Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
1.
Int J Mol Sci ; 22(9)2021 Apr 23.
Article in English | MEDLINE | ID: mdl-33922601

ABSTRACT

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as "undruggable" and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer's disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson's disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.


Subject(s)
Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays/methods , Protein Tyrosine Phosphatases, Non-Receptor/antagonists & inhibitors , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Humans , Molecular Structure
2.
Nat Chem Biol ; 13(5): 486-493, 2017 05.
Article in English | MEDLINE | ID: mdl-28244987

ABSTRACT

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.


Subject(s)
Proteasome Inhibitors/pharmacology , Quinolines/pharmacology , Trans-Activators/antagonists & inhibitors , Dose-Response Relationship, Drug , Humans , Molecular Structure , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/chemistry , Quinolines/chemistry , Structure-Activity Relationship , Trans-Activators/metabolism
3.
Bioorg Med Chem Lett ; 24(3): 1000-1004, 2014 Feb 01.
Article in English | MEDLINE | ID: mdl-24412070

ABSTRACT

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified-one tissue-nonspecific (TNAP) and three tissue-specific-named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.


Subject(s)
Acetanilides/chemistry , Acetanilides/pharmacology , Alkaline Phosphatase/antagonists & inhibitors , Sulfonamides/chemistry , Sulfonamides/pharmacology , Acetanilides/isolation & purification , Animals , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , Humans , Mice , Protein Isoforms/chemistry , Sulfonamides/isolation & purification
4.
Biochem Pharmacol ; 219: 115937, 2024 01.
Article in English | MEDLINE | ID: mdl-37995979

ABSTRACT

Mitochondrial uridine insertion/deletion RNA editing, catalyzed by a multiprotein complex (editosome), is essential for gene expression in trypanosomes and Leishmania parasites. As this process is absent in the human host, a drug targeting this mechanism promises high selectivity and reduced toxicity. Here, we successfully miniaturized our FRET-based full-round RNA editing assay, which replicates the complete RNA editing process, adapting it into a 1536-well format. Leveraging this assay, we screened over 100,000 compounds against purified editosomes derived from Trypanosoma brucei, identifying seven confirmed primary hits. We sourced and evaluated various analogs to enhance the inhibitory and parasiticidal effects of these primary hits. In combination with secondary assays, our compounds marked inhibition of essential catalytic activities, including the RNA editing ligase and interactions of editosome proteins. Although the primary hits did not exhibit any growth inhibitory effect on parasites, we describe eight analog compounds capable of effectively killing T. brucei and/or Leishmania donovani parasites within a low micromolar concentration. Whether parasite killing is - at least in part - due to inhibition of RNA editing in vivo remains to be assessed. Our findings introduce novel molecular scaffolds with the potential for broad antitrypanosomal effects.


Subject(s)
Trypanosoma brucei brucei , Humans , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , High-Throughput Screening Assays , RNA Editing , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA/metabolism
5.
Cancer Cell ; 7(2): 155-65, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15710328

ABSTRACT

Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.


Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Neoplasms/metabolism , Peptide Hydrolases/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Breast Neoplasms/metabolism , Cell Line , Cell Line, Tumor , Chickens , Extracellular Matrix/metabolism , Humans , Melanoma/metabolism , Mice , Microscopy, Fluorescence , NIH 3T3 Cells , Neoplasm Invasiveness , Protein Structure, Tertiary , RNA, Small Interfering/metabolism , src Homology Domains , src-Family Kinases/metabolism
6.
Cancer Res ; 81(6): 1472-1485, 2021 03 15.
Article in English | MEDLINE | ID: mdl-33414172

ABSTRACT

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. SIGNIFICANCE: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.


Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , Endosomes/metabolism , Neoplasm Invasiveness/pathology , Podosomes/drug effects , Protein Serine-Threonine Kinases/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cytoplasmic Dyneins/genetics , Cytoplasmic Dyneins/metabolism , Datasets as Topic , Extracellular Matrix , Female , Gene Expression Profiling , Gene Knockdown Techniques , High-Throughput Screening Assays , Humans , Male , Melanoma/drug therapy , Melanoma/pathology , Mice , Neoplasm Invasiveness/prevention & control , Podosomes/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Skin Neoplasms/drug therapy , Skin Neoplasms/pathology , Time-Lapse Imaging , Xenograft Model Antitumor Assays
7.
Nat Commun ; 12(1): 5397, 2021 09 13.
Article in English | MEDLINE | ID: mdl-34518534

ABSTRACT

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.


Subject(s)
Genetic Predisposition to Disease/genetics , Histone Deacetylase Inhibitors/pharmacology , Leukemia, Myeloid/genetics , Ubiquitin-Protein Ligases/genetics , Xenograft Model Antitumor Assays/methods , Acute Disease , Animals , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , HEK293 Cells , HL-60 Cells , Humans , K562 Cells , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , U937 Cells , Ubiquitin-Protein Ligases/metabolism
8.
Elife ; 82019 10 22.
Article in English | MEDLINE | ID: mdl-31637999

ABSTRACT

High-content phenotypic screening has become the approach of choice for drug discovery due to its ability to extract drug-specific multi-layered data. In the field of epigenetics, such screening methods have suffered from a lack of tools sensitive to selective epigenetic perturbations. Here we describe a novel approach, Microscopic Imaging of Epigenetic Landscapes (MIEL), which captures the nuclear staining patterns of epigenetic marks and employs machine learning to accurately distinguish between such patterns. We validated the MIEL platform across multiple cells lines and using dose-response curves, to insure the fidelity and robustness of this approach for high content high throughput drug discovery. Focusing on noncytotoxic glioblastoma treatments, we demonstrated that MIEL can identify and classify epigenetically active drugs. Furthermore, we show MIEL was able to accurately rank candidate drugs by their ability to produce desired epigenetic alterations consistent with increased sensitivity to chemotherapeutic agents or with induction of glioblastoma differentiation.


Subject(s)
Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/genetics , Drug Discovery/methods , Epigenesis, Genetic/drug effects , High-Throughput Screening Assays , Histones/genetics , Neoplasm Proteins/genetics , Biomarkers, Tumor/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cell Nucleus/metabolism , Dose-Response Relationship, Drug , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Histones/metabolism , Humans , Image Processing, Computer-Assisted/statistics & numerical data , Machine Learning , Microscopy, Fluorescence , Neoplasm Proteins/metabolism
9.
Cancer Lett ; 449: 145-162, 2019 05 01.
Article in English | MEDLINE | ID: mdl-30771432

ABSTRACT

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.


Subject(s)
Melanoma/drug therapy , Nuclear Proteins/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Small Molecule Libraries/administration & dosage , Ubiquitin-Protein Ligases/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Computer Simulation , Down-Regulation , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mass Spectrometry , Melanoma/genetics , Mice , Nuclear Proteins/genetics , Prostatic Neoplasms/genetics , Small Molecule Libraries/isolation & purification , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/genetics , Xenograft Model Antitumor Assays
10.
Eur J Cell Biol ; 87(8-9): 555-67, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18417249

ABSTRACT

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Src-transformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.


Subject(s)
Microfilament Proteins/physiology , Neoplasms/blood supply , Phosphoproteins/physiology , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Cell Line, Tumor , Cell Surface Extensions/chemistry , Humans , Immunohistochemistry , Mice , Microfilament Proteins/metabolism , NIH 3T3 Cells , Neoplasms/metabolism , Neoplasms/pathology , Neovascularization, Pathologic , Phosphate-Binding Proteins , Phosphoproteins/metabolism , Transfection , src Homology Domains
11.
J Biomol Screen ; 20(10): 1232-45, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26265713

ABSTRACT

Endoplasmic reticulum (ER) stress activates three distinct signal transducers on the ER membrane. Inositol-requiring protein 1 (IRE1), the most conserved signal transducer, plays a key role in ER stress-mediated signaling. During ER stress, IRE1 initiates two discrete signaling cascades: the "adaptive" signaling cascade mediated by the XBP1 pathway and the "alarm" signaling cascade mediated by stress-activated protein kinase pathways. Fine-tuning of the balance between the adaptive and alarm signals contributes significantly to cellular fate under ER stress. Thus, we propose that the design of high-throughput screening (HTS) assays to selectively monitor IRE1 mediated-signaling would be desirable for drug discovery. To this end, we report the generation of stable human neural cell lines and development of cell-based HTS luciferase (Luc) reporter gene assays for the identification of pathway-specific chemical modulators of IRE1. We implemented a cell-based Luc assay using a chimeric CHOP-Gal4 transcription factor in 384-well format for monitoring IRE1 kinase-mediated p38MAPK activation and an unfolded response pathway element (URPE)-Luc cell-based assay in 1536-well format for monitoring IRE1's RNase-mediated activation of XBP1. Chemical library screening was successfully conducted with both the CHOP/Gal4-Luc cells and UPRE-Luc engineered cells. The studies demonstrate the feasibility of using these HTS assays for discovery of pathway-selective modulators of IRE1.


Subject(s)
Endoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , Protein Serine-Threonine Kinases/antagonists & inhibitors , Small Molecule Libraries , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Endoplasmic Reticulum Stress , Endoribonucleases/physiology , Enzyme Activation , Genes, Reporter , HeLa Cells , Humans , Luciferases/analysis , Luciferases/genetics , MAP Kinase Signaling System , Neurons , Protein Serine-Threonine Kinases/physiology , Regulatory Factor X Transcription Factors , Thapsigargin/metabolism , Transcription Factors/metabolism , X-Box Binding Protein 1
12.
J Biomol Screen ; 17(2): 163-76, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22034497

ABSTRACT

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.


Subject(s)
High-Throughput Screening Assays/methods , Polyubiquitin/biosynthesis , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Fluorescence Resonance Energy Transfer/methods , Polyubiquitin/chemistry , Small Molecule Libraries , Ubiquitination
13.
Sci Signal ; 2(88): ra53, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19755709

ABSTRACT

Invadopodia are actin-rich membrane protrusions of cancer cells that facilitate pericellular proteolysis and invasive behavior. We show here that reactive oxygen species (ROS) generated by the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) system are necessary for invadopodia formation and function. Knockdown of the invadopodia protein Tks5 [tyrosine kinase substrate with five Src homology 3 (SH3) domains], which is structurally related to the Nox component p47(phox), reduces total ROS abundance in cancer cells. Furthermore, Tks5 and p22(phox) can associate with each other, suggesting that Tks5 is part of the Nox complex. Tyrosine phosphorylation of Tks5 and Tks4, but not other Src substrates, is reduced by Nox inhibition. We propose that Tks5 facilitates the production of ROS necessary for invadopodia formation, and that in turn ROS modulate Tks5 tyrosine phosphorylation in a positive feedback loop.


Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Cell Surface Extensions , Cytochrome b Group/metabolism , NADPH Oxidases/metabolism , Phosphoproteins/physiology , Reactive Oxygen Species/metabolism , Animals , Cell Line , Cell Line, Tumor , Cytochrome b Group/genetics , Feedback, Physiological , Humans , Mice , NADPH Oxidases/genetics , Phosphate-Binding Proteins , Phosphoproteins/antagonists & inhibitors , Phosphorylation , RNA, Small Interfering/pharmacology , Transfection
14.
Mol Biol Cell ; 20(5): 1302-11, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19144821

ABSTRACT

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.


Subject(s)
Cell Membrane Structures/metabolism , Cell Movement/physiology , Phosphoproteins/physiology , Adaptor Proteins, Signal Transducing , Animals , Cell Line , Cell Membrane Structures/physiology , Cell Membrane Structures/ultrastructure , Cloning, Molecular , Humans , Lipid Metabolism , Mice , Phosphate-Binding Proteins , Phosphoproteins/analysis , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphorylation , Protein Structure, Tertiary
15.
J Cell Sci ; 122(Pt 15): 2727-40, 2009 Aug 01.
Article in English | MEDLINE | ID: mdl-19596797

ABSTRACT

Invadopodia are actin-based projections enriched with proteases, which invasive cancer cells use to degrade the extracellular matrix (ECM). The Phox homology (PX)-Src homology (SH)3 domain adaptor protein Tks5 (also known as SH3PXD2A) cooperates with Src tyrosine kinase to promote invadopodia formation but the underlying pathway is not clear. Here we show that Src phosphorylates Tks5 at Y557, inducing it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia. Tks5 mutants unable to bind Nck show reduced matrix degradation-promoting activity and recruit actin to invadopodia inefficiently. Conversely, Src- and Tks5-driven matrix proteolysis and actin assembly in invadopodia are enhanced by Nck1 or Nck2 overexpression and inhibited by Nck1 depletion. We show that clustering at the plasma membrane of the Tks5 inter-SH3 region containing Y557 triggers phosphorylation at this site, facilitating Nck recruitment and F-actin assembly. These results identify a Src-Tks5-Nck pathway in ECM-degrading invadopodia that shows parallels with pathways linking several mammalian and pathogen-derived proteins to local actin regulation.


Subject(s)
Actins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Cell Surface Extensions/physiology , Extracellular Matrix/metabolism , Neoplasms/metabolism , Oncogene Proteins/metabolism , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Animals , Blotting, Western , Cell Surface Extensions/ultrastructure , Fluorescent Antibody Technique , Humans , Immunoprecipitation , Mice , Neoplasms/pathology , Oncogene Proteins/antagonists & inhibitors , Oncogene Proteins/genetics , Phosphate-Binding Proteins , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , RNA, Small Interfering/pharmacology , Transfection , Tumor Cells, Cultured , src Homology Domains
16.
Biochem J ; 371(Pt 3): 947-55, 2003 May 01.
Article in English | MEDLINE | ID: mdl-12534371

ABSTRACT

We investigated the kinetic behaviour and substrate specificity of PTEN (phosphatase and tensin homologue deleted on chromosome 10) using unilamellar vesicles containing substrate lipids in a background of phosphatidylcholine. PTEN displays the characteristics expected of an interfacial enzyme, since the rate of enzyme activity is dependent on the surface concentration of the substrate lipids used (mol fraction), as well as the bulk concentration. Surface-dilution analysis revealed the catalytic efficiency of PTEN for PtdIns(3,4,5) P (3) to be 200-fold greater than for either PtdIns(3,4) P (2) or PtdIns(3,5) P (2), and 1000-fold greater than for PtdIns3 P. The interfacial K (m) value of PTEN for PtdIns(3,4,5) P (3) was very low, reflecting the small proportions of this lipid that are present in cellular membranes. The catalytic-centre activity ( k (cat)) for PtdIns(3,4,5) P (3) was at least 200-fold greater than that for the water-soluble substrate Ins(1,3,4,5) P (4). The preference for lipid substrates may result from an interfacial activation of the enzyme, rather than processive catalysis of vesicular substrates. Moreover, both PtdIns(4,5) P (2) and univalent salts stimulated the activity of PTEN for PtdIns(3,4,5) P (3), but profoundly inhibited activity against Ins(1,3,4,5) P (4). The stimulatory effect of PtdIns(4,5) P (2) was greater in magnitude and more potent in comparison with other anionic phospholipid species. A mutation in the lipid-binding C2 domain (M-CBR3) that is biologically inactive did not alter overall catalytic efficiency in this model, but decreased the efficiency of the interfacial binding step, demonstrating its importance in the catalytic mechanism of PTEN.


Subject(s)
Phospholipids/metabolism , Phosphoric Monoester Hydrolases/metabolism , Tumor Suppressor Proteins/metabolism , Anions , Catalysis , Hydrolysis , Kinetics , PTEN Phosphohydrolase , Phosphatidylinositol Phosphates/metabolism
17.
J Biol Chem ; 278(19): 16844-51, 2003 May 09.
Article in English | MEDLINE | ID: mdl-12615925

ABSTRACT

Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.


Subject(s)
Genes, src/genetics , Metalloendopeptidases/metabolism , Phosphoproteins/metabolism , 3T3 Cells , Animals , COS Cells , Cell Line, Transformed , Membrane Proteins/genetics , Membrane Proteins/metabolism , Metalloendopeptidases/genetics , Mice , Phosphate-Binding Proteins , Phosphoproteins/genetics , Protein Binding , Signal Transduction , src Homology Domains
SELECTION OF CITATIONS
SEARCH DETAIL