ABSTRACT
Background: Based upon preclinical synergy in murine models, we carried out a phase I trial to determine the maximum tolerated dose (MTD), toxicities, pharmacokinetics, and biomarkers of response for the combination of BKM120, a PI3K inhibitor, and olaparib, a PARP inhibitor. Patients and methods: Olaparib was administered twice daily (tablet formulation) and BKM120 daily on a 28-day cycle, both orally. A 3 + 3 dose-escalation design was employed with the primary objective of defining the combination MTD, and secondary objectives were to define toxicities, activity, and pharmacokinetic profiles. Eligibility included recurrent breast (BC) or ovarian cancer (OC); dose-expansion cohorts at the MTD were enrolled for each cancer. Results: In total, 69 of 70 patients enrolled received study treatment; one patient never received study treatment because of ineligibility. Twenty-four patients had BC; 46 patients had OC. Thirty-five patients had a germline BRCA mutation (gBRCAm). Two DLTs (grade 3 transaminitis and hyperglycemia) were observed at DL0 (BKM120 60 mg/olaparib and 100 mg b.i.d.). The MTD was determined to be BKM120 50 mg q.d. and olaparib 300 mg b.i.d. (DL8). Additional DLTs included grade 3 depression and transaminitis, occurring early in cycle 2 (DL7). Anticancer activity was observed in BC and OC and in gBRCAm and gBRCA wild-type (gBRCAwt) patients. Conclusions: BKM120 and olaparib can be co-administered, but the combination requires attenuation of the BKM120 dose. Clinical benefit was observed in both gBRCAm and gBRCAwt pts. Randomized phase II studies will be needed to further define the efficacy of PI3K/PARP-inhibitor combinations as compared with a PARP inhibitor alone.
Subject(s)
Aminopyridines/administration & dosage , BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/drug therapy , Morpholines/administration & dosage , Ovarian Neoplasms/drug therapy , Phthalazines/administration & dosage , Piperazines/administration & dosage , Adult , Aged , Aminopyridines/pharmacokinetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Dose-Response Relationship, Drug , Female , Germ-Line Mutation , Humans , Middle Aged , Morpholines/pharmacokinetics , Neoplasm Grading , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Phosphoinositide-3 Kinase Inhibitors , Phthalazines/pharmacokinetics , Piperazines/pharmacokinetics , Poly(ADP-ribose) Polymerase Inhibitors/administration & dosage , Poly(ADP-ribose) Polymerases/geneticsABSTRACT
Multiple SRC-family kinases (SFKs) are commonly activated in carcinoma and appear to have a role in metastasis through incompletely understood mechanisms. Recent studies have shown that CDCP1 (CUB (complement C1r/C1s, Uegf, Bmp1) Domain-Containing Protein-1) is a transmembrane protein and an SRC substrate potentially involved in metastasis. Here we show that increased SFK and CDCP1 tyrosine phosphorylation is, surprisingly, associated with a decrease in FAK phosphorylation. This appears to be true in human tumors as shown by our correlation analysis of a mass spectrometric data set of affinity-purified phosphotyrosine peptides obtained from normal and cancer lung tissue samples. Induction of tyrosine phosphorylation of CDCP1 in cell culture, including by a mAb that binds to its extracellular domain, promoted changes in SFK and FAK tyrosine phosphorylation, as well as in PKC(TM), a protein known to associate with CDCP1, and these changes are accompanied by increases in adhesion and motility. Thus, signaling events that accompany the CDCP1 tyrosine phosphorylation observed in cell lines and human lung tumors may explain how the CDCP1/SFK complex regulates motility and adhesion.
Subject(s)
Antigens, CD/metabolism , Cell Adhesion Molecules/metabolism , Cell Movement/physiology , Neoplasm Proteins/metabolism , src-Family Kinases/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Neoplasm , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/immunology , Cell Communication/drug effects , Cell Communication/genetics , Cell Communication/physiology , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Enzyme Activation/drug effects , Focal Adhesion Kinase 1/metabolism , HCT116 Cells , Humans , Immunoblotting , Immunoprecipitation , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mutation , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Phosphorylation/drug effects , Protein Binding , Protein Kinase C/metabolism , RNA Interference , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
Proliferating cells adapt metabolism to support the conversion of available nutrients into biomass. How cell metabolism is regulated to balance the production of ATP, metabolite building blocks, and reducing equivalents remains uncertain. Proliferative metabolism often involves an increased rate of glycolysis. A key regulated step in glycolysis is catalyzed by pyruvate kinase to convert phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, there is strong selection for expression of the less active M2 isoform of pyruvate kinase (PKM2) in tumors and other proliferative tissues. Cell growth signals further decrease PKM2 activity, and cells with less active PKM2 use another pathway with separate regulatory properties to convert PEP to pyruvate. One consequence of using this alternative pathway is an accumulation of 3-phosphoglycerate (3PG) that leads to the diversion of 3PG into the serine biosynthesis pathway. In fact, in some cancers a substantial portion of the total glucose flux is directed toward serine synthesis, and genetic evidence suggests that glucose flux into this pathway can promote cell transformation. Environmental conditions can also influence the pathways that cells use to generate biomass with the source of carbon for lipid synthesis changing based on oxygen availability. Together, these findings argue that distinct metabolic phenotypes exist among proliferating cells, and both genetic and environmental factors influence how metabolism is regulated to support cell growth.
Subject(s)
Metabolic Networks and Pathways , Animals , Cell Proliferation , Glucose/metabolism , Glutamine/metabolism , Humans , Pyruvate Kinase/metabolism , Serine/biosynthesisABSTRACT
BACKGROUND: AMP-activated protein kinase (AMPK, PRKA) has central roles in cellular metabolic sensing and energy balance homeostasis, and interacts with various pathways (e.g., TP53 (p53), FASN, MTOR and MAPK3/1 (ERK)). AMP-activated protein kinase activation is cytotoxic to cancer cells, supporting AMPK as a tumour suppressor and a potential therapeutic target. However, no study has examined its prognostic role in colorectal cancers. METHODS: Among 718 colon and rectal cancers, phosphorylated AMPK (p-AMPK) and p-MAPK3/1 expression was detected in 409 and 202 tumours, respectively, by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. RESULTS: Phosphorylated AMPK expression was not associated with survival among all patients. Notably, prognostic effect of p-AMPK significantly differed by p-MAPK3/1 status (P(interaction)=0.0017). Phosphorylated AMPK expression was associated with superior colorectal cancer-specific survival (adjusted HR 0.42; 95% confidence interval (CI), 0.24-0.74) among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases (adjusted HR 1.22; 95% CI: 0.85-1.75). CONCLUSION: Phosphorylated AMPK expression in colorectal cancer is associated with superior prognosis among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases, suggesting a possible interaction between the AMPK and MAPK pathways influencing tumour behaviour.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Aged , Biomarkers, Tumor/metabolism , Class I Phosphatidylinositol 3-Kinases , Colorectal Neoplasms/mortality , DNA Methylation , Female , Humans , Male , Microsatellite Instability , Middle Aged , Mutation , Nerve Tissue Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Prognosis , Proto-Oncogene Proteins B-raf/genetics , RNA-Binding Proteins/metabolismABSTRACT
The high frequency of phosphoinositide 3-kinase (PI3K) pathway alterations in cancer has led to a surge in the development of PI3K inhibitors. Many of these targeted therapies are currently in clinical trials and show great promise for the treatment of PI3K-addicted tumors. These recent developments call for a re-evaluation of the oncogenic mechanisms behind PI3K pathway alterations. This pathway is unique in that every major node is frequently mutated or amplified in a wide variety of solid tumors. Receptor tyrosine kinases upstream of PI3K, the p110 alpha catalytic subunit of PI3K, the downstream kinase, AKT, and the negative regulator, PTEN, are all frequently altered in cancer. In this review, we will examine the oncogenic properties of these genetic alterations to understand whether they are redundant or distinct and propose treatment strategies tailored for these genetic lesions.
Subject(s)
Neoplasms/genetics , Phosphatidylinositol 3-Kinases/physiology , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Enzyme Inhibitors/therapeutic use , Genes, erbB-2/physiology , Genes, ras/physiology , Humans , Models, Biological , Mutation/physiology , Neoplasms/drug therapy , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nuclear Proteins/physiology , PTEN Phosphohydrolase/antagonists & inhibitors , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Signal Transduction/genetics , Signal Transduction/physiology , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
The molecular interplay between the phosphoinositide 3-kinase (PI3K) pathway and mammalian target of rapamycin (mTOR) signalling in the control of cell growth and proliferation has been the subject of much interest and debate amongst cell biologists. A recent escalation of research in this area has come from the discovery of the tuberous sclerosis complex gene products, tuberin and hamartin, as central regulators of mTOR activation. The PI3K effector Akt/protein kinase B has been found to directly phosphorylate tuberin and is thereby thought to activate mTOR through inhibition of the tuberin-hamartin complex. The many recent studies aimed at defining the molecular nature of this revamped PI3K/Akt/mTOR pathway are reviewed here. The collective data discussed have laid the groundwork for important new insights into the many cancers caused by aberrant PI3K activation and the clinically challenging tuberous sclerosis complex disease and have suggested a possible means of treatment for both.
Subject(s)
Phosphatidylinositol 3-Kinases/genetics , Protein Kinases/genetics , Protein Serine-Threonine Kinases , Tuberous Sclerosis/genetics , Genes, Tumor Suppressor , Humans , Protein Kinases/physiology , Protein-Tyrosine Kinases/genetics , Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Repressor Proteins/genetics , Signal Transduction/genetics , TOR Serine-Threonine Kinases , Tuberous Sclerosis/enzymology , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor ProteinsABSTRACT
In vertebrates, the development and integrity of the skeleton requires hydroxyapatite (HA) deposition by osteoblasts. HA deposition is also a marker of, or a participant in, processes as diverse as cancer and atherosclerosis. At present, sites of osteoblastic activity can only be imaged in vivo using gamma-emitting radioisotopes. The scan times required are long, and the resultant radioscintigraphic images suffer from relatively low resolution. We have synthesized a near-infrared (NIR) fluorescent bisphosphonate derivative that exhibits rapid and specific binding to HA in vitro and in vivo. We demonstrate NIR light-based detection of osteoblastic activity in the living animal, and discuss how this technology can be used to study skeletal development, osteoblastic metastasis, coronary atherosclerosis, and other human diseases.
Subject(s)
Fluorescent Dyes/pharmacology , Microscopy, Fluorescence/methods , Osteoblasts/cytology , Animals , Antineoplastic Agents/pharmacology , Biocompatible Materials/pharmacology , Diphosphonates/chemical synthesis , Diphosphonates/pharmacology , Dose-Response Relationship, Drug , Durapatite/pharmacology , Fluorescent Dyes/pharmacokinetics , Humans , Kinetics , Magnetic Resonance Imaging , Male , Mice , Mice, Nude , Models, Chemical , Osteoblasts/metabolism , Pamidronate , Protein Binding , Technetium , Time FactorsABSTRACT
Intra-acinar cell activation of digestive enzyme zymogens including trypsinogen is generally believed to be an early and critical event in acute pancreatitis. We have found that the phosphatidylinositol 3-kinase inhibitor wortmannin can reduce the intrapancreatic activation of trypsinogen that occurs during two dissimilar experimental models of rodent acute pancreatitis, secretagogue- and duct injection-induced pancreatitis. The severity of both models was also reduced by wortmannin administration. In contrast, the NF-kappa B activation that occurs during the early stages of secretagogue-induced pancreatitis is not altered by administration of wortmannin. Ex vivo, caerulein-induced trypsinogen activation is inhibited by wortmannin and LY294002. However, the cytoskeletal changes induced by caerulein were not affected by wortmannin. Concentrations of caerulein that induced ex vivo trypsinogen activation do not significantly increase phosphatidylinositol-3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate levels or induce phosphorylation of Akt/PKB, suggesting that class I phosphatidylinositol 3-kinases are not involved. The concentration of wortmannin that inhibits trypsinogen activation causes a 75% decrease in phosphatidylinositol 3-phosphate, which is implicated in vesicle trafficking and fusion. We conclude that a wortmannin-inhibitable phosphatidylinositol 3-kinase is necessary for intrapancreatic activation of trypsinogen and regulating the severity of acute pancreatitis. Our observations suggest that phosphatidylinositol 3-kinase inhibition might be of benefit in preventing acute pancreatitis.
Subject(s)
Pancreatitis/drug therapy , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/physiology , Trypsinogen/metabolism , Acute Disease , Androstadienes/pharmacology , Animals , Cells, Cultured , Ceruletide/metabolism , Chromones/pharmacology , Cytoskeleton/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/pharmacology , Lysosomes/metabolism , Male , Mice , Morpholines/pharmacology , NF-kappa B/metabolism , Necrosis , Phosphatidylinositol Phosphates/metabolism , Phosphorylation , Rats , Time Factors , WortmanninABSTRACT
Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.
Subject(s)
Phagocytosis/physiology , Phagosomes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Androstadienes/pharmacology , Animals , Cells, Cultured , Enzyme Inhibitors/metabolism , Fibroblasts/metabolism , Genes, Reporter , Humans , Immunoglobulin G/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Lysosomes/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Knockout , Microinjections , Phagosomes/ultrastructure , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , WortmanninABSTRACT
The number of known proteases is increasing at a tremendous rate as a consequence of genome sequencing projects. Although one can guess at the functions of these novel enzymes by considering sequence homology to known proteases, there is a need for new tools to rapidly provide functional information on large numbers of proteins. We describe a method for determining the cleavage site specificity of proteolytic enzymes that involves pooled sequencing of peptide library mixtures. The method was used to determine cleavage site motifs for six enzymes in the matrix metalloprotease (MMP) family. The results were validated by comparison with previous literature and by analyzing the cleavage of individually synthesized peptide substrates. The library data led us to identify the proteoglycan neurocan as a novel MMP-2 substrate. Our results indicate that a small set of libraries can be used to quickly profile an expanding protease family, providing information applicable to the design of inhibitors and to the identification of protein substrates.
Subject(s)
Matrix Metalloproteinases/chemistry , Peptide Library , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Chondroitin Sulfate Proteoglycans/chemistry , Chondroitin Sulfate Proteoglycans/metabolism , Dose-Response Relationship, Drug , Humans , Kinetics , Lectins, C-Type , Matrix Metalloproteinase 2/metabolism , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Neurocan , Peptides/chemistry , Protein Binding , Rats , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.
Subject(s)
Phosphatidylinositol Phosphates/metabolism , Phosphoproteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Lipid Metabolism , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , NADPH Oxidases , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Structure, Tertiary , Sequence Alignment , TransfectionABSTRACT
Here, we investigate the mechanism and function of LKB1, a Ser/Thr kinase mutated in Peutz-Jegher syndrome (PJS). We demonstrate that LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. LKB1 protein is present in both the cytoplasm and nucleus of living cells and translocates to mitochondria during apoptosis. In vivo, LKB1 is highly upregulated in pyknotic intestinal epithelial cells. In contrast, polyps arising in Peutz-Jegher patients are devoid of LKB1 staining and have reduced numbers of apoptotic cells. We propose that a deficiency in apoptosis is a key factor in the formation of multiple benign intestinal polyps in PJS patients, and possibly for the subsequent development of malignant tumors in these patients.
Subject(s)
Apoptosis/physiology , Peutz-Jeghers Syndrome/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , AMP-Activated Protein Kinase Kinases , Cells, Cultured , Gene Expression , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intestine, Small/metabolism , Intestine, Small/pathology , Mitochondria/metabolism , Mutation/physiology , Peutz-Jeghers Syndrome/genetics , Peutz-Jeghers Syndrome/pathology , PhosphorylationSubject(s)
Cell Membrane/metabolism , Cell Nucleus/metabolism , Isoenzymes/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Proteins/metabolism , Transcription Factors/metabolism , Type C Phospholipases/metabolism , Active Transport, Cell Nucleus , Adaptor Proteins, Signal Transducing , Animals , Calcium/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11 , Heterotrimeric GTP-Binding Proteins/metabolism , Hydrolysis , Membrane Lipids/metabolism , Mice , Obesity/genetics , Obesity/metabolism , Phosphatidylinositol Phosphates/metabolism , Phospholipase C beta , Phosphorylation , Protein Structure, Tertiary , Proteins/chemistry , Proteins/genetics , Receptor, Serotonin, 5-HT2C , Receptors, Serotonin/metabolism , Signal Transduction , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
The rapid increase in genomic information requires new techniques to infer protein function and predict protein-protein interactions. Bioinformatics identifies modular signaling domains within protein sequences with a high degree of accuracy. In contrast, little success has been achieved in predicting short linear sequence motifs within proteins targeted by these domains to form complex signaling networks. Here we describe a peptide library-based searching algorithm, accessible over the World Wide Web, that identifies sequence motifs likely to bind to specific protein domains such as 14-3-3, SH2, and SH3 domains, or likely to be phosphorylated by specific protein kinases such as Src and AKT. Predictions from database searches for proteins containing motifs matching two different domains in a common signaling pathway provides a much higher success rate. This technology facilitates prediction of cell signaling networks within proteomes, and could aid in the identification of drug targets for the treatment of human diseases.
Subject(s)
Genome , Signal Transduction , Algorithms , Amino Acid Motifs , Amino Acid Sequence , Animals , Cattle , Databases, Factual , Humans , Internet , Mice , Molecular Sequence Data , Rats , Serine/chemistry , Software , Threonine/chemistry , Tyrosine/chemistryABSTRACT
The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.
Subject(s)
Epidermal Growth Factor/metabolism , Gene Expression/physiology , Neuregulin-1/metabolism , Receptor Protein-Tyrosine Kinases/physiology , Signal Transduction/physiology , Animals , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Cells, CulturedABSTRACT
Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.
Subject(s)
Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Hormones/chemistry , Hormones/metabolism , Kidney/enzymology , Kinetics , Male , Mice , Mice, Inbred C3H , Mice, Inbred ICR , Microvilli/enzymology , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Library , Peptides/chemistry , Peptides/metabolism , Protein Conformation , Protein Subunits , Substrate SpecificityABSTRACT
The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.
Subject(s)
DNA-Binding Proteins/metabolism , Proteins , Transcription Factors/chemistry , Transcription Factors/metabolism , Tyrosine 3-Monooxygenase/metabolism , 14-3-3 Proteins , Acyltransferases , Amino Acid Sequence , Animals , Binding Sites , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Chickens , DNA-Binding Proteins/chemistry , Gene Expression Regulation , HeLa Cells , Humans , Mice , Molecular Sequence Data , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Peptide Library , Peptides/chemistry , Protein-Tyrosine Kinases/antagonists & inhibitors , Amino Acid Sequence , Enzyme Inhibitors/chemical synthesis , Genes, Reporter , Humans , Interleukin-2/genetics , Isoenzymes/metabolism , Jurkat Cells , Kinetics , Lymphocyte Activation , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/pharmacology , Phospholipase C gamma , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Transfection , Type C Phospholipases/metabolism , ZAP-70 Protein-Tyrosine KinaseSubject(s)
Oligopeptides/chemical synthesis , Peptide Library , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Consensus Sequence , Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli , Glutathione Transferase/genetics , Glutathione Transferase/isolation & purification , Molecular Sequence Data , Oligopeptides/chemistry , Peptides/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
Phosphoinositide 3-kinases produce 3'-phosphorylated phosphoinositides that act as second messengers to recruit other signalling proteins to the membrane. Pi3ks are activated by many extracellular stimuli and have been implicated in a variety of cellular responses. The Pi3k gene family is complex and the physiological roles of different classes and isoforms are not clear. The gene Pik3r1 encodes three proteins (p85 alpha, p55 alpha and p50 alpha) that serve as regulatory subunits of class IA Pi3ks (ref. 2). Mice lacking only the p85 alpha isoform are viable but display hypoglycaemia and increased insulin sensitivity correlating with upregulation of the p55 alpha and p50 alpha variants. Here we report that loss of all protein products of Pik3r1 results in perinatal lethality. We observed, among other abnormalities, extensive hepatocyte necrosis and chylous ascites. We also noted enlarged skeletal muscle fibres, brown fat necrosis and calcification of cardiac tissue. In liver and muscle, loss of the major regulatory isoform caused a great decrease in expression and activity of class IA Pi3k catalytic subunits; nevertheless, homozygous mice still displayed hypoglycaemia, lower insulin levels and increased glucose tolerance. Our findings reveal that p55 alpha and/or p50 alpha are required for survival, but not for development of hypoglycaemia, in mice lacking p85 alpha.