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1.
Mol Cell ; 82(22): 4262-4276.e5, 2022 11 17.
Article in English | MEDLINE | ID: mdl-36347258

ABSTRACT

BRAF is frequently mutated in human cancer and the RASopathy syndromes, with RASopathy mutations often observed in the cysteine-rich domain (CRD). Although the CRD participates in phosphatidylserine (PS) binding, the RAS-RAF interaction, and RAF autoinhibition, the impact of these activities on RAF function in normal and disease states is not well characterized. Here, we analyze a panel of CRD mutations and show that they increase BRAF activity by relieving autoinhibition and/or enhancing PS binding, with relief of autoinhibition being the major factor determining mutation severity. Further, we show that CRD-mediated autoinhibition prevents the constitutive plasma membrane localization of BRAF that causes increased RAS-dependent and RAS-independent function. Comparison of the BRAF- and CRAF-CRDs also indicates that the BRAF-CRD is a stronger mediator of autoinhibition and PS binding, and given the increased catalytic activity of BRAF, our studies reveal a more critical role for CRD-mediated autoinhibition in BRAF regulation.


Subject(s)
Cysteine , Proto-Oncogene Proteins B-raf , Humans , Cysteine/genetics , Proto-Oncogene Proteins B-raf/genetics , Protein Domains , Mutation , Syndrome
2.
Mol Cell ; 82(5): 950-968.e14, 2022 03 03.
Article in English | MEDLINE | ID: mdl-35202574

ABSTRACT

A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts.


Subject(s)
GTP Phosphohydrolases , Signal Transduction , Animals , Crystallography, X-Ray , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Guanosine Triphosphate/metabolism , Mammals/metabolism , Nucleotides , Proteins
3.
Mol Cell ; 76(6): 872-884.e5, 2019 12 19.
Article in English | MEDLINE | ID: mdl-31606273

ABSTRACT

The Ras GTPases are frequently mutated in human cancer, and, although the Raf kinases are essential effectors of Ras signaling, the tumorigenic properties of specific Ras-Raf complexes are not well characterized. Here, we examine the ability of individual Ras and Raf proteins to interact in live cells using bioluminescence resonance energy transfer (BRET) technology. We find that C-Raf binds all mutant Ras proteins with high affinity, whereas B-Raf exhibits a striking preference for mutant K-Ras. This selectivity is mediated by the acidic, N-terminal segment of B-Raf and requires the K-Ras polybasic region for high-affinity binding. In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf/C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function.


Subject(s)
Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Neoplasms/enzymology , raf Kinases/metabolism , ras Proteins/metabolism , Animals , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , HeLa Cells , Humans , Male , Mice , Mutation , NIH 3T3 Cells , Neoplasms/genetics , Neoplasms/pathology , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-raf/genetics , Proto-Oncogene Proteins c-raf/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction/genetics , Spheroids, Cellular , raf Kinases/genetics , ras Proteins/genetics
4.
Biochem Soc Trans ; 52(3): 1061-1069, 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38695730

ABSTRACT

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.


Subject(s)
HSP90 Heat-Shock Proteins , Proto-Oncogene Proteins B-raf , Humans , HSP90 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/chemistry , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/chemistry , Protein Multimerization , raf Kinases/metabolism , raf Kinases/chemistry , Animals , Chaperonins/metabolism , Chaperonins/chemistry , Signal Transduction , 14-3-3 Proteins/metabolism , 14-3-3 Proteins/chemistry , Neoplasms/enzymology , Neoplasms/metabolism , Neoplasms/genetics , Proto-Oncogene Proteins c-raf/metabolism , Proto-Oncogene Proteins c-raf/chemistry , Models, Molecular
5.
Mol Cell ; 64(5): 875-887, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27889448

ABSTRACT

Ras pathway signaling plays a critical role in cell growth control and is often upregulated in human cancer. The Raf kinases selectively interact with GTP-bound Ras and are important effectors of Ras signaling, functioning as the initiating kinases in the ERK cascade. Here, we identify a route for the phospho-inhibition of Ras/Raf/MEK/ERK pathway signaling that is mediated by the stress-activated JNK cascade. We find that key Ras pathway components, the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in response to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as rigosertib and paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and may function as a signaling checkpoint to suppress the Ras pathway during conditions of cellular stress.


Subject(s)
Glycine/analogs & derivatives , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System/drug effects , Paclitaxel , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Sulfones , Enzyme Activation/drug effects , Glycine/pharmacokinetics , Glycine/pharmacology , HeLa Cells , Humans , Oxidative Stress , Paclitaxel/pharmacokinetics , Paclitaxel/pharmacology , Phosphorylation , Sulfones/pharmacokinetics , Sulfones/pharmacology , ras Proteins/metabolism
6.
J Nat Prod ; 85(6): 1603-1616, 2022 06 24.
Article in English | MEDLINE | ID: mdl-35696348

ABSTRACT

Seven new peptaibols named tolypocladamides A-G have been isolated from an extract of the fungus Tolypocladium inflatum, which inhibits the interaction between Raf and oncogenic Ras in a cell-based high-throughput screening assay. Each peptaibol contains 11 amino acid residues, an octanoyl or decanoyl fatty acid chain at the N-terminus, and a leucinol moiety at the C-terminus. The peptaibol sequences were elucidated on the basis of 2D NMR and mass spectral fragmentation analyses. Amino acid configurations were determined by advanced Marfey's analyses. Tolypocladamides A-G caused significant inhibition of Ras/Raf interactions with IC50 values ranging from 0.5 to 5.0 µM in a nanobioluminescence resonance energy transfer (NanoBRET) assay; however, no interactions were observed in a surface plasmon resonance assay for binding of the compounds to wild type or G12D mutant Ras constructs or to the Ras binding domain of Raf. NCI 60 cell line testing was also conducted, and little panel selectivity was observed.


Subject(s)
Antineoplastic Agents , Hypocreales , Amino Acids/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Hypocreales/chemistry , Peptaibols/pharmacology
7.
Proc Natl Acad Sci U S A ; 116(9): 3536-3545, 2019 02 26.
Article in English | MEDLINE | ID: mdl-30808747

ABSTRACT

Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell-cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.


Subject(s)
Cell Adhesion/genetics , Cell Movement/genetics , Embryonic Development/genetics , Intracellular Signaling Peptides and Proteins/genetics , Monomeric GTP-Binding Proteins/genetics , Animals , Cadherins/genetics , Gain of Function Mutation/genetics , Gastrulation/genetics , Humans , MAP Kinase Signaling System/genetics , Noonan Syndrome/genetics , Noonan Syndrome/physiopathology , Protein Binding , Zebrafish/genetics
8.
Mol Cell ; 49(4): 751-8, 2013 Feb 21.
Article in English | MEDLINE | ID: mdl-23352452

ABSTRACT

Raf kinases are essential for normal Ras-Raf-MEK-ERK pathway signaling, and activating mutations in components of this pathway are associated with a variety of human cancers, as well as the related developmental disorders Noonan, LEOPARD, and cardiofaciocutaneous syndromes. Although the Raf kinases are known to dimerize during normal and disease-associated Raf signaling, the functional significance of Raf dimerization has not been fully elucidated. Here, using mutational analysis and a peptide inhibitor, we show that dimerization is required for normal Ras-dependent Raf activation and for the biological function of disease-associated Raf mutants with moderate, low, or impaired kinase activity. However, dimerization is not needed for the function of B-Raf mutants with high catalytic activity, such as V600E-B-Raf. Importantly, we find that a dimer interface peptide can effectively block Raf dimerization and inhibit Raf signaling when dimerization is required for Raf function, thus identifying the Raf dimer interface as a therapeutic target.


Subject(s)
MAP Kinase Signaling System , raf Kinases/metabolism , Amino Acid Substitution , Animals , Cell Line , Enzyme Activation , Epidermal Growth Factor/physiology , Humans , Mice , Mutagenesis, Site-Directed , Mutation, Missense , Neoplasms/enzymology , Peptide Fragments/pharmacology , Platelet-Derived Growth Factor/physiology , Protein Interaction Domains and Motifs , Protein Kinase Inhibitors/pharmacology , Protein Multimerization , raf Kinases/antagonists & inhibitors , raf Kinases/chemistry , raf Kinases/genetics , ras Proteins/metabolism
9.
Am J Med Genet A ; 182(4): 866-876, 2020 04.
Article in English | MEDLINE | ID: mdl-31913576

ABSTRACT

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.


Subject(s)
Costello Syndrome/therapy , Ectodermal Dysplasia/therapy , Failure to Thrive/therapy , Heart Defects, Congenital/therapy , Molecular Targeted Therapy , Mutation , Neurofibromatosis 1/therapy , Noonan Syndrome/therapy , ras Proteins/antagonists & inhibitors , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/genetics , Costello Syndrome/genetics , Costello Syndrome/pathology , Ectodermal Dysplasia/genetics , Ectodermal Dysplasia/pathology , Facies , Failure to Thrive/genetics , Failure to Thrive/pathology , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Humans , Intersectoral Collaboration , National Cancer Institute (U.S.) , Neurofibromatosis 1/genetics , Neurofibromatosis 1/pathology , Noonan Syndrome/genetics , Noonan Syndrome/pathology , Research Report , Signal Transduction , United States , ras Proteins/genetics
10.
J Nat Prod ; 83(4): 1288-1294, 2020 04 24.
Article in English | MEDLINE | ID: mdl-32191460

ABSTRACT

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.


Subject(s)
Depsipeptides/pharmacology , Proto-Oncogene Proteins c-raf/antagonists & inhibitors , Theonella/chemistry , ras Proteins/antagonists & inhibitors , Amino Acids/chemistry , Animals , Depsipeptides/chemistry , Depsipeptides/isolation & purification , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Papua New Guinea , Porifera/chemistry , Proto-Oncogene Proteins c-raf/metabolism , Signal Transduction/drug effects , ras Proteins/metabolism
11.
Mol Cell ; 48(2): 298-312, 2012 Oct 26.
Article in English | MEDLINE | ID: mdl-22981863

ABSTRACT

In T cells, the adaptor Bam32 is coupled to Erk activation downstream of the TCR by an unknown mechanism. We characterized in Jurkat cells and primary T lymphocytes a pathway dependent on Bam32-PLC-γ1-Pak1 complexes, in which Pak1 kinase activates Raf-1 and Mek-1, both upstream of Erk. In the Bam32-PLC-γ1-Pak1 complex, catalytically inactive PLC-γ1 is used as a scaffold linking Bam32 to Pak1. PLC-γ1(C-SH2) directly binds S141 of Bam32, preventing LAT-mediated activation of Ras by PLC-γ1. The Bam32-PLC-γ1 interaction enhances the binding of the SH3 domain of the phospholipase with Pak1. The PLC-γ1(SH3)-Pak1 interaction activates Pak1 independently of the small GTPases Rac1/Cdc42, previously described as being the only activators of Pak1 in T cells. Direct binding of the SH3 domain of PLC-γ1 to Pak1 dissociates inactive Pak1 homodimers, a mechanism required for Pak1 activation. We have thus uncovered a LAT/Ras-independent, Bam32-nucleated pathway that activates Erk signaling in T cells.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , CD4-Positive T-Lymphocytes/metabolism , Membrane Proteins/metabolism , Protein Interaction Maps , Signal Transduction , p21-Activated Kinases/metabolism , Binding Sites , GTP Phosphohydrolases/metabolism , Humans , Jurkat Cells , MAP Kinase Kinase 1/metabolism , MAP Kinase Signaling System , Multiprotein Complexes , Phospholipase C gamma/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Type C Phospholipases/metabolism , src Homology Domains
12.
Genes Dev ; 26(7): 641-50, 2012 Apr 01.
Article in English | MEDLINE | ID: mdl-22474259

ABSTRACT

Cancer often arises when normal cellular growth goes awry due to defects in critical signal transduction pathways. A growing number of inhibitors that target specific components of these pathways are in clinical use, but the success of these agents has been limited by the resistance to inhibitor therapy that ultimately develops. Studies have now shown that cancer cells respond to chronic drug treatment by adapting their signaling circuitry, taking advantage of pathway redundancy and routes of feedback and cross-talk to maintain their function. This review focuses on the compensatory signaling mechanisms highlighted by the use of targeted inhibitors in cancer therapy.


Subject(s)
Gene Regulatory Networks , Neoplasms/therapy , Signal Transduction , Animals , Antineoplastic Agents/therapeutic use , Humans , Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/metabolism
13.
Br J Cancer ; 118(1): 3-8, 2018 01.
Article in English | MEDLINE | ID: mdl-29235562

ABSTRACT

The Raf protein kinases are key intermediates in cellular signal transduction, functioning as direct effectors of the Ras GTPases and as the initiating kinases in the ERK cascade. In human cancer, Raf activity is frequently dysregulated due to mutations in the Raf family member B-Raf or to alterations in upstream Raf regulators, including Ras and receptor tyrosine kinases. First-generation Raf inhibitors, such as vemurafenib and dabrafenib, have yielded dramatic responses in malignant melanomas containing B-Raf mutations; however, their overall usefulness has been limited by both intrinsic and acquired drug resistance. In particular, issues related to the dimerisation of the Raf kinases can impact the efficacy of these compounds and are a primary cause of drug resistance. Here, we will review the importance of Raf dimerisation in cell signalling as well as its effects on Raf inhibitor therapy, and we will present the new strategies that are being pursued to overcome the 'Raf Dimer Dilemma'.


Subject(s)
Neoplasms/enzymology , Protein Kinase Inhibitors/pharmacology , raf Kinases/chemistry , Drug Resistance, Neoplasm , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Models, Molecular , Molecular Targeted Therapy , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Oximes/pharmacology , Oximes/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Protein Multimerization , Signal Transduction , Vemurafenib/pharmacology , Vemurafenib/therapeutic use , raf Kinases/antagonists & inhibitors , raf Kinases/genetics
14.
J Nat Prod ; 81(7): 1666-1672, 2018 07 27.
Article in English | MEDLINE | ID: mdl-29979591

ABSTRACT

Six new macrophilone-type pyrroloiminoquines were isolated and identified from an extract of the marine hydroid Macrorhynchia philippina. The proton-deficient and heteroatom-rich structures of macrophilones B-G (2-7) were elucidated by spectroscopic analysis and comparison of their data with those of the previously reported metabolite macrophilone A (1). Compounds 1-7 are the first pyrroloiminoquines to be reported from a hydroid. The macrophilones were shown to inhibit the enzymatic conjugation of SUMO to peptide substrates, and macrophilones A (1) and C (3) exhibit potent and selective cytotoxic properties in the NCI-60 anticancer screen. Bioinformatic analysis revealed a close association of the cytotoxicity profiles of 1 and 3 with two known B-Raf kinase inhibitory drugs. While compounds 1 and 3 showed no kinase inhibitory activity, they resulted in a dramatic decrease in cellular protein levels of selected components of the ERK signal cascade. As such, the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads that target the ERK signal transduction pathway.


Subject(s)
Hydrozoa/chemistry , MAP Kinase Signaling System/drug effects , Pyrroloiminoquinones/isolation & purification , Animals , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Pyrroloiminoquinones/pharmacology , Sumoylation/drug effects
15.
Genes Dev ; 24(14): 1496-506, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20634316

ABSTRACT

Protein scaffolds play an important role in signal transduction, regulating the localization of signaling components and mediating key protein interactions. Here, we report that the major binding partners of the Connector Enhancer of KSR 1 (CNK1) scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors, and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream from insulin and insulin-like growth factor 1 (IGF-1) receptors. We identified a domain located in the C-terminal region of CNK1 that interacts constitutively with the coiled-coil domain of the cytohesins, and found that CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and rescue experiments, we found that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) to generate a PIP(2)-rich microenvironment that is critical for the membrane recruitment of insulin receptor substrate 1 (IRS1) and signal transmission to the PI3K/AKT cascade. These findings identify CNK1 as a new positive regulator of insulin signaling.


Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Insulin/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Signal Transduction , Cell Line , Cell Membrane/metabolism , Guanine Nucleotide Exchange Factors/chemistry , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mass Spectrometry , Phosphatidylinositols/metabolism , Protein Interaction Domains and Motifs
16.
J Biol Chem ; 291(34): 17804-15, 2016 08 19.
Article in English | MEDLINE | ID: mdl-27226552

ABSTRACT

The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Extracellular Signal-Regulated MAP Kinases , MAP Kinase Signaling System , Melanoma , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Casein Kinase II/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Dual Specificity Phosphatase 6/genetics , Dual Specificity Phosphatase 6/metabolism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/genetics , Melanoma/pathology , Phosphorylation/drug effects , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism
17.
Mol Cell ; 34(6): 652-62, 2009 Jun 26.
Article in English | MEDLINE | ID: mdl-19560418

ABSTRACT

Protein scaffolds have emerged as important regulators of MAPK cascades, facilitating kinase activation and providing crucial spatio/temporal control to their signaling outputs. Using a proteomics approach to compare the binding partners of the two mammalian KSR scaffolds, we find that both KSR1 and KSR2 interact with the kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Strikingly, we find that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. Calcineurin dephosphorylates KSR2 on specific sites in response to Ca2+ signals, thus regulating KSR2 localization and activity. Moreover, we find that depletion of endogenous KSR2 impairs Ca2+-mediated ERK activation and ERK-dependent signaling responses in INS1 pancreatic beta-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling.


Subject(s)
Calcineurin/metabolism , Calcium Signaling , Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System , Protein Kinases/physiology , Protein Serine-Threonine Kinases/physiology , Animals , COS Cells , Chlorocebus aethiops , Humans , Mice , Protein Kinases/analysis , Protein Kinases/chemistry , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Rats
18.
J Biol Chem ; 289(26): 18556-68, 2014 Jun 27.
Article in English | MEDLINE | ID: mdl-24825906

ABSTRACT

The Eph receptors and their membrane-bound ligands, ephrins, play important roles in various biological processes such as cell adhesion and movement. The transmembrane ephrinBs transduce reverse signaling in a tyrosine phosphorylation-dependent or -independent, as well as PDZ-dependent manner. Here, we show that ephrinB1 interacts with Connector Enhancer of KSR1 (CNK1) in an EphB receptor-independent manner. In cultured cells, cotransfection of ephrinB1 with CNK1 increases JNK phosphorylation. EphrinB1/CNK1-mediated JNK activation is reduced by overexpression of dominant-negative RhoA. Overexpression of CNK1 alone is sufficient for activation of RhoA; however, both ephrinB1 and CNK1 are required for JNK phosphorylation. Co-immunoprecipitation data showed that ephrinB1 and CNK1 act as scaffold proteins that connect RhoA and JNK signaling components, such as p115RhoGEF and MKK4. Furthermore, adhesion to fibronectin or active Src overexpression increases ephrinB1/CNK1 binding, whereas blocking Src activity by a pharmacological inhibitor decreases not only ephrinB1/CNK1 binding, but also JNK activation. EphrinB1 overexpression increases cell motility, however, CNK1 depletion by siRNA abrogates ephrinB1-mediated cell migration and JNK activation. Moreover, Rho kinase inhibitor or JNK inhibitor treatment suppresses ephrinB1-mediated cell migration. Taken together, our findings suggest that CNK1 is required for ephrinB1-induced JNK activation and cell migration.


Subject(s)
Cell Movement , Ephrin-B1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Cell Line , Cell Line, Tumor , Enzyme Activation , Ephrin-B1/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , JNK Mitogen-Activated Protein Kinases/genetics , Phosphorylation , Protein Binding , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism
19.
Semin Cell Dev Biol ; 22(7): 681-7, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21884813

ABSTRACT

The 14-3-3 proteins were the first phosphoserine/phosphothreonine-binding proteins to be discovered, a finding that provided the foundation for their prominent role in cell signaling. 14-3-3 family members interact with a wide spectrum of proteins including transcription factors, biosynthetic enzymes, cytoskeletal proteins, signaling molecules, apoptosis factors, and tumor suppressors. The interaction with 14-3-3 can have a profound effect on a target protein, altering its localization, stability, conformation, phosphorylation state, activity, and/or molecular interactions. Thus, by modulating the function of a diverse array of binding partners, 14-3-3 proteins have become key regulatory components in many vital cellular processes - processes that are crucial for normal growth and development and that often become dysregulated in human cancer. This review will examine the recent advances that further elucidate the role of 14-3-3 proteins in normal growth and cancer signaling with a particular emphasis on the signaling pathways that impact cell proliferation, cell migration, and epithelial-to-mesenchymal transition.


Subject(s)
14-3-3 Proteins/metabolism , Cell Proliferation , Neoplasms/physiopathology , 14-3-3 Proteins/genetics , Actin Cytoskeleton/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Movement , Disease Progression , Epithelial-Mesenchymal Transition , Gene Expression Regulation , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Binding , Signal Transduction
20.
Nat Chem Biol ; 13(1): 7-8, 2016 12 16.
Article in English | MEDLINE | ID: mdl-27984577
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