Covalent inhibitors of EGFR family protein kinases induce degradation of human Tribbles 2 (TRIB2) pseudokinase in cancer cells

A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule–induced protein down-regulation through drug “off-targets” might be relevant for other inhibitors that serendipitously target pseudokinases.

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Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

10.1101/305243 ◽

2018 ◽

Author(s):

Daniel M Foulkes ◽

Dominic P Byrne ◽

Fiona P Bailey ◽

Samantha Ferries ◽

Claire E Eyers ◽

...

Keyword(s):

Small Molecule ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Catalytic Domain ◽

Growth Factor Receptor ◽

Drug Repurposing ◽

Protein Kinase Inhibitors ◽

Substantial Evidence ◽

Epidermal Growth

ONE SENTENCE SUMMARYA Tribbles 2 pseudokinase small molecule screen led to the identification of known EGFR/HER2 inhibitors that alter the stability of TRIB2in vitroand lead to rapid on-target degradation of TRIB2 in human cancer cells.SHORT ABSTRACTTribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the AKT signaling module. Substantial evidence demonstrates that TRIB2 dysregulation is important in multiple human tumors. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine rich region and interacts with a peptide motif in its own C-terminal tail. We demonstrate that TRIB2 is a target for previously described small molecule protein kinase ‘inhibitors’, which were originally designed to inhibit the catalytic domain of EGFR/HER2 tyrosine kinases. Using thermal-shift assays and drug repurposing, we classify ligands that stabilize or destabilize the TRIB2 pseudokinase domain. TRIB2 destabilizing agents, including the clinical inhibitor afatinib, lead to rapid and on-target TRIB2 protein degradation in tumor cells, eliciting tractable effects on cell signaling and survival. Our data identifies leads for further development of TRIB2-degrading drugs and highlights compound-induced TRIB2 downregulation, which might be mechanistically relevant for other catalytically-deficient (pseudo)kinases targeted by small molecules.FULL ABSTRACTA major challenge associated with biochemical and cellular analysis of pseudokinases is the lack of target-validated small molecule ligands with which to probe molecular function. Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, which includes the canonical AKT signaling module. There is substantial evidence that human TRIB2 is a therapeutic target in both solid tumors and blood cancers. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine-rich region and interacts with a peptide motif in its own C-terminal tail, which was previously shown to drive interaction with cellular E3 ubiquitin ligases. In this study we demonstrate that TRIB2 is a target for previously described small molecule protein kinase inhibitors, which were originally designed to inhibit the canonical catalytic domain of the tyrosine kinases EGFR/HER2. Using a thermal-shift assay, we discovered TRIB2 ligands within the Published Kinase Inhibitor Set (PKIS), and employed a drug repurposing approach to classify compounds that either stabilize or destabilize TRIB2in vitro. Remarkably, TRIB2 destabilizing agents, including the clinical covalent drug afatinib, lead to rapid and on-target TRIB2 degradation in human cells, eliciting tractable effects on signaling and survival. Our data reveal the first drug-leads for development of TRIB2-degrading ligands, which will also be invaluable for unravelling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein downregulation through drug ‘off-targets’ might be relevant for other inhibitors that serendipitously target pseudokinases.ABBREVIATIONSDSFDifferential Scanning FluorimetryEGFREpidermal Growth Factor ReceptorHER2Human Epidermal Growth Factor Receptor 2MSMass spectrometryMSTMicroScale ThermophoresisPKISPublished Kinase Inhibitors SetTRIB2Tribbles 2TSAThermal Stability Assay

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New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors

Biochemical Journal ◽

10.1042/bcj20180265 ◽

2018 ◽

Vol 475 (15) ◽

pp. 2417-2433 ◽

Cited By ~ 8

Author(s):

Dominic P. Byrne ◽

Yong Li ◽

Krithika Ramakrishnan ◽

Igor L. Barsukov ◽

Edwin A. Yates ◽

...

Keyword(s):

Protein Kinase ◽

Heparan Sulfate ◽

Small Molecule ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Protein Kinase Inhibitors ◽

Fluorescent Substrate ◽

Raf Kinase ◽

Shift Analysis

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

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New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

10.1101/296533 ◽

2018 ◽

Author(s):

Dominic P Byrne ◽

Yong Li ◽

Krithika Ramakrishnan ◽

Igor L Barsukov ◽

Edwin A Yates ◽

...

Keyword(s):

Protein Kinase ◽

Small Molecule ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Heparan Sulphate ◽

Protein Kinase Inhibitors ◽

Fluorescent Substrate ◽

Shift Analysis ◽

In Cells

ABSTRACTSulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3’-phosphoadenosine 5’-phosphosulphate) to the 2-Oposition of α-L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compoundsin vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.SUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

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New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

10.1101/296707 ◽

2018 ◽

Author(s):

Dominic P Byrne ◽

Yong Li ◽

Pawin Ngamlert ◽

Krithika Ramakrishnan ◽

Claire E Eyers ◽

...

Keyword(s):

Protein Kinase ◽

Small Molecule ◽

Protein Interactions ◽

Chemical Biology ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Protein Kinase Inhibitors ◽

Post Translational Modification ◽

Protein Tyrosine

ABSTRACTProtein tyrosine sulphation is a post-translational modification (PTM) best known for regulating extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from the co-factor PAPS (3’-phosphoadenosine 5’-phosphosulphate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation. In this paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulphation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and 2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set (PKIS), we identified oxindole-based inhibitors of the Ser/Thr kinase RAF as low micromolar inhibitors of TPST1/2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulphotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulphation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.SUMMARY STATEMENTWe develop new assays to quantify tyrosine sulphation by the human tyrosine sulphotransferases TPST1 and 2. TPST1 and 2 catalytic activities are inhibited by protein kinase inhibitors, suggesting new starting points to synthesise (or repurpose) small molecule compounds to evaluate biological TPST using chemical biology.

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Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis

International Journal of Molecular Sciences ◽

10.3390/ijms19092599 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2599 ◽

Cited By ~ 6

Author(s):

Martin Sramek ◽

Jakub Neradil ◽

Petra Macigova ◽

Peter Mudry ◽

Kristyna Polaskova ◽

...

Keyword(s):

Protein Kinase ◽

Cell Line ◽

Tumor Cells ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Growth Factor Receptor ◽

Tumor Cell Line ◽

Protein Kinase Inhibitors ◽

Infantile Myofibromatosis ◽

Pdgfr Beta

Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta.

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Spironolactone, a Classic Potassium-Sparing Diuretic, Reduces Survivin Expression and Chemosensitizes Cancer Cells to Non-DNA-Damaging Anticancer Drugs

Cancers ◽

10.3390/cancers11101550 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1550 ◽

Cited By ~ 2

Author(s):

Tomomi Sanomachi ◽

Shuhei Suzuki ◽

Keita Togashi ◽

Asuka Sugai ◽

Shizuka Seino ◽

...

Keyword(s):

Cancer Cells ◽

Kinase Inhibitors ◽

Xenograft Model ◽

Growth Factor Receptor ◽

Survivin Expression ◽

Mouse Xenograft Model ◽

Anti Cancer ◽

Underlying Mechanisms

Spironolactone, a classical diuretic drug, is used to treat tumor-associated complications in cancer patients. Spironolactone was recently reported to exert anti-cancer effects by suppressing DNA damage repair. However, it currently remains unclear whether spironolactone exerts combinational effects with non-DNA-damaging anti-cancer drugs, such as gemcitabine and epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Using the cancer cells of lung cancer, pancreatic cancer, and glioblastoma, the combinational effects of spironolactone with gemcitabine and osimertinib, a third-generation EGFR-TKI, were examined in vitro with cell viability assays. To elucidate the underlying mechanisms, we investigated alterations induced in survivin, an anti-apoptotic protein, by spironolactone as well as the chemosensitization effects of the suppression of survivin by YM155, an inhibitor of survivin, and siRNA. We also examined the combinational effects in a mouse xenograft model. The results obtained revealed that spironolactone augmented cell death and the suppression of cell growth by gemcitabine and osimertinib. Spironolactone also reduced the expression of survivin in these cells, and the pharmacological and genetic suppression of survivin sensitized cells to gemcitabine and osimertinib. This combination also significantly suppressed tumor growth without apparent adverse effects in vivo. In conclusion, spironolactone is a safe candidate drug that exerts anti-cancer effects in combination with non-DNA-damaging drugs, such as gemcitabine and osimertinib, most likely through the suppression of survivin.

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Doxazosin, a Classic Alpha 1-Adrenoceptor Antagonist, Overcomes Osimertinib Resistance in Cancer Cells via the Upregulation of Autophagy as Drug Repurposing

Biomedicines ◽

10.3390/biomedicines8080273 ◽

2020 ◽

Vol 8 (8) ◽

pp. 273 ◽

Cited By ~ 1

Author(s):

Shuhei Suzuki ◽

Masahiro Yamamoto ◽

Tomomi Sanomachi ◽

Keita Togashi ◽

Asuka Sugai ◽

...

Keyword(s):

Cancer Cells ◽

Safety Profile ◽

Kinase Inhibitor ◽

Drug Repositioning ◽

Growth Factor Receptor ◽

Drug Repurposing ◽

Adrenoceptor Antagonist ◽

Anticancer Effects ◽

Epidermal Growth ◽

Excellent Safety Profile

Osimertinib, which is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, is an important anticancer drug because of its high efficacy and excellent safety profile. However, resistance against osimertinib is inevitable; therefore, therapeutic strategies to overcome the resistance are needed. Doxazosin, a classic quinazoline-based alpha 1-adrenoceptor antagonist is used to treat hypertension and benign prostatic hyperplasia with a known safety profile. The anticancer effects of doxazosin have been examined in various types of malignancies from the viewpoint of drug repositioning or repurposing. However, it currently remains unclear whether doxazosin sensitizes cancer cells to osimertinib. Herein, we demonstrated that doxazosin induced autophagy and enhanced the anticancer effects of osimertinib on the cancer cells and cancer stem cells of non-small cell lung cancer, pancreatic cancer, and glioblastoma at a concentration at which the growth of non-tumor cells was not affected. The osimertinib-sensitizing effects of doxazosin were suppressed by 3-methyladenine, an inhibitor of autophagy, which suggested that the effects of doxazosin were mediated by autophagy. The present study provides evidence for the efficacy of doxazosin as a combination therapy with osimertinib to overcome resistance against osimertinib.

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CXCL12 Enhances Survival of Chronic Lymphocytic Leukemia Cells Via a RAF-Dependent Pathway That Can Be Inhibited by Sorafenib

Blood ◽

10.1182/blood.v116.21.2445.2445 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2445-2445

Author(s):

Davorka Messmer ◽

Jessie-F Fecteau ◽

Morgan O'Hayre ◽

Tracy Handel ◽

Thomas J. Kipps

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Survival ◽

Small Molecule ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Growth Factor Receptor ◽

Lymphocytic Leukemia ◽

Increased Risk ◽

Dependent Pathway

Abstract Abstract 2445 The cellular microenvironment is critical for the survival of Chronic Lymphocytic Leukemia (CLL) cells. CLL cells die rapidly in vitro unless they receive survival signals from stromal cells or “nurse-like” cells (NLCs). CLL cell survival is in part mediated by the stromal cell-derived factor-1 (SDF-1alpha, designated as CXCL12), which is expressed by NLCs. CXCL12 is a highly conserved chemokine that can promote CLL-cell survival through its receptor CXCR4. Prior studies showed that treatment of CLL cells with CXCL12 induced activation of Extracellular Signal-Regulated Kinase (ERK). In this study, we examined CXCL12 signaling in CLL cells to characterize the mechanism (s) accounting for its ability to enhance CLL-cell survival. For this we examined CLL cells with high- or low- level expression of the zeta-associated protein of 70 kD (ZAP-70), a tyrosine kinase that is expressed by CLL cells of patients who have an increased risk for early disease progression and short survival. We found that CXCL12 induced a robust intracellular Ca2+ flux in ZAP-70+ CLL cells but only modest-to-poor Ca2+ flux in ZAP-70-negative CLL cells. Furthermore, ZAP-70+ CLL cells (n=10) responded to CXCL12 stimulation with increased and prolonged phosphorylation of ERK and MEK compared to ZAP-70-negative CLL cells (n=9). To investigate the underlying mechanism for MEK activation in ZAP-70+ CLL, we used small molecule inhibitors and found that CXCL12-induced phosphorylation of ERK and MEK could be blocked by sorafenib, a small molecule inhibitor of RAF. The role of RAF was further supported using KG5, a kinase inhibitor of RAF signaling through B-RAF and C-RAF in addition to platelet-derived-growth-factor-receptor (PDGFR) alpha and beta, Flt3, and Kit. As a control, we used a kinase inhibitor that targets all of these kinases except B- and C-RAF (KG1) and found it could not inhibit MEK activation. The involvement of Raf was further substantiated using GW5074, an inhibitor of B-RAF and C-RAF. Both KG5 and GW5074 could inhibit CXCL12-induced MEK activation in ZAP-70+ CLL samples. CXCL12-induced activation of MEK/ERK was not affected by sunitinib, an inhibitor of non-RAF kinases that also are inhibited by sorafenib, including VEGFR, PDGFR, Flt3, and c-Kit. Sorafenib not only inhibited MEK/ERK activation but also caused apoptosis of CLL cells whereby ZAP-70+ CLL cells showed incresed sensitivity to lower doses of sorafenib. Consistent with these results we found that ZAP-70+ CLL cells had a greater responsiveness to CXCL12 for survival in vitro than did ZAP-70-negative CLL cells. We conclude that CXCL12 can enhance survival particularly of ZAP-70+ CLL cells via a RAF dependent pathway, which can be targeted by the kinase inhibitor sorafenib. As such, sorafenib might be effective in blocking the protective influence of the microenvironment on CLL cells, suggesting that this drug could have activity in the treatment of patients with this disease. Disclosures: No relevant conflicts of interest to declare.

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A human-based multi-gene signature enables quantitative drug repurposing for metabolic disease

eLife ◽

10.7554/elife.68832 ◽

2022 ◽

Vol 11 ◽

Author(s):

James A Timmons ◽

Andrew Anighoro ◽

Robert J Brogan ◽

Jack Stahl ◽

Claes Wahlestedt ◽

...

Keyword(s):

Kinase Inhibitors ◽

Growth Factor Receptor ◽

Drug Repurposing ◽

Disease Status ◽

Gene Signature ◽

Genome Wide ◽

Epidermal Growth ◽

Ir Signature

Insulin resistance (IR) contributes to the pathophysiology of diabetes, dementia, viral infection, and cardiovascular disease. Drug repurposing (DR) may identify treatments for IR; however, barriers include uncertainty whether in vitro transcriptomic assays yield quantitative pharmacological data, or how to optimise assay design to best reflect in vivo human disease. We developed a clinical-based human tissue IR signature by combining lifestyle-mediated treatment responses (>500 human adipose and muscle biopsies) with biomarkers of disease status (fasting IR from >1200 biopsies). The assay identified a chemically diverse set of >130 positively acting compounds, highly enriched in true positives, that targeted 73 proteins regulating IR pathways. Our multi-gene RNA assay score reflected the quantitative pharmacological properties of a set of epidermal growth factor receptor-related tyrosine kinase inhibitors, providing insight into drug target specificity; an observation supported by deep learning-based genome-wide predicted pharmacology. Several drugs identified are suitable for evaluation in patients, particularly those with either acute or severe chronic IR.

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Live dynamics of GFP-calponin: isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences

Journal of Cell Science ◽

10.1242/jcs.113.21.3725 ◽

2000 ◽

Vol 113 (21) ◽

pp. 3725-3736 ◽

Cited By ~ 3

Author(s):

C. Danninger ◽

M. Gimona

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Actin Cytoskeleton ◽

Actin Polymerization ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Muscle Cells ◽

Protein Kinase Inhibitors

The calponin family of F-actin-, tropomyosin- and calmodulin-binding proteins currently comprises three genetic variants. Their functional roles implicated from in vitro studies include the regulation of actomyosin interactions in smooth muscle cells (h1 calponin), cytoskeletal organisation in non-muscle cells (h2 calponin) and the control of neurite outgrowth (acidic calponin). We have now investigated the effects of calponin (CaP) isoforms and their C-terminal deletion mutants on the actin cytoskeleton by time lapse video microscopy of GFP fusion proteins in living smooth muscle cells and fibroblasts. It is shown that h1 CaP associates with the actin stress fibers in the more central part of the cell, whereas h2 CaP localizes to the ends of stress fibres and in the motile lamellipodial protrusions of spreading cells. Cells expressing h2 CaP spread more efficiently than those expressing h1 CaP and expression of GFP h1 CaP resulted in reduced cell motility in wound healing experiments. Notably, expression of GFP h1 CaP, but not GFP h2 CaP, conferred increased resistance of the actin cytoskeleton to the actin polymerization antagonists cytochalasin B and latrunculin B, as well as to the protein kinase inhibitors H7-dihydrochloride and rho-kinase inhibitor Y-27632. These data point towards a dual role of CaP in the stabilization and regulation of the actin cytoskeleton in vivo. Deletion studies further identify an autoregulatory role for the unique C-terminal tail sequences in the respective CaP isoforms.

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