Akt1 is the principal Akt isoform regulating apoptosis in limiting cytokine concentrations.

The activation of the Akt signalling in response to cytokine receptor signalling promotes protein synthesis, cellular growth and proliferation. To determine the role of Akt in interleukin-3 (IL-3) signalling, we generated IL-3-dependent myeloid cell lines from mice lacking Akt1, Akt2 or Akt3. Akt1 deletion resulted in accelerated apoptosis at low concentrations of IL-3. Expression of constitutively active Akt1 was sufficient to delay apoptosis in response to IL-3 withdrawal, but not sufficient to induce proliferation in the absence of IL-3. Akt1 prolonged survival of Bim- or Bad-deficient cells, but not cells lacking Puma, indicating that Akt1-dependent repression of apoptosis was in part dependent on Puma and independent of Bim or Bad. Our data show that a key role of Akt1 during IL-3 signalling is to repress p53-dependent apoptosis pathways, including transcriptional upregulation of Puma. Moreover, our data indicate that regulation of BH3-only proteins by Akt is dispensable for Akt-dependent cell survival.

Mer receptor tyrosine kinase promotes invasion and survival in glioblastoma multiforme.

The infiltration of glioma cells into adjacent tissue is one of the major obstacles in the therapeutic management of malignant brain tumours, in most cases precluding complete surgical resection. Consequently, malignant glioma patients almost invariably experience tumour recurrences. Within the brain, glioma cells migrate rapidly either amoeboidly or mesenchymally to invade surrounding structures, in dependence on the extracellular environment. In addition, radiotherapy, frequently applied as adjuvant therapeutic modality, may enhance tumour cell mobility. Here, we show that the receptor tyrosine kinase Mer (MerTK) is overexpressed in glioblastoma multiforme (GBM) and that this is accompanied with increased invasive potential. MerTK expression is maintained in primary GBM-derived tumour spheres under stem cell culture conditions but diminishes significantly in serum-containing cultures with concomitant downregulation of Nestin and Sox2. Depletion of MerTK disrupts the rounded morphology of glioma cells and decreases their invasive capacity. Furthermore, the expression and phosphorylation of myosin light chain 2 are strongly associated with MerTK activity, indicating that the effect of MerTK on glioma cell invasion is mediated by actomyosin contractility. Finally, DNA damage robustly triggers the upregulation and phosphorylation of MerTK, which protects cells from apoptosis. This effect is strongly impaired upon MerTK depletion or overexpression of an inactive MerTK mutant. Collectively, our data suggests that MerTK is a novel therapeutic target in the treatment of the malignant gliomas.

Constitutive Notch2 signaling in neural stem cells promotes tumorigenic features and astroglial lineage entry.

Recent studies identified a highly tumorigenic subpopulation of glioma stem cells (GSCs) within malignant gliomas. GSCs are proposed to originate from transformed neural stem cells (NSCs). Several pathways active in NSCs, including the Notch pathway, were shown to promote proliferation and tumorigenesis in GSCs. Notch2 is highly expressed in glioblastoma multiforme (GBM), a highly malignant astrocytoma. It is therefore conceivable that increased Notch2 signaling in NSCs contributes to the formation of GBM. Here, we demonstrate that mice constitutively expressing the activated intracellular domain of Notch2 in NSCs display a hyperplasia of the neurogenic niche and reduced neuronal lineage entry. Neurospheres derived from these mice show increased proliferation, survival and resistance to apoptosis. Moreover, they preferentially differentiate into astrocytes, which are the characteristic cellular population of astrocytoma. Likewise, we show that Notch2 signaling increases proliferation and resistance to apoptosis in human GBM cell lines. Gene expression profiling of GBM patient tumor samples reveals a positive correlation of Notch2 transcripts with gene transcripts controlling anti-apoptotic processes, stemness and astrocyte fate, and a negative correlation with gene transcripts controlling proapoptotic processes and oligodendrocyte fate. Our data show that Notch2 signaling in NSCs produces features of GSCs and induces astrocytic lineage entry, consistent with a possible role in astrocytoma formation.

PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage.

Protein kinase B (PKB/Akt) is ubiquitously expressed in cells. Phosphorylation of its multiple targets in response to various stimuli, including growth factors or cytokines, promotes cell survival and inhibits apoptosis. PKB is upregulated in many different cancers and a significant amount of the enzyme is present in its activated form. Here we show that PKB phosphorylates one of the anti-apoptotic proteins--transcription factor Twist-1 at Ser42. Cells expressing Twist-1 displayed inefficient p53 upregulation in response to DNA damage induced by gamma-irradiation or the genotoxic drug adriamycin. This influenced the activation of p53 target genes such as p21(Waf1) and Bax and led to aberrant cell-cycle regulation and the inhibition of apoptosis. The impaired induction of these p53 effector molecules is likely to be mediated by PKB-dependent phosphorylation of Twist-1 because, unlike the wild-type mutant, the Twist-1 S42A mutant did not confer cell resistance to DNA damage. Moreover, phosphorylation of Twist-1 at Ser42 was shown in vivo in various human cancer tissues, suggesting that this post-translational modification ensures functional activation of Twist-1 after promotion of survival during carcinogenesis.

Stress appraisals, emotions, and coping among international adolescent golfers.

The aims of this study were to (a) explore the emotions generated during or as a consequence of stress appraisals, after coping, and after the event outcome, (b) explore whether multiple emotions were generated from the initial stress appraisal through to event outcome, and (c) to explore whether outcomes that were perceived as favorable resulted in positive emotions and outcomes that were perceived as unfavorable or neither favorable nor unfavorable resulted in negatively toned emotions. Participants were 10 male English international adolescent golfers (mean+/-SD; age 16.7+/-1.6 years), who were interviewed regarding their experiences of stress appraisals, emotions, and coping during competitive golf. Results revealed that emotions were generated within or as a consequence of stress appraisals, after coping, and after the event outcome. Additionally, multiple emotions were generated from the stressful appraisal to the event outcome. Positively toned emotions were cited more frequently than negatively toned emotions after favorable events, whereas negatively toned emotions were reported more frequently than positively toned emotions after unfavorable events. Coping appears important in generating positively toned emotions.

PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage.

The promyelocytic leukemia (PML) tumor suppressor protein, a central regulator of cell proliferation and apoptosis, is frequently fused to the retinoic acid receptor-alpha (RARalpha) in acute PML. Here we show the interaction of PML with another tumor suppressor protein, the serine/threonine kinase homeodomain-interacting protein kinase (HIPK2). In response to DNA damage, HIPK2 phosphorylates PML at serines 8 and 38. Although HIPK2-mediated phosphorylation of PML occurs early during the DNA damage response, the oncogenic PML-RARalpha fusion protein is phosphorylated with significantly delayed kinetics. DNA damage or HIPK2 expression leads to the stabilization of PML and PML-RARalpha proteins. The N-terminal phosphorylation sites contribute to the DNA damage-induced PML SUMOylation and are required for the ability of PML to cooperate with HIPK2 for the induction of cell death.

Transcriptional profiling of myotubes from patients with type 2 diabetes: no evidence for a primary defect in oxidative phosphorylation genes.

AIMS/HYPOTHESIS: Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independently of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes. METHODS: Using cDNA microarray technology and global pathway analysis with the Gene Map Annotator and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes. RESULTS: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either method of global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis (PPARGC1A and NRF1) or in mitochondrial mass between diabetic and control myotubes. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that impaired mitochondrial biogenesis is not a primary defect in the sequence of events leading to insulin resistance and type 2 diabetes.

Protein kinases, from B to C.

The PKB (protein kinase B) and PKC (protein kinase C) families display highly related catalytic domains that require a largely conserved series of phosphorylations for the expression of their optimum activities. However, in cells, the dynamics of these modifications are quite distinct. Based on experimental evidence, it is argued that the underlying mechanisms determining these divergent behaviours relate to the very different manner in which their variant regulatory domains interact with their respective catalytic domains. It is concluded that the distinct behaviours of PKB and PKC proteins are defined by the typical ground states of these proteins.

Physiological roles of PKB/Akt isoforms in development and disease.

PKB (protein kinase B, also known as Akt) is a serine/threonine protein kinase that is important in various signalling cascades and acts as a major signal transducer downstream of activated phosphoinositide 3-kinase. There are three closely related isoforms of PKB in mammalian cells, PKBalpha (Akt1), PKBbeta (Akt2) and PKBgamma (Akt3), and this review discusses recent advances in our understanding of the functions of these isoforms in the regulation of adipocyte differentiation, glucose homoeostasis and tumour development.

Physiological functions of protein kinase B/Akt.

The genetic manipulation of mice has become an essential and elegant method for studying the function of proteins in physiology, and for testing the veracity of information obtained from cell culture experiments. During the past few years, a variety of transgenic and knockout mouse models of PKB (protein kinase B)/Akt have been generated and investigated. In this paper, we focus on the phenotypes of these PKB/Akt overexpression and mutant mice that may help to elucidate the functions exerted by PKB/Akt in mammals.

Views of chartered physiotherapists on the psychological content of their practice: a preliminary study in the United Kingdom.

BACKGROUND: Although research into the psychological aspects of sports injury is increasing and psychological interventions have been identified as important in the rehabilitation process, few studies have focused on how sports medicine practitioners deal with psychological problems. OBJECTIVE: To investigate the perceptions of English chartered physiotherapists on the psychological content of their practice. METHODS: The Physiotherapist and Sport Psychology Questionnaire (PSPQ) was adapted slightly from the Athletic Trainer and Sport Psychology Questionnaire and used in this study. A survey package comprising a PSPQ, introductory letter, and self addressed envelope was mailed to 179 chartered physiotherapists registered in the England Eastern Region Sports Medicine Directory. RESULTS: In total, 90 (50% response rate) questionnaires were returned. The sample consisted of 67 women and 23 men with a mean (SD) age of 40.1 (5.4) years and 9.2 (3.1) years of experience as chartered physiotherapists. Descriptive statistical and qualitative analysis showed that physiotherapists believed athletes were often psychologically affected by injury. The physiotherapists also reported often using psychological techniques when treating injured athletes, but few reported having access to a sport psychologist for referral. CONCLUSIONS: This research indicates that future physiotherapy education may need more emphasis on the psychological aspect of injury, and seek to increase knowledge on the potential of using psychological interventions within a physiotherapy rehabilitation programme. Furthermore, some form of referral network should be established between chartered physiotherapists and sport psychologists.

The phosphatidylinositide 3'-kinase/Akt survival pathway is a target for the anticancer and radiosensitizing agent PKC412, an inhibitor of protein kinase C.

Activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt survival pathway protects against apoptotic stress stimuli. Therefore, compounds that down-regulate this pathway are of clinical interest for single and combined anticancer treatment modalities. Here we demonstrate that the cytotoxic effect of the protein kinase C (PKC)-inhibitor N-benzoylated staurosporine (PKC412) is mediated via the PI3K/Akt pathway. Dose-dependent down-regulation of the proliferative activity, activation of the apoptotic machinery, and cell killing by PKC412 (0-1 microM) in Rat1a-fibroblasts and H-ras-oncogene-transformed fibroblasts correlated with a decrease of Akt phosphorylation and a reduced phosphorylation of the endogenous Akt-substrate GSK3-alpha. Expression of the dominant-active myristoylated form of Akt abrogated this cytotoxic effect of PKC412. Experiments with Apaf-1-deficient cells revealed that PKC412-induced cytotoxicity depends on an intact apoptosome but that the decrease of Akt phosphorylation is not attributable to apoptosis execution. Comparative experiments indicate that PKC412 and the parent-compound staurosporine down-regulate this survival pathway upstream or at the level of Akt but by a different mechanism than the PI3K-inhibitor LY294002. Furthermore, inhibition of this pathway by PKC412 is relevant for sensitization to ionizing radiation. These results demonstrate the specific role of this signaling pathway for the PKC412-mediated down-regulation of an apoptotic threshold and its cytotoxicity.

Ten years of protein kinase B signalling: a hard Akt to follow.

It is ten years since the publication of three papers describing the cloning of a new proto-oncogene serine/threonine kinase termed protein kinase B (PKB)/Akt. Key roles for this protein kinase in cellular processes such as glucose metabolism, cell proliferation, apoptosis, transcription and cell migration are now well established. The explosion of publications involving PKB/Akt in the past three years emphasizes the high level of current interest in this signalling molecule. This review focuses on tracing the characterization of this kinase, through the elucidation of its mechanism of regulation, to its role in regulating physiological and pathophysiological processes, to our current understanding of the biology of PKB/Akt, and prospects for the future.

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane.

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKBalpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKBalpha at the plasma membrane. Binding of CTMP reduces the activity of PKBalpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.

Phosphatidylinositol 3-kinase confers resistance to encephalomyocarditis and herpes simplex virus-induced cell death through the activation of distinct downstream effectors.

The Janus kinase/STAT pathway has emerged as the paradigm of IFN-induced protection from viral infections. However, the possible participation of other signaling proteins in this protection is not clearly understood. In this report, we demonstrate that activation of phosphatidylinositol 3-kinase (PI3K) by either serum factors or IFNs blocks cell death induced by encephalomyocarditis virus (EMCV) and HSV. This increased resistance to virus-induced cell death does not involve the activation of the STAT pathway and occurs in the presence of normal viral replication. Interestingly, the cell uses two different PI3K regulated pathways to block EMCV- and HSV-induced cell death. The increased sensitivity of p85alpha(-/-) embryonic fibroblasts to EMCV-induced cell death is specifically corrected by overexpression of an activated allele of Akt/protein kinase B, but not activated mitogen-activated protein kinase extracellular kinase. Conversely, the augmented sensitivity of p85alpha(-/-) cells to HSV-induced cell death was compensated for by expression of an activated form of mitogen-activated protein kinase extracellular kinase, but not by activated Akt/protein kinase B. We conclude from these data that PI3K-activated pathways function in parallel with the Janus kinase/STAT pathway to protect cells from the lethal effects of viruses.

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation.

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased approximately 6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

Reduced protein phosphatase 2A activity induces hyperphosphorylation and altered compartmentalization of tau in transgenic mice.

Hyperphosphorylated isoforms of the microtubule-associated protein tau are the major components of neurofibrillary lesions in Alzheimer's disease (AD). Protein phosphatase (PP) 2A is a major phosphatase implicated in tau dephosphorylation in vitro. Dephosphorylation of tau can be blocked in vivo by okadaic acid, a potent inhibitor of PP2A. Moreover, activity of PP2A is reduced in AD brains. To elucidate the role of PP2A in tau phosphorylation and pathogenesis, we expressed a dominant negative mutant form of the catalytic subunit Calpha of PP2A, L199P, in mice by using a neuron-specific promoter. We obtained mice with high expression levels of Calpha L199P in cortical, hippocampal, and cerebellar neurons. PP2A activity in brain homogenates of transgenic mice was reduced to 66%. Endogenous tau protein was hyperphosphorylated at distinct sites including the AT8 epitope Ser-202/Thr-205, a major AD-associated tau phosphoepitope. AT8-positive tau aggregates accumulated in the soma and dendrites of cortical pyramidal cells and cerebellar Purkinje cells and co-localized with ubiquitin. Our data establish that PP2A plays a crucial role in tau phosphorylation. Our results also show that reduced PP2A activity is associated with altered compartmentalization and ubiquitination of tau, resembling a key pathological finding in AD.

Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site serine 472 in the carboxyl-terminal hydrophobic domain.

We have reported previously the cloning and characterization of human and mouse protein kinase B gamma (PKB gamma), the third member of the PKB family of second messenger-regulated serine/threonine kinases (Brodbeck, D., Cron, P., and Hemmings, B. A. (1999) J. Biol. Chem. 274, 9133--9136). Here we report the isolation of human and mouse PKB gamma 1, a splice variant lacking the second regulatory phosphorylation site Ser-472 in the hydrophobic C-terminal domain. Expression of PKB gamma 1 is low compared with PKB gamma, and it is regulated in different human tissues. We show that PKB gamma and PKB gamma 1 differ in their response to stimulation by insulin, pervanadate, peroxide, or okadaic acid. Activation of PKB gamma 1 requires phosphorylation at a single regulatory site Thr-305. Interestingly, this site is phosphorylated to a higher extent in PKB gamma compared with PKB gamma 1 upon maximal stimulation by pervanadate, and this is reflected in the respective specific kinase activities. Furthermore, upon insulin stimulation of transfected cells, PKB gamma 1 translocates to the plasma membrane to a lesser extent than PKB gamma. Taken together, these results suggest that phosphorylation of the hydrophobic motif at the extreme C terminus of PKB gamma may facilitate translocation of the kinase to the membrane and/or its phosphorylation on the activation loop site by phosphoinositide-dependent protein kinase-1.

Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells.

Members of the phosphoprotein phosphatase (PPP) family of protein serine/threonine phosphatases, including protein phosphatase (PP)1, PP2A and PP2B, share invariant active-site residues that are critical for catalytic function [Zhuo, Clemens, Stone and Dixon (1994) J. Biol. Chem. 269, 26234-26238]. Mutation of the active-site residues Asp(88) or His(118) within the human PP2A catalytic subunit (PP2Ac)alpha impaired catalytic activity in vitro; the D88N and H118N substitutions caused a 9- and 23-fold reduction in specific activity respectively, when compared with wild-type recombinant PP2Ac, indicating an important role for these residues in catalysis. Consistent with this, the D88N and H118N substituted forms failed to provide PP2A function in vivo, because, unlike wild-type human PP2Acalpha, neither substituted for the endogenous PP2Ac enzyme of budding yeast. Relative to wild-type PP2Ac, the active-site mutants were dramatically overexpressed in High Five insect cells using the baculovirus system. Milligram quantities of PP2Ac were purified from 1x10(9) High Five cells and the kinetic constants for dephosphorylation of the peptide RRA(pT)VA (single-letter amino-acid notation) by PP2Ac (K(m)=337.5 microM; k(cat)=170 s(-1)) and D88N (K(m)=58.4 microM; k(cat)=2 s(-1)) were determined. The results show that the substitution impairs catalysis severely without a significant effect on substrate binding, consistent with the PPP catalytic mechanism. Combination of the baculovirus and yeast systems provides a strategy whereby the structure-function of PP2Ac may be fully explored, a goal which has previously proven difficult, owing to the stringent auto-regulatory control of PP2Ac protein levels in vivo.