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1.
Int J Mol Sci ; 25(13)2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-39000229

RESUMO

Binding affinity is a fundamental parameter in drug design, describing the strength of the interaction between a molecule and its target protein. Accurately predicting binding affinity is crucial for the rapid development of novel therapeutics, the prioritization of promising candidates, and the optimization of their properties through rational design strategies. Binding affinity is determined by the mechanism of recognition between proteins and ligands. Various models, including the lock and key, induced fit, and conformational selection, have been proposed to explain this recognition process. However, current computational strategies to predict binding affinity, which are based on these models, have yet to produce satisfactory results. This article explores the connection between binding affinity and these protein-ligand interaction models, highlighting that they offer an incomplete picture of the mechanism governing binding affinity. Specifically, current models primarily center on the binding of the ligand and do not address its dissociation. In this context, the concept of ligand trapping is introduced, which models the mechanisms of dissociation. When combined with the current models, this concept can provide a unified theoretical framework that may allow for the accurate determination of the ligands' binding affinity.


Assuntos
Desenho de Fármacos , Ligação Proteica , Proteínas , Ligantes , Proteínas/química , Proteínas/metabolismo , Conformação Proteica , Modelos Moleculares , Sítios de Ligação , Humanos
2.
Int J Mol Sci ; 25(6)2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38542228

RESUMO

Recently, we identified a novel mechanism of enzyme inhibition in N-myristoyltransferases (NMTs), which we have named 'inhibitor trapping'. Inhibitor trapping occurs when the protein captures the small molecule within its structural confines, thereby preventing its free dissociation and resulting in a dramatic increase in inhibitor affinity and potency. Here, we demonstrate that inhibitor trapping also occurs in the kinases. Remarkably, the drug imatinib, which has revolutionized targeted cancer therapy, is entrapped in the structure of the Abl kinase. This effect is also observed in p38α kinase, where inhibitor trapping was found to depend on a 'magic' methyl group, which stabilizes the protein conformation and increases the affinity of the compound dramatically. Altogether, these results suggest that inhibitor trapping is not exclusive to N-myristoyltransferases, as it also occurs in the kinase family. Inhibitor trapping could enhance the binding affinity of an inhibitor by thousands of times and is as a key mechanism that plays a critical role in determining drug affinity and potency.


Assuntos
Piperazinas , Pirimidinas , Pirimidinas/farmacologia , Piperazinas/farmacologia , Benzamidas/farmacologia , Mesilato de Imatinib/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Quinases da Família src/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico
3.
Int J Mol Sci ; 24(14)2023 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-37511367

RESUMO

Predicting inhibitor potency is critical in drug design and development, yet it has remained one of computational biology's biggest unresolved challenges. Here, we show that in the case of the N-myristoyltransferase (NMT), this problem could be traced to the mechanisms by which the NMT enzyme is inhibited. NMT adopts open or closed conformations necessary for orchestrating the different steps of the catalytic process. The results indicate that the potency of the NMT inhibitors is determined by their ability to stabilize the enzyme conformation in the closed state, and that in this state, the small molecules themselves are trapped and locked inside the structure of the enzyme, creating a significant barrier for their dissociation. By using molecular dynamics simulations, we demonstrate that the conformational stabilization of the protein molecule in its closed form is highly correlated with the ligands activity and can be used to predict their potency. Hence, predicting inhibitor potency in silico might depend on modeling the conformational changes of the protein molecule upon binding of the ligand rather than estimating the changes in free binding energy that arise from their interaction.


Assuntos
Aciltransferases , Simulação de Dinâmica Molecular , Aciltransferases/metabolismo
4.
Molecules ; 27(17)2022 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-36080246

RESUMO

N-myristoyltransferase (NMT) inhibitors that were initially developed for treatment of parasitic protozoan infections, including sleeping sickness, malaria, and leismaniasis, have also shown great promise as treatment for oncological diseases. The successful transition of NMT inhibitors, which are currently at preclinical to early clinical stages, toward clinical approval and utilization may depend on the development and design of a diverse set of drug molecules with particular selectivity or pharmacological properties. In our study, we report that a common feature in the inhibitory mechanism of NMT is the formation of a salt bridge between a positively charged chemical group of the small molecule and the negatively charged C-terminus of an enzyme. Based on this observation, we designed a virtual screening protocol to identify novel ligands that mimic this mode of interaction. By screening over 1.1 million structures downloaded from the ZINC database, several hits were identified that displayed NMT inhibitory activity. The stability of the inhibitor-NMT complexes was evaluated by molecular dynamics simulations. The ligands from the stable complexes were tested in vitro and some of them appear to be promising leads for further optimization.


Assuntos
Aciltransferases , Inibidores Enzimáticos , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Simulação de Acoplamento Molecular
5.
Proc Natl Acad Sci U S A ; 114(33): E6912-E6921, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28760953

RESUMO

It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6-CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Encefalomielite Autoimune Experimental/imunologia , Glicoproteínas de Membrana/imunologia , Células A549 , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Ligantes , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
6.
Nat Cancer ; 4(2): 240-256, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36759733

RESUMO

BRAFV600E mutation confers a poor prognosis in metastatic colorectal cancer (CRC) despite combinatorial targeted therapies based on the latest understanding of signaling circuitry. To identify parallel resistance mechanisms induced by BRAF-MEK-EGFR co-targeting, we used a high-throughput kinase activity mapping platform. Here we show that SRC kinases are systematically activated in BRAFV600E CRC following targeted inhibition of BRAF ± EGFR and that coordinated targeting of SRC with BRAF ± EGFR increases treatment efficacy in vitro and in vivo. SRC drives resistance to BRAF ± EGFR targeted therapy independently of ERK signaling by inducing transcriptional reprogramming through ß-catenin (CTNNB1). The EGFR-independent compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E2 loop that can be blocked with cyclooxygenase-2 (COX2) inhibitors. Co-targeting of COX2 with BRAF + EGFR promotes durable suppression of tumor growth in patient-derived tumor xenograft models. COX2 inhibition represents a drug-repurposing strategy to overcome therapeutic resistance in BRAFV600E CRC.


Assuntos
Neoplasias Colorretais , Proteínas Proto-Oncogênicas B-raf , Humanos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores ErbB/genética , Quinases da Família src/genética , Quinases da Família src/uso terapêutico
7.
Front Mol Biosci ; 9: 1066029, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36703920

RESUMO

The salt bridge is the strongest non-covalent interaction in nature and is known to participate in protein folding, protein-protein interactions, and molecular recognition. However, the role of salt bridges in the context of drug design has remained not well understood. Here, we report that a common feature in the mechanism of inhibition of the N-myristoyltransferases (NMT), promising targets for the treatment of protozoan infections and cancer, is the formation of a salt bridge between a positively charged chemical group of the small molecule and the negatively charged C-terminus of the enzyme. Substituting the inhibitor positively charged amine group with a neutral methylene group prevents the formation of the salt bridge and leads to a dramatic activity loss. Molecular dynamics simulations have revealed that salt bridges stabilize the NMT-ligand complexes by functioning as molecular clips that stabilize the conformation of the protein structure. As such, the creation of salt bridges between the ligands and their protein targets may find an application as a valuable tool in rational drug design.

8.
Mol Cancer Res ; 19(6): 957-967, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33727342

RESUMO

The Src family kinases (SFK) are homologs of retroviral oncogenes, earning them the label of proto-oncogenes. Their functions are influenced by positive and negative regulatory tyrosine phosphorylation events and inhibitory and activating intramolecular and extramolecular interactions. This regulation is disrupted in their viral oncogene counterparts. However, in contrast to most other proto-oncogenes, the genetic alteration of these genes does not seem to occur in human tumors and how and whether their functions are altered in human cancers remain to be determined. To look for proteomic-level alterations, we took a more granular look at the activation states of SFKs based on their two known regulatory tyrosine phosphorylations, but found no significant differences in their activity states when comparing immortalized epithelial cells with cancer cells. SFKs are known to have other less well-studied phosphorylations, particularly within their unstructured N-terminal unique domains (UD), although their role in cancers has not been explored. In comparing panels of epithelial cells with cancer cells, we found a decrease in S17 phosphorylation in the UD of Src in cancer cells. Dephosphorylated S17 favors the dimerization of Src that is mediated through the UD and suggests increased Src dimerization in cancers. These data highlight the important role of the UD of Src and suggest that a deeper understanding of proteomic-level alterations of the unstructured UD of SFKs may provide considerable insights into how SFKs are deregulated in cancers. IMPLICATIONS: This work highlights the role of the N-terminal UD of Src kinases in regulating their signaling functions and possibly in their deregulation in human cancers.


Assuntos
Proteoma/metabolismo , Proteômica/métodos , Serina/metabolismo , Quinases da Família src/metabolismo , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Ativação Enzimática , Células Epiteliais/metabolismo , Humanos , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Mutação , Fosforilação , Ligação Proteica , Serina/genética , Quinases da Família src/genética
9.
Am J Pathol ; 174(5): 1756-65, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19349359

RESUMO

The roles of epithelial cells encompass both cellular- and tissue-level functions that involve numerous cell-cell and cell-matrix interactions, which ultimately mediate the highly structured arrangement of cells on a basement membrane. Although maintaining this basic structure is critical for preserving tissue integrity, plasticity in epithelial cell behavior is also critical for processes such as cell migration during development or wound repair, mitotic cell detachment, and physiological shedding. The mechanisms that mediate epithelial cell plasticity are only beginning to be understood. We previously identified Trask, a transmembrane protein that is phosphorylated by src kinases during mitosis. In this study, we report that the phosphorylation of Trask is associated with anchorage loss in epithelial cells. Phosphorylation of Trask is seen during the cell-detachment phase of mitosis, in experimentally induced interphase detachment, and during cell migration in experimental epithelial models. An analysis of human tissues shows that Trask is widely expressed in many epithelial tissues but not in most tissues of mesenchymal origin, except for a subset of early hematopoietic cells. Trask is not phosphorylated in epithelial tissues in vivo; however, its phosphorylation is seen in epithelial cells undergoing mitosis or physiological shedding. Trask is a novel epithelial membrane protein that is phosphorylated by src kinases when epithelial cells disengage from their tissue framework, identifying an important new regulator of epithelial tissue dynamics.


Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Células Epiteliais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Antígenos CD/genética , Antígenos de Neoplasias , Mama/citologia , Mama/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/genética , Movimento Celular/fisiologia , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Interfase/fisiologia , Mesoderma/citologia , Mesoderma/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias/genética , Fosforilação , Transdução de Sinais
10.
Clin Cancer Res ; 15(7): 2311-22, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19318475

RESUMO

PURPOSE: The frequently elevated activities of the c-src and c-yes products in human epithelial tumors suggest that these activated tyrosine kinases have tumorigenic functions analogous to the v-src and v-yes oncogene products. Studies of v-src-transformed fibroblasts have identified many of the effectors of this potent oncogene; however, because c-src and c-yes lack the mutational and promiscuous activities of their retroviral oncogene homologues, their presumptive tumorigenic functions in human epithelial tumors are more subtle, less well-defined, and await identification of possible effectors more directly relevant to epithelial cells. EXPERIMENTAL DESIGN: We recently identified a transmembrane glycoprotein named Trask that is expressed in epithelial tissues but not fibroblasts and is phosphorylated by SRC kinases in mitotic epithelial cells. In this study, we have surveyed the expression and phosphorylation of Trask in many human epithelial cancer cell lines and surgical tissues and tumors. RESULTS: Trask is widely expressed in human epithelial tissues, but its phosphorylation is tightly regulated and restricted to detached mitotic cells or cells undergoing physiologic shedding. However, abberant Trask phosphorylation is seen in many epithelial tumors from all stages including preinvasive, invasive, and metastatic tumors. Trask phosphorylation requires SRC kinases, and is also aberrantly hyperphosphorylated in the SRC-activated PyMT mouse epithelial tumors and dephosphorylated by the SRC inhibitor treatment of these tumors. CONCLUSIONS: The widespread phosphorylation of Trask in many human epithlelial cancers identifies a new potential effector of SRC kinases in human epithelial tumorigenesis.


Assuntos
Antígenos CD/metabolismo , Carcinoma/enzimologia , Moléculas de Adesão Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Antígenos de Neoplasias , Neoplasias da Mama/enzimologia , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/enzimologia , Células Epiteliais/enzimologia , Feminino , Humanos , Fosforilação , Quinases da Família src/metabolismo
11.
Stem Cells Dev ; 17(2): 343-53, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18447649

RESUMO

The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated intracellular form of c-kit receptor, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine-induced differentiation of the HSC/MPP-like cell line EML into myeloerythroid lineages. These findings prompted us to analyze tr-kit expression in purified murine fetal liver and bone marrow cell populations containing long-term repopulating (LTR) HSCs, short-term repopulating (STR) HSCs, MPPs, lineage-committed progenitors, and immature blood cells. Remarkably, these studies have revealed that in contrast to more widespread expression of c-kit, tr-kit is transcribed solely in cell populations enriched for LTR-HSCs, STR-HSCs, and MPPs. On the other hand, cell populations in which HSCs and MPPs are either present at a much lower frequency or are absent altogether, cells representing more advanced stages of differentiation into lymphoid and myeloid lineages do not express tr-kit. The observation that tr-kit is co-expressed with c-kit only in more primitive HSC- and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of the stem cell factor (SCF)/c-kit pathway or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. Taken together, these findings necessitate functional characterization of tr-kit and analysis of its potential role in the self-renewal, proliferation, and/or differentiation of HSC and multipotent progenitors.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Multipotentes/metabolismo , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas c-kit/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Dados de Sequência Molecular , Células-Tronco Multipotentes/fisiologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-kit/química , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/fisiologia , Homologia de Sequência de Aminoácidos
12.
Cell Rep ; 25(2): 449-463.e4, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30304684

RESUMO

The mode of regulation of Src kinases has been elucidated by crystallographic studies identifying conserved structured protein modules involved in an orderly set of intramolecular associations and ligand interactions. Despite these detailed insights, much of the complex behavior and diversity in the Src family remains unexplained. A key missing piece is the function of the unstructured N-terminal region. We report here the function of the N-terminal region in binding within a hydrophobic pocket in the kinase domain of a dimerization partner. Dimerization substantially enhances autophosphorylation and phosphorylation of selected substrates, and interfering with dimerization is disruptive to these functions. Dimerization and Y419 phosphorylation are codependent events creating a bistable switch. Given the versatility inherent in this intrinsically disordered region, its multisite phosphorylations, and its divergence within the family, the unique domain likely functions as a central signaling hub overseeing much of the activities and unique functions of Src family kinases.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Multimerização Proteica , Domínios de Homologia de src , Quinases da Família src/química , Quinases da Família src/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/química , Ligantes , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Transdução de Sinais
13.
Oncogene ; 37(21): 2817-2836, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29511352

RESUMO

Tumor metastasis depends on the dynamic regulation of cell adhesion through ß1-integrin. The Cub-Domain Containing Protein-1, CDCP1, is a transmembrane glycoprotein which regulates cell adhesion. Overexpression and loss of CDCP1 have been observed in the same cancer types to promote metastatic progression. Here, we demonstrate reduced CDCP1 expression in high-grade, primary prostate cancers, circulating tumor cells and tumor metastases of patients with castrate-resistant prostate cancer. CDCP1 is expressed in epithelial and not mesenchymal cells, and its cell surface and mRNA expression declines upon stimulation with TGFß1 and epithelial-to-mesenchymal transition. Silencing of CDCP1 in DU145 and PC3 cells resulted in 3.4-fold higher proliferation of non-adherent cells and 4.4-fold greater anchorage independent growth. CDCP1-silenced tumors grew in 100% of mice, compared to 30% growth of CDCP1-expressing tumors. After CDCP1 silencing, cell adhesion and migration diminished 2.1-fold, caused by loss of inside-out activation of ß1-integrin. We determined that the loss of CDCP1 reduces CDK5 kinase activity due to the phosphorylation of its regulatory subunit, CDK5R1/p35, by c-SRC on Y234. This generates a binding site for the C2 domain of PKCδ, which in turn phosphorylates CDK5 on T77. The resulting dissociation of the CDK5R1/CDK5 complex abolishes the activity of CDK5. Mutations of CDK5-T77 and CDK5R1-Y234 phosphorylation sites re-establish the CDK5/CDKR1 complex and the inside-out activity of ß1-integrin. Altogether, we discovered a new mechanism of regulation of CDK5 through loss of CDCP1, which dynamically regulates ß1-integrin in non-adherent cells and which may promote vascular dissemination in patients with advanced prostate cancer.


Assuntos
Antígenos CD/genética , Antígenos CD/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Regulação para Baixo , Integrina beta1/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Animais , Antígenos de Neoplasias , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Gradação de Tumores , Metástase Neoplásica , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo
14.
Gene ; 299(1-2): 195-204, 2002 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-12459267

RESUMO

Drosophila gene Pumilio (Pum) is a founder member of an evolutionarily conserved family of RNA-binding proteins that are present from yeast to mammals, and act as translational repressors during embryo development and cell differentiation. The human genome contains two Pumilio related genes, PUM1 and PUM2, that encode 127 and 114 kDa proteins with evolutionarily highly conserved Pum RNA-binding domain (86 and 88% homology with the fly Pum protein). PUM1 and PUM2 proteins share 83% overall similarity, with RNA-binding domain being 91% identical. Both PUM1 and PUM2 show relatively widespread and mostly overlapping expression in human tissues, and are very large genes with highly conserved gene structure. PUM1 consists of 22 exons, spanning about 150 kb on chromosome 1p35.2, whereas PUM2 consists of 20 exons and spans at least 80 kb on chromosome 2p23-24. Extremely high evolutionary conservation of the RNA-binding domain from yeast to humans, and conserved function of Pumilio proteins in invertebrates and lower vertebrates suggest that mammalian Pumilio proteins could also play an important role in translational regulation of embryogenesis and cell development and differentiation.


Assuntos
Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 2/genética , Clonagem Molecular , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , Evolução Molecular , Éxons , Feminino , Expressão Gênica , Genes/genética , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
15.
Cancer Res ; 73(3): 1168-79, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23243018

RESUMO

The cell surface glycoprotein Trask/CDCP1 is phosphorylated during anchorage loss in epithelial cells in which it inhibits integrin clustering, outside-in signaling, and cell adhesion. Its role in cancer has been difficult to understand, because of the lack of a discernible pattern in its various alterations in cancer cells. To address this issue, we generated mice lacking Trask function. Mammary tumors driven by the PyMT oncogene and skin tumors driven by the SmoM2 oncogene arose with accelerated kinetics in Trask-deficient mice, establishing a tumor suppressing function for this gene. Mechanistic investigations in mammary tumor cell lines derived from wild-type or Trask-deficient mice revealed a derepression of integrin signaling and an enhancement of integrin-growth factor receptor cross-talk, specifically in unanchored cell states. A similar restrictive link between anchorage and growth in untransformed epithelial cells was observed and disrupted by elimination of Trask. Together our results establish a tumor-suppressing function in Trask that restricts epithelial cell growth to the anchored state.


Assuntos
Antígenos de Neoplasias/fisiologia , Integrinas/fisiologia , Neoplasias Mamárias Experimentais/patologia , Glicoproteínas de Membrana/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Animais , Moléculas de Adesão Celular , Feminino , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Tamoxifeno/farmacologia
16.
Cell Cycle ; 10(8): 1225-32, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21490433

RESUMO

Phosphotyrosine signaling in anchored epithelial cells constitutes a spacially ordained signaling program that largely functions to promote integrin-linked focal adhesion complexes, serving to secure cell anchorage to matrix and as a bidirectional signaling hub that coordinates the physical state of the cell and its environment with cellular functions including proliferation and survival. Cells release their adhesions during processes such as mitosis, migration, or tumorigenesis, but the fate of signaling through tyrosine phosphorylation in unanchored cells remains poorly understood. In an examination of epithelial cells in the unanchored state, we find abundant phosphotyrosine signaling, largely recommitted to an anti-adhesive function mediated through the Src family phosphorylation of their transmembrane substrate Trask/CDCP1/gp140. Src-Trask phosphorylation inhibits integrin clustering and focal adhesion assembly and signaling, defining an active phosphotyrosine signaling program underlying the unanchored state. Src-Trask signaling and Src-focal adhesion signaling inactivate each other, constituting two opposing modes of phosphotyrosine signaling that define a switch underline cell anchorage state. Src kinases are prominent drivers of both signaling modes, identifying their position at the helm of adhesion signaling capable of specifying anchorage state through substrate selection. These experimental studies along with concurring phylogenetic evidence suggest that phosphorylation on tyrosine is a signaling function fundamentally linked with the regulation of integrins.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfotirosina/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Antígenos CD/genética , Antígenos de Neoplasias , Adesão Celular/fisiologia , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/genética , Adesões Focais/genética , Adesões Focais/metabolismo , Regulação da Expressão Gênica , Humanos , Integrinas/genética , Integrinas/metabolismo , Masculino , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Quinases da Família src/genética
17.
PLoS One ; 6(4): e19154, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21559459

RESUMO

Trask/CDCP1 is a transmembrane protein with a large extracellular and small intracellular domains. The intracellular domain (ICD) undergoes tyrosine phosphorylation by Src kinases during anchorage loss and, when phosphorylated, Trask functions to inhibit cell adhesion. The extracellular domain (ECD) undergoes proteolytic cleavage by serine proteases, although the functional significance of this remains unknown. There is conflicting evidence regarding whether it functions to signal the phosphorylation of the ICD. To better define the structural determinants that mediate the anti-adhesive functions of Trask, we generated a series of deletion mutants of Trask and expressed them in tet-inducible cell models to define the structural elements involved in cell adhesion signaling. We find that the ECD is dispensable for the phosphorylation of the ICD or for the inhibition of cell adhesion. The anti-adhesive functions of Trask are entirely embodied within its ICD and are specifically due to tyrosine phosphorylation of the ICD as this function is completely lost in a phosphorylation-defective tyrosine-phenylalanine mutant. Both full length and cleaved ECDs are fully capable of phosphorylation and undergo phosphorylation during anchorage loss and cleavage is not an upstream signal for ICD phosphorylation. These data establish that the anti-adhesive functions of Trask are mediated entirely through its tyrosine phosphorylation. It remains to be defined what role, if any, the Trask ECD plays in its adhesion functions.


Assuntos
Antígenos CD/química , Antígenos CD/fisiologia , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/fisiologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Antígenos de Neoplasias , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência/métodos , Microscopia de Contraste de Fase/métodos , Mutação , Fases de Leitura Aberta , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Transfecção , Tirosina/química
18.
Mol Cell Biol ; 31(4): 766-82, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21189288

RESUMO

Trask is a recently described transmembrane substrate of Src kinases whose expression and phosphorylation has been correlated with the biology of some cancers. Little is known about the molecular functions of Trask, although its phosphorylation has been associated with cell adhesion. We have studied the effects of Trask phosphorylation on cell adhesion, integrin activation, clustering, and focal adhesion signaling. The small hairpin RNA (shRNA) knockdown of Trask results in increased cell adhesiveness and a failure to properly inactivate focal adhesion signaling, even in the unanchored state. On the contrary, the experimentally induced phosphorylation of Trask results in the inhibition of cell adhesion and inhibition of focal adhesion signaling. This is mediated through the inhibition of integrin clustering without affecting integrin affinity state or ligand binding activity. Furthermore, Trask signaling and focal adhesion signaling inactivate each other and signal in exclusion with each other, constituting a switch that underlies cell anchorage state. These data provide considerable insight into how Trask functions to regulate cell adhesion and reveal a novel pathway through which Src kinases can oppose integrin-mediated cell adhesion.


Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Proteínas de Neoplasias/metabolismo , Quinases da Família src/metabolismo , Antígenos CD/genética , Antígenos de Neoplasias , Sequência de Bases , Adesão Celular/fisiologia , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Integrinas/química , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fosforilação , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Especificidade por Substrato
19.
Blood Cells Mol Dis ; 30(1): 55-69, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12667987

RESUMO

Self-renewal is the common functional property of all types of stem cells and is thought to be regulated by unknown conserved intrinsic and extrinsic molecular mechanisms. Recently, an evolutionarily conserved Pumilio family of RNA-binding proteins that regulate asymmetric cell division was found to be essential for stem cell maintenance and self-renewal in Drosophila and Caenorhabditis elegans. Based on conserved function in invertebrates and lower vertebrates it was recently proposed that an ancestral function of Pumilio proteins is to support proliferation and self-renewal of stem cells. This raises an interesting possibility that Pumilio could be part of evolutionarily conserved intrinsic molecular mechanism that regulates self-renewal of mammalian stem cells. Here we describe cloning and comparative sequence analysis of Pum1 and Pum2 genes, mouse members of the Pumilio family, and for the first time demonstrate expression of Pumilio genes in mammalian hematopoietic stem cells (HSC). Pum1 and Pum2 share 51 and 55% overall similarity with the fly Pum, whereas their RNA-binding domains show a very high degree of evolutionary conservation (86-88% homology). Both genes are expressed in a variety of tissues suggesting that they have widespread function. During blood cell development Pum1 and Pum2 exhibit differential expression in cell populations enriched for HSC and progenitors. Both genes are highly transcribed in populations of adult HSC (Rho-123(low)Sca-1(+)c-kit(+)Lin(-) cells). In a more heterogeneous population of HSC (Lin(-)Sca-1(+)) and in progenitors (Lin(-)Sca-1(-) cells) Pum1 is not transcribed, whereas Pum2 expression is significantly down-regulated. Ongoing in vitro and in vivo functional analysis of mouse Pumilio genes will help to elucidate the biological role of mammalian Pumilio genes and determine whether they play any role in maintenance of mammalian stem cells, such as HSC.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Mapeamento Cromossômico , Cromossomos/genética , Clonagem Molecular , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , Proteínas de Drosophila/genética , Evolução Molecular , Éxons , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Células-Tronco Hematopoéticas/citologia , Íntrons , Masculino , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
20.
IUBMB Life ; 55(7): 359-66, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-14584586

RESUMO

Drosophila Pumilio (Pum) protein is a founder member of a novel family of RNA-binding proteins, known as the PUF family. The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants, and are characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats. The conserved biochemical features and genetic function of PUF family members have emerged from studies of model organisms. PUF proteins bind to related sequence motifs in the 3' untranslated region (3'UTR) of specific target mRNAs and repress their translation. Frequently, PUF proteins function asymmetrically to create protein gradients, thus causing asymmetric cell division and regulating cell fate specification. Thus, it was recently proposed that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells. Here we review the evolution, conserved genetic and biochemical properties of PUF family of proteins, and discuss protein interactions, upstream regulators and downstream targets of PUF proteins. We also suggest that a conserved mechanism of PUF function extends to the newly described mammalian members of the PUF family (human PUM1 and PUM2, and mouse Pum1 and Pum2), that show extensive homology to Drosophila Pum, and could have an important role in cell development, fate specification and differentiation.


Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/fisiologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Caenorhabditis elegans , Divisão Celular , Linhagem da Célula , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Evolução Molecular , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
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