RESUMO
Recent studies identified cyclase-associated proteins (CAPs) as important regulators of actin dynamics that control assembly and disassembly of actin filaments (F-actin). While these studies significantly advanced our knowledge of their molecular functions, the physiological relevance of CAPs largely remained elusive. Gene targeting in mice implicated CAP2 in heart physiology and skeletal muscle development. Heart defects in CAP2 mutant mice were associated with altered activity of serum response factor (SRF), a transcription factor involved in multiple biological processes including heart function, but also skeletal muscle development. By exploiting mouse embryonic fibroblasts (MEFs) from CAP2 mutant mice, we aimed at deciphering the CAP2-dependent mechanism relevant for SRF activity. Reporter assays and mRNA quantification by qPCR revealed reduced SRF-dependent gene expression in mutant MEFs. Reduced SRF activity in CAP2 mutant MEFs was associated with altered actin turnover, a shift in the actin equilibrium towards monomeric actin (G-actin) as well as and reduced nuclear levels of myocardin-related transcription factor A (MRTF-A), a transcriptional SRF coactivator that is shuttled out of the nucleus and, hence, inhibited upon G-actin binding. Moreover, pharmacological actin manipulation with jasplakinolide restored MRTF-A distribution in mutant MEFs. Our data are in line with a model in which CAP2 controls the MRTF-SRF pathway in an actin-dependent manner. While MRTF-A localization and SRF activity was impaired under basal conditions, serum stimulation induced nuclear MRTF-A translocation and SRF activity in mutant MEFs similar to controls. In summary, our data revealed that in MEFs CAP2 controls basal MRTF-A localization and SRF activity, while it was dispensable for serum-induced nuclear MRTF-A translocation and SRF stimulation.
Assuntos
Proteínas de Transporte/metabolismo , Fibroblastos/citologia , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Transporte/análise , Células Cultivadas , Fibroblastos/metabolismo , Camundongos , Fator de Resposta Sérica/análise , Transativadores/análiseRESUMO
Microtubule-associated serine/threonine-protein kinase-like (MASTL) is a mitosis-accelerating kinase with emerging roles in cancer progression. However, possible cell cycle-independent mechanisms behind its oncogenicity remain ambiguous. Here, we identify MASTL as an activator of cell contractility and MRTF-A/SRF (myocardin-related transcription factor A/serum response factor) signaling. Depletion of MASTL increased cell spreading while reducing contractile actin stress fibers in normal and breast cancer cells and strongly impairing breast cancer cell motility and invasion. Transcriptome and proteome profiling revealed MASTL-regulated genes implicated in cell movement and actomyosin contraction, including Rho guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and MRTF-A target genes tropomyosin 4.2 (TPM4), vinculin (VCL), and nonmuscle myosin IIB (NM-2B, MYH10). Mechanistically, MASTL associated with MRTF-A and increased its nuclear retention and transcriptional activity. Importantly, MASTL kinase activity was not required for regulation of cell spreading or MRTF-A/SRF transcriptional activity. Taken together, we present a previously unknown kinase-independent role for MASTL as a regulator of cell adhesion, contractility, and MRTF-A/SRF activity.
Assuntos
Citoesqueleto de Actina/enzimologia , Adesão Celular/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Humanos , Integrinas/genética , Integrinas/metabolismo , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteoma/metabolismo , RNA Interferente Pequeno , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fibras de Estresse/genética , Fibras de Estresse/metabolismo , Transativadores/genética , Transcriptoma/genética , Tropomiosina/genética , Tropomiosina/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMO
Entosis is a nonapoptotic form of cell death initiated by actomyosin-dependent homotypic cell-in-cell invasion that can be observed in malignant exudates during tumor progression. We previously demonstrated formin-mediated actin dynamics at the rear of the invading cell as well as nonapoptotic plasma membrane (PM) blebbing in this cellular motile process. Although the contractile actin cortex involved in bleb-driven motility is well characterized, a role for transcriptional regulation in this process has not been studied. Here, we explore the impact of the actin-controlled MRTF-SRF (myocardin-related transcription factor-serum response factor) pathway for sustained PM blebbing and entotic invasion. We find that cortical blebbing is tightly coupled to MRTF nuclear shuttling to promote the SRF transcriptional activity required for entosis. Furthermore, PM blebbing triggered SRF-mediated up-regulation of the metastasis-associated ERM protein Ezrin. Notably, Ezrin is sufficient and important to sustain bleb dynamics for cell-in-cell invasion when SRF is suppressed. Our results highlight the critical role of the actin-regulated MRTF transcriptional pathway for bleb-associated invasive motility, such as during entosis.