RESUMO
BACKGROUND: The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them. METHODS: Based on a phosphoproteomics screen for novel PIM substrates, we have used kinase assays and fluorescence-based imaging techniques to validate actin capping proteins as PIM1 substrates and interaction partners. We have analysed the functional consequences of capping protein phosphorylation on cell migration and adhesion by using wound healing and real-time impedance-based assays. We have also investigated phosphorylation-dependent effects on actin polymerization by analysing the protective role of capping protein phosphomutants in actin disassembly assays. RESULTS: We have identified capping proteins CAPZA1 and CAPZB2 as PIM1 substrates, and shown that phosphorylation of either of them leads to increased adhesion and migration of human prostate cancer cells. Phosphorylation also reduces the ability of the capping proteins to protect polymerized actin from disassembly. CONCLUSIONS: Our data suggest that PIM kinases are able to induce changes in actin dynamics to support cell adhesion and movement. Thus, we have identified a novel mechanism through which PIM kinases enhance motility and metastatic behaviour of cancer cells. Video abstract.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Movimento Celular , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Extensões da Superfície Celular/metabolismo , Citoplasma/metabolismo , Humanos , Masculino , Camundongos , Fosforilação , Multimerização Proteica , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidoresRESUMO
BACKGROUND: Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. METHODS: We used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets. RESULTS: Here we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens. CONCLUSIONS: Based on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer.
Assuntos
Movimento Celular , Fatores de Transcrição NFATC/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proliferação de Células , Humanos , Masculino , Espectrometria de Massas , Células PC-3 , Fosforilação , Neoplasias da Próstata/patologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The ability of cells to migrate and form metastases is one of the fatal hallmarks of cancer that can be conquered only with better understanding of the molecules and regulatory mechanisms involved. The oncogenic PIM kinases have been shown to support cancer cell survival and motility, but the PIM-regulated pathways stimulating cell migration and invasion are less well characterized than those affecting cell survival. Here we have identified the glycogen synthase kinase 3ß (GSK3B) and the forkhead box P3 (FOXP3) transcription factor as direct PIM targets, whose tumour-suppressive effects in prostate cancer cells are inhibited by PIM-induced phosphorylation, resulting in increased cell migration. Targeting GSK3B is also essential for the observed PIM-enhanced expression of the prostaglandin-endoperoxide synthase 2 (PTGS2), which is an important regulator of both cell migration and adhesion. Accordingly, selective inhibition of PIM activity not only reduces cell migration, but also affects integrin-mediated cell adhesion. Taken together, these data provide novel mechanistic insights on how and why patients with metastatic prostate cancer may benefit from therapies targeting PIM kinases, and how such approaches may also be applicable to inflammatory conditions.
Assuntos
Adenocarcinoma/enzimologia , Movimento Celular , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-pim-1/fisiologia , Adenocarcinoma/patologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de SinaisRESUMO
Oncogenic Pim family kinases are often overexpressed in human hematopoietic malignancies as well as in solid tumours. These kinases contribute to tumorigenesis by promoting cell survival and by enhancing resistance against chemotherapy and radiation therapy. Furthermore, we have recently shown that they increase the metastatic potential of adherent cancer cells. Here we describe identification of tricyclic benzo[cd]azulenes and their derivatives as effective and selective inhibitors of Pim kinases. These compounds inhibit Pim autophosphorylation and abrogate the anti-apoptotic effects of Pim kinases. They also reduce cancer cell motility and suppress proliferation of lymphoblastoid cell lines infected and immortalized by the Epstein-Barr virus. Thus, these novel Pim-selective inhibitors provide promising compounds for both research and therapeutic purposes.
Assuntos
Azulenos/farmacologia , Transformação Celular Viral/efeitos dos fármacos , Herpesvirus Humano 4/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Herpesvirus Humano 4/metabolismo , Humanos , Camundongos , Células Mieloides/virologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/metabolismoRESUMO
Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from ß-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites.
Assuntos
Fosfoproteínas/química , Compostos de Estanho/química , Sequência de Aminoácidos , Animais , Células COS , Caseínas/química , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/química , Vidro , Limite de Detecção , Fatores de Transcrição NFATC/biossíntese , Fatores de Transcrição NFATC/química , Fosfoproteínas/isolamento & purificação , Radioisótopos de Fósforo , Fosforilação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9), which we had recently identified as a potent and selective inhibitor for all Pim family members. RESULTS: We now demonstrate that the Pim kinase inhibitor DHPCC-9 is very effective also in cell-based assays. DHPCC-9 impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid cells and inhibits intracellular phosphorylation of Pim substrates such as Bad. Moreover, DHPCC-9 slows down migration and invasion of cancer cells derived from either prostate cancer or squamocellular carcinoma patients. Silencing of Pim expression reduces cell motility, while Pim overexpression enhances it, strongly suggesting that the observed effects of DHPCC-9 are dependent on Pim kinase activity. Interestingly, DHPCC-9 also abrogates NFATc-dependent migration of cancer cells, implying that NFATc factors mediate at least part of the pro-migratory effects of Pim kinases. CONCLUSIONS: Altogether, our data indicate that DHPCC-9 is not only a powerful tool to investigate physiological effects of the oncogenic Pim family kinases, but also an attractive molecule for drug development to inhibit invasiveness of Pim-overexpressing cancer cells.
Assuntos
Movimento Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Humanos , Camundongos , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/genéticaRESUMO
Host signal-transduction pathways are intimately involved in the switch between latency and productive infection of herpes viruses. As with other herpes viruses, infection by Kaposi's sarcoma herpesvirus (KSHV) displays these two phases. During latency only few viral genes are expressed, while in the productive infection the virus is reactivated with initiation of extensive viral DNA replication and gene expression, resulting in production of new viral particles. Viral reactivation is crucial for KSHV pathogenesis and contributes to the progression of KS. We have recently identified Pim-1 as a kinase reactivating KSHV upon over-expression. Here we show that another Pim family kinase, Pim-3, also induces viral reactivation. We demonstrate that expression of both Pim-1 and Pim-3 is induced in response to physiological and chemical reactivation in naturally KSHV-infected cells, and we show that they are required for KSHV reactivation under these conditions. Furthermore, our data indicate that Pim-1 and Pim-3 contribute to viral reactivation by phosphorylating the KSHV latency-associated nuclear antigen (LANA) on serine residues 205 and 206. This counteracts the LANA-mediated repression of the KSHV lytic gene transcription. The identification of Pim family kinases as novel cellular regulators of the gammaherpesvirus life cycle facilitates a deeper understanding of virus-host interactions during reactivation and may represent potential novel targets for therapeutic intervention.
Assuntos
Antígenos Virais/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ativação Viral , Latência Viral , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-pim-1/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Células Vero , Replicação ViralRESUMO
Latent Epstein-Barr virus (EBV) infection is strongly associated with B-cell proliferative diseases such as Burkitt's lymphoma. Here we show that the oncogenic serine/threonine kinases Pim-1 and Pim-2 enhance the activity of the viral transcriptional activator EBNA2. During EBV infection of primary B-lymphocytes, the mRNA expression levels of pim genes, especially of pim-2, are upregulated and remain elevated in latently infected B-cell lines. Thus, EBV-induced upregulation of Pim kinases and Pim-stimulated EBNA2 transcriptional activity may contribute to the ability of EBV to immortalize B-cells and predispose them to malignant growth.
Assuntos
Infecções por Vírus Epstein-Barr/enzimologia , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos B/enzimologia , Linfócitos B/virologia , Sequência de Bases , Linhagem Celular , DNA/genética , Endonucleases , Infecções por Vírus Epstein-Barr/etiologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Humanos , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-pim-1 , Ativação Transcricional , Regulação para Cima , Proteínas ViraisRESUMO
Pim-1 is an oncogenic serine/threonine kinase implicated in cytokine-induced signal transduction and in development of lymphoid malignancies. However, its precise function as well as physiological substrates have remained unknown. In this study we demonstrate that Pim-1 can physically interact with the NFATc1 transcription factor and phosphorylate it in vitro on several serine residues. In contrast to previously recognized NFATc kinases, wild-type Pim-1 enhances NFATc-dependent transactivation and IL-2 production in Jurkat T cells, while kinase-deficient Pim-1 mutants inhibit them in a dominant negative fashion. Our results reveal a novel, phosphorylation-dependent regulatory mechanism targeting NFATc1 through which Pim-1 acts as a downstream effector of Ras to facilitate IL-2-dependent proliferation and/or survival of lymphoid cells.