RESUMO
Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention.
Assuntos
Proteína Substrato Associada a Crk , Peptídeos , Fosfotirosina , Proteínas Proto-Oncogênicas c-crk , Domínios de Homologia de src , Proteína Substrato Associada a Crk/metabolismo , Proteína Substrato Associada a Crk/química , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Proto-Oncogênicas c-crk/química , Humanos , Fosfotirosina/metabolismo , Fosfotirosina/química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Ligação Proteica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Simulação de Acoplamento Molecular/métodos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/químicaRESUMO
Destruction of erythropoiesis process leads to various diseases, including thrombocytopenia, anaemia, and leukaemia. miR-429-CT10 regulation of kinase-like (CRKL) axis involved in development, progression and metastasis of cancers. However, the exact role of miR-429-CRKL axis in leukaemic cell differentiation are still unknown. The current work aimed to uncover the effect of miR-429-CRKL axis on erythropoiesis. In the present study, CRKL upregulation was negatively correlated with miR-429 downregulation in both chronic myeloid leukaemia (CML) patient and CR patient samples. Moreover, CRKL expression level was significantly decreased while miR-429 expression level was increased during the erythroid differentiation of K562 cells following hemin treatment. Functional investigations revealed that overexpression and knockdown of CRKL was remarkably effective in suppressing and promoting hemin-induced erythroid differentiation of K562 cells, whereas, miR-429 exhibited opposite effects to CRKL. Mechanistically, miR-429 regulates erythroid differentiation of K562 cells by downregulating CRKL via selectively targeting CRKL-3'-untranslated region (UTR) through Raf/MEK/ERK pathway. Conversely, CRKII had no effect on erythroid differentiation of K562 cells. Taken together, our data demonstrated that CRKL (but not CRKII) and miR-429 contribute to development, progression and erythropoiesis of CML, miR-429-CRKL axis regulates erythropoiesis of K562 cells via Raf/MEK/ERK pathway, providing novel insights into effective diagnosis and therapy for CML patients.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular , Células Eritroides , Hemina , Leucemia Mielogênica Crônica BCR-ABL Positiva , MicroRNAs , Proteínas Proto-Oncogênicas c-crk , Humanos , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Diferenciação Celular/efeitos dos fármacos , Células Eritroides/metabolismo , Células Eritroides/efeitos dos fármacos , Células Eritroides/patologia , Células Eritroides/citologia , Eritropoese/genética , Eritropoese/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Hemina/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Proto-Oncogênicas c-crk/genéticaRESUMO
Protein-protein interactions (PPIs) are known to be involved in most cellular functions, and a detailed knowledge of such interactions is essential for studying their role in normal and pathological conditions. Significant progress is being made in the identification of PPIs through advances in computational methods. In particular, the AlphaFold2 machine learning-based model has been shown to accelerate drug discovery process by predicting the 3D structure of protein complexes. In this chapter, a straightforward protocol for predicting interprotein interactions between PAR-3 and its protein partner adapter molecule crk is provided. Such artificial intelligence-based and publicly available approaches can provide a resource for further investigation of therapeutic drug targets.
Assuntos
Inteligência Artificial , Mapeamento de Interação de Proteínas , Mapeamento de Interação de Proteínas/métodos , Humanos , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Proto-Oncogênicas c-crk/química , Ligação Proteica , Biologia Computacional/métodos , SoftwareRESUMO
CRKL (CRK Like Proto-Oncogene) belongs to the Crk family and is a 39-kDa adapter protein that encodes SH2 and SH3 (src homologs) domains. To identify its oncogenic role in malignant melanoma, we investigated the association between CRKL and mutation, prognosis, tumor mutation burden, immune cell infiltration of melanoma, and explored the associations between CRKL and immunotherapy response. Our results showed that abnormal CRKL expression is associated with poor prognosis in melanoma and is significantly correlated with immune-activated pathways and processes, immune cell infiltrations, and expression of immunoregulators. Importantly, we found that CRKL expression is a predictive biomarker for anti-PD1 therapy response in melanoma patients. Furthermore, inhibiting CRKL expression in melanoma cell lines suppressed their proliferation and metastasis, as well as activated the pyroptosis-related pathway. Our study provides potential mechanisms of melanoma pathogenesis, which may suggest new avenues for targeted therapy in this disease.
Assuntos
Melanoma , Proteínas Nucleares , Humanos , Biomarcadores , Imunoterapia , Proteínas Nucleares/genética , Prognóstico , Proteínas Proto-Oncogênicas c-crk/metabolismoRESUMO
OBJECTIVE: To detect the expression of Growth factor binding protein 2 associated binding protein 2 (Gab2) and CT10 regulator of kinase II (CrkII) in ovarian cancer and analyze their clinical significance. To explore the effects of Gab2 and CrkII on the biological behavior of ovarian cancer cells. To analyze the possible molecular mechanism of Gab2 in the development of ovarian cancer. METHODS: Immunohistochemistry was used to detect the expression of Gab2 and CrkII in ovarian cancer. Chi square test was used to analyze the correlation between Gab2, CrkII and clinical parameters. Using Cox regression model to evaluate the risk factors affecting the prognosis. To analyze the correlation between Gab2, CrkII and survival rate by Kaplan-Meier. Cell experiments were preformed to explore the effects of Gab2 and CrkII on the biological behavior of cells. The interaction between Gab2 and CrkII was explored by immunoprecipitation. RESULTS: Immunohistochemistry revealed that high expression of Gab2 and CrkII in ovarian cancer. Patients with high expression of Gab2 or CrkII had higher International Federation of Gynecology and Obstetrics (FIGO) stage, grade and platinum-resistance recurrence. Multivariate analysis showed that Gab2 and CrkII were independent prognostic factors. Kaplan-Meier curve showed that the higher Gab2 and CrkII were, the poor prognosis the patients had. We observed that the overexpression of Gab2 and CrkII promoted the proliferation, metastasis and reduced chemosensitivity of cells. Conversely, the knockdown of Gab2 and CrkII resulted in the opposite results. In CrkII-knockdown cells, we found that Gab2 mediates biological behavior through CrkII. CONCLUSIONS: The expression of Gab2 and CrkII increase in ovarian cancer. The higher expression of Gab2 and CrkII predict the poor prognosis of patients. Gab2 and CrkII promote the proliferation and migration and reduce the chemosensitivity of cells. Gab2 regulates the biological behaviors of ovarian cancer cells through CrkII.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Neoplasias Ovarianas , Proteínas Proto-Oncogênicas c-crk , Feminino , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Carcinogênese/genéticaRESUMO
BACKGROUND: C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. METHODS: We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. RESULTS: CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. CONCLUSION: Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.
Assuntos
Fator 2 de Liberação do Nucleotídeo Guanina , Proteínas Nucleares , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Células HEK293 , Proteínas Nucleares/metabolismo , Nucleotídeos/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Domínios de Homologia de src , Tirosina/metabolismoRESUMO
CRK and CRKL encode cytoplasmic adaptors that contribute to the etiology of congenital heart disease. Neural crest cells (NCCs) are required for cardiac outflow tract (OFT) septation and aortic arch formation. The roles of Crk/Crkl in NCCs during mouse cardiovascular development remain unknown. To test this, we inactivated Crk and/or Crkl in NCCs. We found that the loss of Crk, rather than Crkl, in NCCs resulted in double outlet right ventricle, while loss of both Crk/Crkl in NCCs resulted in severe defects with earlier lethality due to failed OFT septation and severe dilation of the pharyngeal arch arteries (PAAs). We found that these defects are due to altered cell morphology resulting in reduced localization of NCCs to the OFT and failed integrity of the PAAs, along with reduced expression of Integrin signaling genes. Further, molecular studies identified reduced differentiation of vascular smooth muscle cells that may in part be due to altered Notch signaling. Additionally, there is increased cellular stress that leads to modest increase in apoptosis. Overall, this explains the mechanism for the Crk/Crkl phenotype.
Assuntos
Cardiopatias Congênitas , Crista Neural , Animais , Diferenciação Celular/genética , Cardiopatias Congênitas/metabolismo , Camundongos , Músculo Liso Vascular/metabolismo , Crista Neural/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Transdução de Sinais/genéticaRESUMO
PURPOSE: Compelling evidence has unveiled the importance of long noncoding RNAs (lncRNAs) in malignant behavior of Wilms' tumor (WT). Hereon, we intend to assess the function and associated molecular mechanism of lncRNA maternally expressed gene 8 (MEG8) in WT cells. METHODS: Expression levels of MEG8, miR-23a-3p, and CT10 regulator of kinase (CRK) were determined by quantitative real-time polymerase chain reaction. Cell viability was assessed by MTT assay. Besides, wound healing assay and transwell assay were applied to examine abilities of cell migration and invasion, respectively. Dual-luciferase reporter assay was employed to test the interplay among MEG8, miR-23a-3p, and CRK. Western blot was used to detect relative protein expression of CRK. RESULTS: MEG8 and CRK expression was elevated, while miR-23a-3p expression was decreased in WT tissues and cells. The histologic type, lymphatic metastasis, and National Wilms Tumor Study (NWTS) stage were associated with the expression of MEG8, miR-23a-3p, and CRK in WT patients. MEG8 knockdown or miR-23a-3p overexpression restrained WT cells in cell viability, migration, and invasiveness in vitro. As to mechanism exploration, MEG8 could directly bind to miR-23a-3p and then miR-23a-3p targeted CRK. MEG8 was inversely correlated with miR-23a-3p and positively correlated with CRK in WT tissues. Meantime, miR-23a-3p was inversely correlated with CRK in WT tissues. Additionally, MEG8 knockdown-mediated suppressive impacts on cell viability, migration, and invasiveness were reversed by overexpression of CRK or repression of miR-23a-3p in WT cells. CONCLUSIONS: The cell viability, migration, and invasiveness of WT cells were repressed by MEG8 knockdown via targeting the miR-23a-3p/CRK axis.
Assuntos
Neoplasias Renais , MicroRNAs , RNA Longo não Codificante , Tumor de Wilms , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Tumor de Wilms/genéticaRESUMO
During angiogenesis, cell adhesion molecules expressed on the endothelial cell surface promote the growth and survival of newly forming vessels. Hence, elucidation of the signaling pathways activated by cell-to-matrix adhesion may assist in the discovery of new targets to be used in antiangiogenic therapy. In proliferating endothelial cells, the single-pass transmembrane glycoprotein CD93 has recently emerged as an important endothelial cell adhesion molecule regulating vascular maturation. In this study, we unveil a signaling pathway triggered by CD93 that regulates actin cytoskeletal dynamics responsible of endothelial cell adhesion. We show that the Src-dependent phosphorylation of CD93 and the adaptor protein Cbl leads to the recruitment of Crk, which works as a downstream integrator in the CD93-mediated signaling. Moreover, confocal microscopy analysis of FRET-based biosensors shows that CD93 drives the coordinated activation of Rac1 and RhoA at the cell edge of spreading cells, thus promoting the establishment of cell polarity and adhesion required for cell motility.
Assuntos
Citoesqueleto de Actina/metabolismo , Moléculas de Adesão Celular/metabolismo , Movimento Celular/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Complemento/metabolismo , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Adesão Celular/genética , Moléculas de Adesão Celular/genética , Polaridade Celular/genética , Células Cultivadas , Humanos , Glicoproteínas de Membrana/genética , Fosforilação/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Interferência de RNA , Receptores de Complemento/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
Focal adhesion kinase (FAK) is well established as a regulator of cell migration, but whether and how the closely related proline-rich tyrosine kinase 2 (Pyk2) regulates fibroblast motility is still under debate. Using mouse embryonic fibroblasts (MEFs) from Pyk2-/- mice, we show here, for the first time, that lack of Pyk2 significantly impairs both random and directed fibroblast motility. Pyk2-/- MEFs show reduced cell-edge protrusion dynamics, which is dependent on both the kinase and protein-protein binding activities of Pyk2. Using bioinformatics analysis of in vitro high- throughput screens followed by text mining, we identified CrkI/II as novel substrates and interactors of Pyk2. Knockdown of CrkI/II shows altered dynamics of cell-edge protrusions, which is similar to the phenotype observed in Pyk2-/- MEFs. Moreover, epistasis experiments suggest that Pyk2 regulates the dynamics of cell-edge protrusions via direct and indirect interactions with Crk that enable both activation and down-regulation of Crk-mediated cytoskeletal signaling. This complex mechanism may enable fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation of cell motility, a process that should be strictly limited to specific time and context in normal cells, on the other hand.
Assuntos
Movimento Celular/genética , Fibroblastos/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Animais , Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Citoesqueleto/metabolismo , Quinase 2 de Adesão Focal/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Wilm's tumor is a common renal malignancy in childhood with unsatisfactory prognosis. microRNA-215-5p (miR-215-5p) has been reported as a tumor-suppressive miRNA in different types of human cancers, but rarely in the Wilm's tumor. In light of this, we tried to investigate the regulatory role and underlying mechanism of miR-215-5p in the Wilm's tumor. METHODS: After sample collection and cell culture, the expression of miR-215-5p and CT10 Regulator of Kinase (CRK) was detected. Then rhabdoid tumor cell lines (formerly classified as Wilms' tumor cell lines), G401 and WT-CLS1 cells were transfected with pcDNA3.1, pcDNA3.1-CRK, sh-NC, sh-CRK, agomir NC, miR-215-5p agomir, antagomir NC or miR-215-5p antagomir to explore the function of miR-215-5p and CRK in the Wilm's tumor cell proliferation and migration. Moreover, the relationship between miR-215-5p and CRK was analyzed by dual luciferase reporter gene assay. RESULTS: Lowly-expressed miR-215-5p and highly-expressed CRK were observed in the Wilm's tumor tissues and cells. Transfection of pcDNA3.1-CRK or miR-215-5p antagomir could promote G401 and WT-CLS1 cell proliferation and enhance migration ability, while transfection of sh-CRK or miR-215-5p agomir led to opposite results. Additionally, miR-215-5p may bind to CRK. Moreover, transfection of pcDNA3.1-CRK in G401 and WT-CLS1 cells could partially reverse the inhibitory effect of miR-215-5p agomir on the proliferation and migration of Wilm's tumor cells. CONCLUSION: Our study highlighted that miR-215-5p could suppress the proliferation and migration of Wilm's tumor cells by regulating the expression of CRK, providing new ideas for molecular targeted therapy for Wilm's tumor.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Tumor de Wilms/patologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-crk/genética , Células Tumorais Cultivadas , Tumor de Wilms/genética , Tumor de Wilms/metabolismoRESUMO
Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.
Assuntos
Proteínas Musculares/genética , Mutação , Síndromes Miastênicas Congênitas/tratamento farmacológico , Síndromes Miastênicas Congênitas/genética , Envelhecimento , Agrina/genética , Agrina/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/imunologia , Modelos Animais de Doenças , Feminino , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Masculino , Camundongos , Terapia de Alvo Molecular , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Síndromes Miastênicas Congênitas/imunologia , Fosforilação , Fosfotirosina/genética , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Receptores Proteína Tirosina Quinases/agonistas , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Recidiva , Sinapses/metabolismoRESUMO
Crk and CrkL are cellular counterparts of the viral oncoprotein v-Crk. Crk and CrkL are overexpressed in many types of human cancer, correlating with poor prognosis. Furthermore, gene knockdown and knockout of Crk and CrkL in tumor cell lines suppress tumor cell functions, including cell proliferation, transformation, migration, invasion, epithelial-mesenchymal transition, resistance to chemotherapy drugs, and in vivo tumor growth and metastasis. Conversely, overexpression of tumor cells with Crk or CrkL enhances tumor cell functions. Therefore, Crk and CrkL have been proposed as therapeutic targets for cancer treatment. However, it is unclear whether Crk and CrkL make distinct or overlapping contributions to tumor cell functions in various cancer types because Crk or CrkL have been examined independently in most studies. Two recent studies using colorectal cancer and glioblastoma cells clearly demonstrated that Crk and CrkL need to be ablated individually and combined to understand distinct and overlapping roles of the two proteins in cancer. A comprehensive understanding of individual and overlapping roles of Crk and CrkL in tumor cell functions is necessary to develop effective therapeutic strategies. This review systematically discusses crucial functions of Crk and CrkL in tumor cell functions and provides new perspectives on targeting Crk and CrkL in cancer therapy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Animais , Citoesqueleto/metabolismo , Transição Epitelial-Mesenquimal , HumanosRESUMO
The expression levels of CT10 regulator of kinase (Crk) and Crk-like (CrkL) are elevated in many human cancers, including glioblastoma (GBM), and are believed to contribute to poor prognosis. Although Crk and CrkL have been proposed as therapeutic targets in these tumors, the lack of a reliable, quantitative assay to measure Crk and CrkL activity has hindered development of inhibitors. Here, we knocked down Crk, CrkL, or both using siRNAs in a human GBM cell line, U-118MG, to determine the respective, quantitative contributions of Crk and CrkL to cellular phenotypes. The combined use of specific and potent Crk and CrkL siRNAs induced effective knockdown of CrkII, CrkI, and CrkL. Whereas Crk knockdown did not affect cell morphology, proliferation, adhesion, or invasion, CrkL knockdown caused shrinkage of cells and inhibition of cell proliferation, adhesion, and invasion. Crk/CrkL double knockdown resulted in more pronounced morphological alterations and more robust inhibition of proliferation, adhesion, and invasion. Furthermore, Crk/CrkL double knockdown completely blocked cell migration, and this effect was rescued by transient overexpression of CrkL but not of Crk. Quantification of protein levels indicated that CrkL is expressed more abundantly than CrkII and CrkI in U-118MG cells. These results demonstrate both the predominant role of CrkL and the essential overlapping functions of Crk and CrkL in U-118MG cells. Furthermore, our study indicates that migration of U-118MG cells depends entirely on Crk and CrkL. Thus, impedance-based, real-time measurement of tumor cell migration represents a robust assay for monitoring Crk and CrkL activities.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Humanos , Técnicas In Vitro , Fenótipo , Proteínas Proto-Oncogênicas c-crk/genéticaRESUMO
Opportunistic parasites of the Apicomplexa phylum use a variety of division modes built on two types of cell cycles that incorporate two distinctive mechanisms of mitosis: uncoupled from and coupled to parasite budding. Parasites have evolved novel factors to regulate such unique replication mechanisms that are poorly understood. Here, we have combined genetics, quantitative fluorescence microscopy, and global proteomics approaches to examine endodyogeny in Toxoplasma gondii dividing by mitosis coupled to cytokinesis. In the current study, we focus on the steps controlled by the recently described atypical Cdk-related kinase T. gondii Crk6 (TgCrk6). While inspecting protein complexes, we found that this previously orphaned TgCrk6 kinase interacts with a parasite-specific atypical cyclin, TgCyc1. We built conditional expression models and examined primary cell cycle defects caused by the lack of TgCrk6 or TgCyc1. Quantitative microscopy assays revealed that tachyzoites deficient in either TgCrk6 or the cyclin partner TgCyc1 exhibit identical mitotic defects, suggesting cooperative action of the complex components. Further examination of the mitotic structures indicated that the TgCrk6/TgCyc1 complex regulates metaphase. This novel finding confirms a functional spindle assembly checkpoint (SAC) in T. gondii. Measuring global changes in protein expression and phosphorylation, we found evidence that canonical activities of the Toxoplasma SAC are intertwined with parasite-specific tasks. Analysis of phosphorylation motifs suggests that Toxoplasma metaphase is regulated by CDK, mitogen-activated kinase (MAPK), and Aurora kinases, while the TgCrk6/TgCyc1 complex specifically controls the centromere-associated network. IMPORTANCE The rate of Toxoplasma tachyzoite division directly correlates with the severity of the disease, toxoplasmosis, which affects humans and animals. Thus, a better understanding of the tachyzoite cell cycle would offer much-needed efficient tools to control the acute stage of infection. Although tachyzoites divide by binary division, the cell cycle architecture and regulation differ significantly from the conventional binary fission of their host cells. Unlike the unidirectional conventional cell cycle, the Toxoplasma budding cycle is braided and is regulated by multiple essential Cdk-related kinases (Crks) that emerged in the place of missing conventional cell cycle regulators. How these novel Crks control apicomplexan cell cycles is largely unknown. Here, we have discovered a novel parasite-specific complex, TgCrk6/TgCyc1, that orchestrates a major mitotic event, the spindle assembly checkpoint. We demonstrated that tachyzoites incorporated parasite-specific tasks in the canonical checkpoint functions.
Assuntos
Proteínas de Protozoários , Toxoplasma , Toxoplasmose , Animais , Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Toxoplasmose/genética , Toxoplasmose/metabolismo , Toxoplasmose/parasitologiaRESUMO
The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cápsula do Cristalino/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cápsula do Cristalino/crescimento & desenvolvimento , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-crk/genética , Fatores de TempoRESUMO
Podocytes are actin-rich epithelial cells whose effacement and detachment are the main cause of glomerular disease. Crk family proteins: Crk1/2 and CrkL are reported to be important intracellular signaling proteins that are involved in many biological processes. However, the roles of them in maintaining podocyte morphology and function remain poorly understood. In this study, specific knocking down of Crk1/2 and CrkL in podocytes caused abnormal cell morphology, actin cytoskeleton rearrangement and dysfunction in cell adhesion, spreading, migration, and viability. The p130Cas, focal adhesion kinase, phosphatidylinositol 3-kinase/Akt, p38 and JNK signaling pathways involved in these alterations. Furthermore, knocking down CrkL alone conferred a more modest phenotype than did the Crk1/2 knockdown and the double knockdown. Kidney biopsy specimens from patients with focal segmental glomerulosclerosis and minimal change nephropathy showed downregulation of Crk1/2 and CrkL in glomeruli. In zebrafish embryos, Crk1/2 and CrkL knockdown compromised the morphology and caused abnormal glomerular development. Thus, our results suggest that Crk1/2 and CrkL expression are important in podocytes; loss of either will cause podocyte dysfunction, leading to foot process effacement and podocyte detachment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Proteínas Proto-Oncogênicas c-crk/metabolismo , Citoesqueleto de Actina/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Fosfatidilinositol 3-Quinase/metabolismoRESUMO
The Crk adaptor protein, a critical modifier of multiple signaling pathways, is overexpressed in many cancers where it contributes to tumor progression and metastasis. Recently, we have shown that Crk interacts with the peptidyl prolyl cis-trans isomerase, Cyclophilin A (CypA; PP1A) via a G219P220Y221 (GPY) motif in the carboxyl-terminal linker region of Crk, thereby delaying pY221 phosphorylation and preventing downregulation of Crk signaling. Here, we investigate the physiologic significance of the CypA/Crk interaction and query whether CypA inhibition affects Crk signaling in vitro and in vivo. We show that CypA, when induced under conditions of hypoxia, regulates Crk pY221 phosphorylation and signaling in cancer cell lines. Using nuclear magnetic resonance spectroscopy, we show that CypA binds to the Crk GPY motif via the catalytic PPII domain of CypA, and small-molecule nonimmunosuppressive inhibitors of CypA (Debio-025) disrupt the CypA-CrkII interaction and restores phosphorylation of Crk Y221. In cultured cell lines, Debio-025 suppresses cell migration, and when administered in vivo in an orthotopic model of triple-negative breast cancer, Debio-025 showed antitumor efficacy either alone or in combination with anti-PD-1 mAb, reducing both tumor volume and metastatic lung dispersion. Furthermore, when analyzed by NanoString immune profiling, treatment of Debio-025 with anti-PD-1 mAb increased both T-cell signaling and innate immune signaling in tumor microenvironment. IMPLICATIONS: These data suggest that pharmacologic inhibition of CypA may provide a promising and unanticipated consequence in cancer biology, in part by targeting the CypA/CrkII axis that regulates cell migration, tumor metastasis, and host antitumor immune evasion.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ciclosporina/administração & dosagem , Inibidores de Checkpoint Imunológico/administração & dosagem , Peptidilprolil Isomerase/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ciclosporina/farmacologia , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Camundongos , Modelos Moleculares , Metástase Neoplásica , Peptidilprolil Isomerase/química , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteínas Proto-Oncogênicas c-crk/química , Análise de Sequência de RNA , Microambiente Tumoral/efeitos dos fármacosRESUMO
Proline-rich motifs (PRMs) are widely used for mediating protein-protein interactions with weak binding affinities. Because they are intrinsically disordered when unbound, conformational entropy plays a significant role for the binding. However, residue-level differences of the entropic contribution in the binding of different ligands remain not well understood. We use all-atom molecular dynamics simulation and the maximal information spanning tree formalism to analyze conformational entropy associated with the binding of two PRMs, one from the Abl kinase and the other from the nonstructural protein 1 of the 1918 Spanish influenza A virus, to the N-terminal SH3 (nSH3) domain of the CrkII protein. Side chains of the stably folded nSH3 experience more entropy change upon ligand binding than the backbone, whereas PRMs involve comparable but heterogeneous entropy changes among the backbone and side chains. In nSH3, two conserved nonpolar residues forming contacts with the PRM experience the largest side-chain entropy loss. In contrast, the C-terminal charged residues of PRMs that form polar contacts with nSH3 experience the greatest side-chain entropy loss, although their "fuzzy" nature is attributable to the backbone that remains relatively flexible. Thus, residues that form high-occupancy contacts between nSH3 and PRM do not reciprocally contribute to entropy loss. Furthermore, certain surface residues of nSH3 distal to the interface with PRMs gain entropy, indicating a nonlocal effect of ligand binding. Comparing between the PRMs from cAbl and nonstructural protein 1, the latter involves a larger side-chain entropy loss and forms more contacts with nSH3. Consistent with experiments, this indicates stronger binding of the viral ligand at the expense of losing the flexibility of side chains, whereas the backbone experiences less entropy loss. The entropy "hotspots" as identified in this study will be important for tuning the binding affinity of various ligands to a receptor.
Assuntos
Influenza Humana , Entropia , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas c-crk/metabolismoRESUMO
The 1918 influenza A virus (IAV) caused the worst flu pandemic in human history. Non-structural protein 1 (NS1) is an important virulence factor of the 1918 IAV and antagonizes host antiviral immune responses. NS1 increases virulence by activating phosphoinositide 3-kinase (PI3K) via binding to the p85ß subunit of PI3K. Intriguingly, unlike the NS1 of other human IAV strains, 1918 NS1 hijacks another host protein, CRK, to form a ternary complex with p85ß, resulting in hyperactivation of PI3K. However, the molecular basis of the ternary interaction between 1918 NS1, CRK, and PI3K remains elusive. Here, we report the structural and thermodynamic bases of the ternary interaction. We find that the C-terminal tail (CTT) of 1918 NS1 remains highly flexible in the complex with p85ß. Thus, the CTT of 1918 NS1 in the complex with PI3K can efficiently hijack CRK. Notably, our study indicates that 1918 NS1 enhances its affinity to p85ß in the presence of CRK, which might result in enhanced activation of PI3K. Our results provide structural insight into how 1918 NS1 hijacks two host proteins simultaneously.