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1.
BJOG ; 121 Suppl 7: 58-66, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25488090

ABSTRACT

OBJECTIVE: To test whether supervised pelvic floor exercises antenatally will reduce the incidence of postpartum stress incontinence in at-risk primigravidae with bladder neck mobility, ultrasonically proven. DESIGN: Single blind, randomised controlled trial. SETTING: Antenatal clinic in a UK NHS Trust Hospital. SAMPLE: Two hundred and sixty-eight primigravidae attending an antenatal clinic at approximately 20 weeks of gestation with bladder neck mobility, on standardised valsalva, of 5 mm or more linear movement. The median age was 28, ranging from 16 to 47 years. INTERVENTION: Patients randomised to supervised pelvic floor exercises (n = 139) attended a physiotherapist at monthly intervals from 20 weeks until delivery. The exercises comprised three repetitions of eight contractions each held for six seconds, with two minutes rest between repetitions. These were repeated twice daily. At 34 weeks of gestation the number of contractions per repetition was increased to 12. Both the untreated control group and the study group received verbal advice on pelvic floor exercises from their midwives antenatally. MAIN OUTCOME MEASURES: Subjective reporting of stress incontinence at three months postpartum. Pelvic floor strength, using perineometry, and bladder neck mobility measured by perineal ultrasound. RESULTS: Of the 268 women enrolled, information on the main outcome variable was available for 110 in the control group and 120 in the study group. Fewer women in the supervised pelvic floor exercise group reported postpartum stress incontinence, 19.2% compared with 32.7% in the control group (RR 0.59 [0.37-0.92]). There was no change in bladder neck mobility and no difference in pelvic floor strength between groups after exercise, although all those developing postpartum stress incontinence had significantly poorer perineometry scores than those who were continent. CONCLUSIONS: The findings suggest that antenatal supervised pelvic floor exercises are effective in reducing the risk of postpartum stress incontinence in primigravidae with bladder neck mobility.


Subject(s)
Delivery, Obstetric/adverse effects , Exercise Therapy , Obstetric Labor Complications/physiopathology , Pelvic Floor/physiopathology , Postpartum Period , Urinary Bladder/physiopathology , Urinary Incontinence, Stress/prevention & control , Adolescent , Adult , Exercise Therapy/methods , Female , Humans , Middle Aged , Muscle Contraction , Obstetric Labor Complications/therapy , Parity , Patient Compliance , Pregnancy , Single-Blind Method , State Medicine , Surveys and Questionnaires , United Kingdom/epidemiology , Urinary Incontinence, Stress/epidemiology , Urinary Incontinence, Stress/physiopathology
3.
Int Urogynecol J ; 25(2): 181-7, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24126727

ABSTRACT

INTRODUCTION AND HYPOTHESIS: This study was performed to determine whether anatomical recurrence of cystocoele 1 year after anterior vaginal repair is related to biomechanical properties and/or the content of collagen in the vaginal wall and epithelial tissues. METHODS: In this prospective, observational study in a UK teaching hospital, we assessed women undergoing surgery for symptomatic anterior compartment prolapse. Outcome measures were anatomical recurrence, biomechanical strength and collagen content in vaginal tissues. In part one of the study, 42 women underwent biomechanical testing of full-thickness anterior vaginal wall tissue samples to determine the elastic moduli and yield stress. In part two, 59 women underwent immunohistochemical testing of anterior vaginal wall tissue samples to determine tissue content of procollagen I; collagen types I, III, V; and matrix metalloproteinases 1 and 2 (MMP-1 and 2). Results were then compared with anatomical outcome at 1 year postsurgery. RESULTS: Differences in yield strain in all outcome groups (optimal, satisfactory and unsatisfactory) were not statistically significant. Considerable variation was found in collagen type I in both satisfactory and unsatisfactory groups. There was no difference or correlation with procollagen, collagen types III and V, and MMP-1 and recurrence of pelvic organ prolapse (POP) between groups. There was a weak correlation between collagen type I and higher yield stress in both groups. CONCLUSIONS: Anatomical failure of anterior repair does not appear to be related to the biomechanical strength or collagen content of the anterior vaginal wall.


Subject(s)
Gynecologic Surgical Procedures , Uterine Prolapse/etiology , Uterine Prolapse/surgery , Vagina/metabolism , Vagina/physiopathology , Adult , Aged , Aged, 80 and over , Biomechanical Phenomena/physiology , Collagen/metabolism , Female , Follow-Up Studies , Humans , Immunohistochemistry , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 2/metabolism , Middle Aged , Prospective Studies , Recurrence , Treatment Outcome , United Kingdom , Uterine Prolapse/epidemiology , Vagina/surgery
4.
Int Urogynecol J ; 24(3): 377-84, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22864764

ABSTRACT

INTRODUCTION AND HYPOTHESIS: This prospective multi-centre true two-sided equivalence trial was designed to test the clinical equivalence of open (ASCP) and laparoscopic (LSCP) sacrocolpopexy using objective and subjective outcomes METHODS: The study was carried out in three urogynaecology units in England, UK and the patient population consisted of women referred with symptomatic and bothersome post-hysterectomy vaginal vault prolapse at least 1 cm above or beyond the hymeneal remnants. The interventions were either abdominal or laparoscopic sacrocolpopexy following randomisation to one of the types of surgery. RESULTS: For the primary outcome (point C on the POP-Q) the results at 1 year were -6.63 cm for the open ASCP and -6.67 cm for the LSCP respectively. Subjective outcomes at 1 year showed that 90% of the ASCP group and 80% of the LSCP group were "much better". There were improvements with regard to blood loss, haemoglobin and shorter length of stay in the LSCP group compared with the ASCP group. CONCLUSION: This fully powered randomised controlled trial comparing open and laparoscopic sacrocolpopexy has shown clinical equivalence.


Subject(s)
Abdomen/surgery , Colposcopy/methods , Laparoscopy/methods , Pelvic Organ Prolapse/surgery , Aged , Blood Loss, Surgical , England , Female , Follow-Up Studies , Humans , Length of Stay , Middle Aged , Prospective Studies , Treatment Outcome
5.
BJOG ; 115(8): 985-90, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18651881

ABSTRACT

OBJECTIVE: To determine the long-term effectiveness of antenatal pelvic floor muscle training (PFMT) on stress urinary incontinence (SUI). DESIGN: Eight-year follow up of a randomised controlled trial (RCT). SETTING: Acute NHS Teaching Trust. POPULATION: Participants in an RCT of antenatal PFMT 8 years previously. METHOD: Participants were asked about the presence of SUI, impact on quality of life, frequency of performance of PFMT and details of subsequent deliveries. MAIN OUTCOME MEASURE: The prevalence of SUI at 8 years. RESULTS: One hundred and sixty-four (71%) of the original 230 women responded. The significant improvement in postnatal SUI originally shown in the PFMT group compared with controls (19.2 versus 32.7%, P = 0.02) at 3 months was not evident 8 years later (35.4 versus 38.8%, P = 0.7). On direct questioning, 68.4% of the study group claimed that they still performed PFMT as taught during the study, with 38.0% of them performing this twice or more per week. There was no difference in outcome between those who performed PFMT twice or more per week compared with those performing PFMT less frequently. There were no differences in quality-of-life domains between the study and the control groups at 8 years. CONCLUSION: The initially beneficial effect of supervised antenatal PFMT on SUI did not continue for a long term despite the majority claiming to still perform PFMT. These findings are in keeping with those of other studies and raise concerns about the long-term efficacy of PFMT. Strategies to improve compliance with PFMT are required.


Subject(s)
Exercise Therapy/methods , Pelvic Floor , Pregnancy Complications/prevention & control , Urinary Incontinence, Stress/prevention & control , Adolescent , Adult , Female , Follow-Up Studies , Humans , Middle Aged , Patient Compliance , Pregnancy , Prenatal Care/methods , Treatment Outcome , Urinary Incontinence, Stress/etiology
6.
Biochem Soc Trans ; 35(Pt 1): 1-6, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17212578

ABSTRACT

Over the last 45 years, I have been working on growth factors, their receptors and signal transduction mechanisms. This period has seen a tremendous growth in knowledge and technology, and all of this, together with a focus interest in oncology, has steered me along a path designed to understand growth factor signalling so that we can see how drugs that target signalling pathways might be able to control cancer. The knowledge that we already have is likely to lead to cures for many common cancers within the next 25 years.


Subject(s)
Antineoplastic Agents/therapeutic use , ErbB Receptors/metabolism , Medical Oncology/trends , Neoplasms/drug therapy , Animals , Drug Therapy/methods , Humans , Immunotherapy/methods , Intercellular Signaling Peptides and Proteins/metabolism , Medical Oncology/methods , Models, Biological , Neoplasms/metabolism , Signal Transduction
7.
Int Urogynecol J Pelvic Floor Dysfunct ; 17(6): 636-41, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16552618

ABSTRACT

OBJECTIVE: To test whether cannabinoids reduce urge incontinence episodes without affecting voiding in patients with multiple sclerosis. This was part of the multicentre trial of the Cannabinoids in Multiple Sclerosis (CAMS) study. SUBJECTS AND METHODS: The CAMS study randomised 630 patients to receive oral administration of cannabis extract, Delta(9)-tetrahydrocannabinol (THC) or matched placebo. For this substudy subjects completed incontinence diaries. RESULTS: All three groups showed a significant reduction, p<0.01, in adjusted episode rate (i.e. correcting for baseline imbalance) from baseline to the end of treatment: cannabis extract, 38%; THC, 33%; and placebo, 18%. Both active treatments showed significant effects over placebo (cannabis extract, p=0.005; THC, p=0.039). CONCLUSION: The findings are suggestive of a clinical effect of cannabis on incontinence episodes in patients with MS. This is in contrast to the negative finding of the CAMS study, where no difference was seen in the primary outcome of spasticity.


Subject(s)
Analgesics, Non-Narcotic/pharmacology , Dronabinol/pharmacology , Multiple Sclerosis/epidemiology , Urinary Incontinence, Urge/prevention & control , Urination/drug effects , Analgesics, Non-Narcotic/therapeutic use , Cannabidiol , Cannabinoids , Comorbidity , Dronabinol/therapeutic use , Female , Humans , Middle Aged , Multiple Sclerosis/drug therapy , Quality of Life , Urinary Incontinence, Urge/drug therapy , Urinary Incontinence, Urge/epidemiology , Urodynamics
8.
Br J Cancer ; 90(1): 173-81, 2004 Jan 12.
Article in English | MEDLINE | ID: mdl-14710226

ABSTRACT

Microarray analysis offers a powerful tool for studying the mechanisms of cellular transformation, although the correlation between mRNA and protein expression is largely unknown. In this study, a microarray analysis was performed to compare transcription in response to overexpression of the ErbB-2 receptor tyrosine kinase in a model mammary luminal epithelial cell system, and in response to the ErbB-specific growth factor heregulin beta1. We sought to validate mRNA changes by monitoring changes at the protein level using a parallel proteomics strategy, and report a surprisingly high correlation between transcription and translation for the subset of genes studied. We further characterised the identified targets and relate differential expression to changes in the biological properties of ErbB-2-overexpressing cells. We found differential regulation of several key cell cycle modulators, including cyclin D2, and downregulation of a large number of interferon-inducible genes, consistent with increased proliferation of the ErbB-2-overexpressing cells. Furthermore, differential expression of genes involved in extracellular matrix modelling and cellular adhesion was linked to altered adhesion of these cells. Finally, we provide evidence for enhanced autocrine activation of MAPK signalling and the AP-1 transcription complex. Together, we have identified changes that are likely to drive proliferation and anchorage-independent growth of ErbB-2- overexpressing cancer cells.


Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Genes, erbB-2 , Mammary Glands, Human/cytology , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , Receptor, ErbB-2/biosynthesis , Cell Cycle , Cell Division , Cell Transformation, Neoplastic , Epithelial Cells/physiology , Female , Humans , Protein Biosynthesis , Proteomics , RNA, Messenger/analysis , Signal Transduction , Transcription, Genetic
9.
J Am Soc Mass Spectrom ; 13(9): 1042-51, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12322952

ABSTRACT

We have revisited the direct analysis experiments reported by Tomer and co-workers in the MALDI-TOFMS analysis of phosphopeptide-loaded immobilized metal ion affinity chromatography (IMAC) beads (Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282). The results described herein provide no evidence to support a laser-induced direct desorption of phosphopeptides chelated on IMAC beads. However, we have established that solubilization of mono-phosphopeptides from their immobilized Fe3+-NTA chelates does occur effectively in solutions containing certain MALDI matrices. Particularly effective is 2,5-dihydroxybenzoic acid (2,5-DHB), which apparently forms a stronger chelation complex with Fe3+-NTA than mono-phosphopeptides. With regard to the disparity observed between the low pH value of MALDI matrices (saturated 2,5-DHB(aq) approximately pH 2) and the high pH values of conventional IMAC eluents (typically above pH 7), we have also investigated the influence of eluent pH on the recovery of phosphopeptides from IMAC media. Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono- and all poly-phosphopeptide species from Fe3+-NTA IMAC resin.


Subject(s)
Metals , Phosphopeptides/analysis , Amino Acid Sequence , Hydrogen-Ion Concentration , Indicators and Reagents , Iron , Molecular Sequence Data , Reproducibility of Results , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
10.
BJOG ; 109(1): 68-76, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11845813

ABSTRACT

OBJECTIVE: To test whether supervised pelvic floor exercises antenatally will reduce the incidence of postpartum stress incontinence in at-risk primigravidae with bladder neck mobility, ultrasonically proven. DESIGN: Single blind, randomised controlled trial. SETTING: Antenatal clinic in a UK NHS Trust Hospital. SAMPLE: Two hundred and sixty-eight primigravidae attending an antenatal clinic at approximately 20 weeks of gestation with bladder neck mobility, on standardised valsalva, of 5 mm or more linear movement. The median age was 28, ranging from 16 to 47 years. INTERVENTION: Patients randomised to supervised pelvic floor exercises (n = 139) attended a physiotherapist at monthly intervals from 20 weeks until delivery. The exercises comprised three repetitions of eight contractions each held for six seconds, with two minutes rest between repetitions. These were repeated twice daily. At 34 weeks of gestation the number of contractions per repetition was increased to 12. Both the untreated control group and the study group received verbal advice on pelvic floor exercises from their midwives antenatally. MAIN OUTCOME MEASURES: Subjective reporting of stress incontinence at three months postpartum. Pelvic floor strength, using perineometry, and bladder neck mobility measured by perineal ultrasound. RESULTS: Of the 268 women enrolled, information on the main outcome variable was available for 110 in the control group and 120 in the study group. Fewer women in the supervised pelvic floor exercise group reported postpartum stress incontinence, 19.2% compared with 32.7% in the control group (RR 0.59 [0.37-0.92]). There was no change in bladder neck mobility and no difference in pelvic floor strength between groups after exercise, although all those developing postpartum stress incontinence had significantly poorer perineometry scores than those who were continent. CONCLUSIONS: The findings suggest that antenatal supervised pelvic floor exercises are effective in reducing the risk of postpartum stress incontinence in primigravidae with bladder neck mobility.


Subject(s)
Exercise Therapy/methods , Puerperal Disorders/prevention & control , Urinary Bladder/physiology , Urinary Incontinence, Stress/prevention & control , Adolescent , Adult , Body Mass Index , Delivery, Obstetric/methods , Female , Humans , Incontinence Pads , Joint Instability/physiopathology , Middle Aged , Obstetric Labor Complications/physiopathology , Patient Compliance , Pelvic Floor/physiology , Pregnancy , Prenatal Care/methods , Puerperal Disorders/physiopathology , Quality of Life , Risk Factors , Single-Blind Method , Treatment Outcome , Urinary Incontinence, Stress/physiopathology , Valsalva Maneuver
11.
EMBO J ; 20(23): 6805-15, 2001 Dec 03.
Article in English | MEDLINE | ID: mdl-11726516

ABSTRACT

IkappaB kinase (IKK) is a key mediator of NF-kappaB activation induced by various immunological signals. In T cells and most other cell types, the primary target of IKK is a labile inhibitor of NF-kappaB, IkappaBalpha, which is responsible for the canonical NF-kappaB activation. Here, we show that in T cells infected with the human T-cell leukemia virus (HTLV), IKKalpha is targeted to a novel signaling pathway that mediates processing of the nfkappab2 precursor protein p100, resulting in active production of the NF-kappaB subunit, p52. This pathogenic action is mediated by the HTLV-encoded oncoprotein Tax, which appears to act by physically recruiting IKKalpha to p100, triggering phosphorylation-dependent ubiquitylation and processing of p100. These findings suggest a novel mechanism by which Tax modulates the NF-kappaB signaling pathway.


Subject(s)
Gene Products, tax/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , T-Lymphocytes/metabolism , Amino Acid Sequence , Cell Line , Enzyme Activation , Genes, Dominant , Genes, Reporter , Green Fluorescent Proteins , Human T-lymphotropic virus 1/metabolism , Humans , I-kappa B Kinase , Immunoblotting , Jurkat Cells , Luciferases/metabolism , Luminescent Proteins/metabolism , Molecular Sequence Data , NF-kappa B p52 Subunit , Phosphorylation , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , Retroviridae/genetics , Retroviridae/metabolism , Signal Transduction , Time Factors , Transfection , Ubiquitin/metabolism , Viral Envelope Proteins/metabolism
12.
Annu Rev Cell Dev Biol ; 17: 615-75, 2001.
Article in English | MEDLINE | ID: mdl-11687500

ABSTRACT

The phosphoinositide 3-kinase (PI3K) family of enzymes is recruited upon growth factor receptor activation and produces 3' phosphoinositide lipids. The lipid products of PI3K act as second messengers by binding to and activating diverse cellular target proteins. These events constitute the start of a complex signaling cascade, which ultimately results in the mediation of cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. Therefore, PI3Ks play a central role in many cellular functions. The factors that determine which cellular function is mediated are complex and may be partly attributed to the diversity that exists at each level of the PI3K signaling cascade, such as the type of stimulus, the isoform of PI3K, or the nature of the second messenger lipids. Numerous studies have helped to elucidate some of the key factors that determine cell fate in the context of PI3K signaling. For example, the past two years has seen the publication of many transgenic and knockout mouse studies where either PI3K or its signaling components are deregulated. These models have helped to build a picture of the role of PI3K in physiology and indeed there have been a number of surprises. This review uses such models as a framework to build a profile of PI3K function within both the cell and the organism and focuses, in particular, on the role of PI3K in cell regulation, immunity, and development. The evidence for the role of deregulated PI3K signaling in diseases such as cancer and diabetes is reviewed.


Subject(s)
Cell Differentiation/physiology , Cell Transformation, Neoplastic/genetics , Homeostasis/physiology , Immunity , Phosphatidylinositol 3-Kinases/physiology , Signal Transduction/immunology , Animals , Enzyme Activation , Gene Expression Regulation, Enzymologic , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/physiology , Lipid Metabolism , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/genetics , Second Messenger Systems , Signal Transduction/physiology
13.
J Biol Chem ; 276(44): 40385-8, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11553609

ABSTRACT

The Tax oncoprotein encoded by human T-cell leukemia virus induces both T-cell activation and apoptosis. The mechanism by which Tax induces apoptosis has remained unclear. Using genetically manipulated T-cell lines, we demonstrate that Tax-induced T-cell death is dependent on NF-kappaB signaling. Tax fails to induce apoptosis in T cells lacking IkappaB kinase gamma (IKKgamma), an essential component of the NF-kappaB signaling pathway. This defect was rescued when the mutant cells were reconstituted with exogenous IKKgamma. We further demonstrate that the Tax-induced T-cell death is mediated by TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), because this event can be effectively inhibited by a TRAIL-blocking antibody. Consistent with this functional aspect, Tax stimulates the expression of TRAIL mRNA. Finally, we provide genetic evidence demonstrating that the NF-kappaB signaling pathway is essential for TRAIL gene induction by both Tax and T-cell activation signals. These studies reveal a novel function of the NF-kappaB signaling pathway and suggest a key mechanism by which Tax induces T-cell death.


Subject(s)
Apoptosis/physiology , Gene Expression Regulation/physiology , Gene Products, tax/physiology , Membrane Glycoproteins/genetics , NF-kappa B/metabolism , Signal Transduction , T-Lymphocytes/cytology , Tumor Necrosis Factor-alpha/genetics , Apoptosis Regulatory Proteins , Base Sequence , DNA Primers , Humans , Jurkat Cells , NF-kappa B/physiology , RNA, Messenger/genetics , TNF-Related Apoptosis-Inducing Ligand , Transcriptional Activation
14.
Trends Pharmacol Sci ; 22(7): 376-84, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11431033

ABSTRACT

The completion of the genomic sequences of numerous organisms from human and mouse to Caenorhabditis elegans and many microorganisms, and the definition of their genes provides a database to interpret cellular protein-expression patterns and relate them to protein function. Proteomics technologies that are dependent on mass spectrometry and involve two-dimensional gel electrophoresis are providing the main window into the world of differential protein-expression analysis. In this article, the limitations and expectations of this research field are examined and the future of the analytical needs of proteomics is explored.


Subject(s)
Peptide Mapping/methods , Proteome/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Animals , Electrophoresis, Gel, Two-Dimensional/methods , Electrophoresis, Gel, Two-Dimensional/trends , Humans , Mass Spectrometry/methods , Mass Spectrometry/trends , Peptide Mapping/trends , Sequence Tagged Sites , Signal Transduction/physiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/trends
15.
Annu Rev Biochem ; 70: 535-602, 2001.
Article in English | MEDLINE | ID: mdl-11395417

ABSTRACT

The 3-phosphorylated inositol lipids fulfill roles as second messengers by interacting with the lipid binding domains of a variety of cellular proteins. Such interactions can affect the subcellular localization and aggregation of target proteins, and through allosteric effects, their activity. Generation of 3-phosphoinositides has been documented to influence diverse cellular pathways and hence alter a spectrum of fundamental cellular activities. This review is focused on the 3-phosphoinositide lipids, the synthesis of which is acutely triggered by extracellular stimuli, the enzymes responsible for their synthesis and metabolism, and their cell biological roles. Much knowledge has recently been gained through structural insights into the lipid kinases, their interaction with inhibitors, and the way their 3-phosphoinositide products interact with protein targets. This field is now moving toward a genetic dissection of 3-phosphoinositide action in a variety of model organisms. Such approaches will reveal the true role of the 3-phosphoinositides at the organismal level in health and disease.


Subject(s)
Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositols/metabolism , 1-Phosphatidylinositol 4-Kinase/chemistry , 1-Phosphatidylinositol 4-Kinase/metabolism , Actins/metabolism , Androstadienes/chemistry , Androstadienes/pharmacology , Animals , Apoptosis/physiology , Binding Sites , Blood Proteins/chemistry , Catalytic Domain , Cell Division/physiology , Chromones/chemistry , Chromones/pharmacology , Cytoskeleton/metabolism , Enzyme Inhibitors/pharmacology , Humans , Morpholines/chemistry , Morpholines/pharmacology , PTEN Phosphohydrolase , Phosphatidylinositols/chemistry , Phosphoinositide-3 Kinase Inhibitors , Phosphoproteins/chemistry , Phosphoric Monoester Hydrolases/metabolism , Sequence Homology, Amino Acid , Tumor Suppressor Proteins/metabolism , Wortmannin
16.
J Biol Chem ; 276(24): 21544-54, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11278889

ABSTRACT

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.


Subject(s)
Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/physiology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Amino Acid Substitution , Androstadienes/pharmacology , Animals , Binding Sites , COS Cells , Cell Line , Chlorocebus aethiops , Conserved Sequence , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphatidylinositol 3-Kinases/genetics , Point Mutation , Protein Conformation , Protein Kinases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Sirolimus/pharmacology , Software , Substrate Specificity , Transfection , Wortmannin
17.
Biochem J ; 352 Pt 2: 425-33, 2000 Dec 01.
Article in English | MEDLINE | ID: mdl-11085936

ABSTRACT

Phosphorylation of protein kinase C (PKC) provides an amplitude control that operates in conjunction with allosteric effectors. Under many conditions, PKC isotypes appear to be highly phosphorylated; however, the cellular inputs that maintain these phosphorylations are not characterized. In the present work, it is shown that there is a differential phosphorylation of PKCdelta in adherent versus suspension cultures of transfected HEK-293 cells. It is established that integrin activation is sufficient to trigger PKCdelta phosphorylation and that this signals through phosphoinositide 3-kinase (PI3-kinase) to stimulate the phosphorylation of two sites, T505 and S662. The loss of signal input to PKCdelta in suspension culture is dependent on the tumour suppressor gene PTEN, which encodes a bi-functional phosphotyrosine/phosphoinositide 3-phosphate phosphatase. In the PTEN(-/-) UM-UC-3 bladder carcinoma cell line grown in suspension, transfected PKCdelta no longer accumulates in a dephospho-form on serum removal. By contrast, in a UM-UC-3-derivative cell line stably expressing PTEN, PKCdelta does become dephosphorylated under these conditions. Employing the PTEN Gly(129)-->Glu mutant, which is selectively defective in lipid phosphatase activity, it was established that it is the lipid phosphatase activity that controls PKCdelta phosphorylation. The evidence indicates that PKCdelta phosphorylation and its latent activity are maintained in serum-deprived adherent cultures through integrin-matrix interactions. This control acts through a pathway involving a lipid product of PI3-kinase in a manner that can be suppressed by PTEN.


Subject(s)
Integrin beta1/metabolism , Isoenzymes/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Kinase C/metabolism , Tumor Suppressor Proteins , Cell Line , Enzyme Activation , Humans , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Kinase C-delta
18.
Mol Med Today ; 6(9): 347-57, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10954868

ABSTRACT

The phosphoinositide 3-kinases (PI3-kinases) are a ubiquitously expressed enzyme family that, through the generation of phospholipid second messengers, play a key role in the regulation of many cellular processes. These include motility, proliferation and survival, and carbohydrate metabolism. Members of the PI3-kinase family and related kinases, their mechanism of activation and the cellular events that they influence are described in this review. As knowledge of their involvement in disease processes increases, the PI3-kinases appear to be an increasingly attractive target for drug development, particularly in the fields of cancer and other proliferative diseases, and in the treatment of inflammatory and immunological conditions. Evidence of the functional specialization of PI3-kinase isoforms suggests that selective inhibition with acceptable toxicity might be possible.


Subject(s)
Enzyme Inhibitors/therapeutic use , Phosphoinositide-3 Kinase Inhibitors , Animals , Drug Design , Humans , Lipid Metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/physiology
19.
EMBO J ; 19(15): 4015-25, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10921882

ABSTRACT

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.


Subject(s)
Neoplasm Proteins , Nerve Tissue Proteins/metabolism , Phosphoinositide-3 Kinase Inhibitors , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Apoptosis , Binding Sites , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression Regulation, Enzymologic , Humans , Mice , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Neurons, Afferent/cytology , Phosphatidylinositol 3-Kinases/genetics , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Signal Transduction , U937 Cells , src Homology Domains
20.
Mol Cell Biol ; 20(11): 3817-30, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10805725

ABSTRACT

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.


Subject(s)
ErbB Receptors/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Binding Sites , Calcium/metabolism , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/pharmacology , Humans , Phosphates/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphotyrosine/metabolism , Platelet-Derived Growth Factor/metabolism , Receptor, ErbB-2/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Tumor Cells, Cultured
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