with type IIb hyperlipidaemia.

BACKGROUND: Postprandial hyperlipidaemia is known to be a high-risk factor for atherosclerotic disease because of rapid and lasting accumulations of triglyceride-rich lipoproteins and remnants. The Niemann-Pick C1-Like 1 (NPC1L1) protein acts as an intestinal cholesterol transporter and ezetimibe, which inhibits NPC1L1, has been used in patients with hypercholesterolaemia. We investigated effects of ezetimibe on fasting lipid and lipoprotein profiles and postprandial hyperlipidaemia in patients with type IIb hyperlipidaemia. MATERIALS AND METHODS: Ezetimibe 10 mg per day was administered in ten patients with type IIb hyperlipidaemia for 2 months, and lipid and lipoprotein profiles were examined during fasting and after an oral fat loading (OFL) test. RESULTS: In the fasting state, ezetimibe significantly decreased not only total cholesterol, low density lipoprotein (LDL)-cholesterol and apolipoproteinB-100 (apoB-100) levels but triglycerides (TG), apoB-48 and remnant lipoprotein cholesterol (RemL-C) levels. High performance liquid chromatography analysis showed that ezetimibe decreased cholesterol and TG levels in the very low density lipoprotein (VLDL) and LDL size ranges as well as apoB-100 levels, suggesting a decrease in numbers of VLDL and LDL particles. After OFL, ezetimibe decreased the area under the curve for TG, apoB-48 and RemL-C. Ezetimibe decreased postprandial elevations of cholesterol and TG levels in the chylomicrons (CM) size range, suggesting that the postprandial production of CM particles was suppressed by ezetimibe. CONCLUSIONS: These findings suggest that ezetimibe improves fasting lipoprotein profiles and postprandial hyperlipidaemia by suppressing intestinal CM production in patients with type IIb hyperlipidaemia and such treatment may prove to be effective in reducing atherosclerosis.

Detection of protein kinase activity specifically activated at metaphase-anaphase transition.

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle-dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.

Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor.

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.

Roles of Rho-associated kinase in cytokinesis; mutations in Rho-associated kinase phosphorylation sites impair cytokinetic segregation of glial filaments.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, regulates formation of stress fibers and focal adhesions, myosin fiber organization, and neurite retraction through the phosphorylation of cytoskeletal proteins, including myosin light chain, the ERM family proteins (ezrin, radixin, and moesin) and adducin. Rho-kinase was found to phosphorylate a type III intermediate filament (IF) protein, glial fibrillary acidic protein (GFAP), exclusively at the cleavage furrow during cytokinesis. In the present study, we examined the roles of Rho-kinase in cytokinesis, in particular organization of glial filaments during cytokinesis. Expression of the dominant-negative form of Rho-kinase inhibited the cytokinesis of Xenopus embryo and mammalian cells, the result being production of multinuclei. We then constructed a series of mutant GFAPs, where Rho-kinase phosphorylation sites were variously mutated, and expressed them in type III IF-negative cells. The mutations induced impaired segregation of glial filament (GFAP filament) into postmitotic daughter cells. As a result, an unusually long bridge-like cytoplasmic structure formed between the unseparated daughter cells. Alteration of other sites, including the cdc2 kinase phosphorylation site, led to no remarkable defect in glial filament separation. These results suggest that Rho-kinase is essential not only for actomyosin regulation but also for segregation of glial filaments into daughter cells which in turn ensures correct cytokinetic processes.

Mitosis-specific phosphorylation of vimentin by protein kinase C coupled with reorganization of intracellular membranes.

Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed form changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.

Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.

Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.

Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD.

Keratin 8 and 18 (K8/18) are the major components of intermediate filament (IF) proteins of simple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tumor necrosis factor (TNF)-induced cell death. We have now identified human TNF receptor type 1 (TNFR1)-associated death domain protein (TRADD) to be the K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound K18 and K14, type I (acidic) keratins. The COOH-terminal region of TRADD interacted with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in human mammary epithelial cells. Overexpression of the NH2 terminus (amino acids 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of keratins, rendered the cells more resistant to killing by TNF. We also showed that overexpressed NH2 termini of K18 and K8/18 were associated with endogenous TRADD in SW13 cells, resulting in the inhibition of caspase-8 activation. These results indicate that K18 may sequester TRADD to attenuate interactions between TRADD and activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells.

Bronchovascular cuff formation and lung lymph flow in edema formation of anesthetized sheep.

The relationship between bronchovascular cuff formation and lung lymph flow in hydrostatic edema was evaluated. After a balloon was inserted into the left atrium to increase left atrial pressure for 5 hrs, peripheral lung tissues were resected for analysis of the wet-dry ratio and cuff formation. The degree of cuff formation was expressed as the cuff ratio (outer diameter of cuff/outer diameter of microvessel or airway) in three size categories: 80-200, 200-400, and 400-750 microm in diameter. The amount of excess lung lymph (Ex LL) for 5 hrs was calculated from the recorded data for the whole lymph flow wave. The wet-dry ratio showed a significant correlation with ALAP and lung lymph flow increased significantly (flow rate, 0.67 +/- 0.46 ml/min (mean +/- SD); Ex LL, 56.4 +/- 47.6 ml). Cuff formation was found at all levels of the bronchovascular tree, with a larger cuff ratio (> 1.3) observed at arteries and veins of 80-200 microm in diameter, but a significant correlation with Ex LL was found only for arteries of 80-200 microm. Fluid accumulation in lung interstitium first occurred at smaller extra-alveolar arteries even under mild hydrostatic pressure elevation with a significant increase in lymph flow.

[Lipofibroadenoma of the thymus].

Here we report a case of a rare thymic tumors histologically diagnosed as lipofibroadenoma. The patient was a 32-year-old male who displayed an anterior mediastinal tumor on a chest computed tomography (CT) scan while being treated for pneumonia. The tumor was localized within the thymus, and the diameter was 3 cm. No significant change was observed in the tumor on a CT scan taken 6 months after the 1st scan. Suspecting a thymoma from the CT and magnetic resonance imaging (MRI) findings, we performed a thymothymectomy via a median sternotomy. The histopathological diagnosis was a lipofibroadenoma of the thymus. The findings resembled fibroadenoma of the breast. Lymphocytes were scarce within the tumor with abundant interstitial stroma, and the tumor epithelial cells displayed restiform and dendritic structures. The epithelial cells were mostly negative for Ki-67 immunohistochemical staining. A very small amount of calcification was detected within the tumor using alizarin red staining. Based on the histopathological findings, it was considered to be a benign tumor with little growth potential, and which had been present for a long period of time.

Spatiotemporal distribution of protein kinase and phosphatase activities.

Reversible protein phosphorylation plays an essential role in controlling cellular functions. Accumulating evidence indicates that the subcellular distribution of protein kinase and phosphatase activities is organized dynamically. The spatiotemporal dynamics of kinase and phosphatase activities appears to account for the elaborate coordination of the cellular functions achieved by protein phosphorylation and dephosphorylation.

[Bloody sputum caused by hemorrhage around staple-lines 5 years after surgery; report of a case].

23 year-old non-smoking male who had underwent bilateral video-assisted thoracoscopic surgery (VATS) bullectomy for spontaneous pneumothorax using surgical stapler (Endo GIA, Tyco Healthcare) 5 years before, referred to our hospital due to hemoptysis. Chest computed tomography (CT) revealed infiltrative shadow surrounding stapled-line at right pulmonary apex. Aspiration-shadows were scattered in right lung parenchyma. Bronchoscopy revealed bloody clot extended from right B1 to main bronchi. These findings suggested that the cause of bloody sputum was bleeding from the tissue around staples used in VATS bullectomy. On admission he treated with hemostatic agents, and bloody sputum and abnormal CT shadows disappeared. Metallic surgical staplers may cause airway bleeding after surgery in its chronic stage, although complications due to them are rare.

Activation of Rac by cadherin through the c-Src-Rap1-phosphatidylinositol 3-kinase-Vav2 pathway.

Cadherin first forms homo-cis-dimers on the cell surface of the same cells, followed by formation of homo-trans-dimers (trans-interactions) in a Ca2+-dependent manner, eventually causing adherens junctions. In addition, trans-interacting cadherin induces activation of Rac small G protein, which stabilizes non-trans-interacting cadherin on the plasma membrane by inhibiting its endocytosis through the reorganization of the actin cytoskeleton. However, it has not fully been understood how cadherin induces the activation of Rac. We examined here the molecular mechanism of the activation of Rac by trans-interacting cadherin in fibroblasts and epithelial cells. Trans-interacting cadherin induced activation of c-Src locally at the cadherin-based cell-cell adhesion sites. c-Src then tyrosine-phosphorylated Vav2, one of the Rac-GDP/GTP exchange factors (GEFs), and induced activation of C3G, one of the Rap1-GEFs, through Crk adaptor protein, resulting in the activation of Rap1 locally at the cadherin-based cell-cell adhesion sites. The c-Src-catalysed tyrosine phosphorylation was not sufficient for the activation of Vav2 and the c-Src-induced activation of Rap1 was additionally necessary for it, although activated Rap1 alone was not sufficient for the activation of non-tyrosine-phosphorylated Vav2. This effect of Rap1 on Vav2 was mediated by phosphatidylinositol 3-kinase. We describe here the signaling pathway from trans-interacting cadherin to the activation of Rac.

Phase transformation of plasma-sprayed hydroxyapatite coating with preferred crystalline orientation.

Highly oriented hydroxyapatite coatings (HACs) were obtained on titanium substrates through a radio-frequency thermal plasma spraying (TPS) method. XRD patterns showed that the HACs had crystallites with [001] preferred orientation vertical to the coating's surface. XRD results also indicated that tetracalcium phosphate crystallites in the as-sprayed HAC were oriented in the (100) direction. XRD peaks corresponding to tetracalcium phosphate, tricalcium phosphate and calcium oxide were absent after heat and hydrothermal treatment. The orientation degree of the HAC was influenced little by such post-heat treatments. Considering the crystallographic relationship between the tetracalcium phosphate in the as-sprayed HAC and the HA crystallites formed in the heat-treated HAC, these XRD results indicate that the tetracalcium phosphate in the as-prepared coatings transformed topotaxially into HA during the post-heat treatment. TEM and SEM analyses of the highly oriented HAC were conducted. The characteristic lamellar structure of TPS deposits was observed in cross-sections of the HAC. A prismatic texture was also observed in magnified SEM images. TEM observation showed that 200-800-nm-wide prismatic crystallites were formed in HA splats, and their longitudinal axis was oriented vertically to the coating's surface. SAD patterns showed that the longitudinal axis of the prismatic crystallites corresponded to the [001] zone axis of the HA crystal.

PDK1 homologs activate the Pkc1-mitogen-activated protein kinase pathway in yeast.

PDK1 (phosphoinositide-dependent kinase 1) is a mammalian growth factor-regulated serine/threonine kinase. Using a genetic selection based on a mutant form of the yeast MAP kinase kinase Ste7, we isolated a gene, PKH2, encoding a structurally and functionally conserved yeast homolog of PDK1. Yeast cells lacking both PKH2 and PKH1, encoding another PDK1 homolog, were nonviable, indicating that Pkh1 and Pkh2 share an essential function. A temperature-sensitive mutant, pkh1(D398G) pkh2, was phenotypically similar to mutants defective in the Pkc1-mitogen-activated protein kinase (MAPK) pathway. Genetic epistasis analyses, the phosphorylation of Pkc1 by Pkh2 in vitro, and reduced Pkc1 activity in the pkh1(D398G) pkh2 mutant indicate that Pkh functions upstream of Pkc1. The Pkh2 phosphorylation site in Pkc1 (Thr-983) is part of a conserved PDK1 target motif and essential for Pkc1 function. Thus, the yeast PDK1 homologs activate Pkc1 and the Pkc1-effector MAPK pathway.