RESUMO
Caveolae are specialized domains of the plasma membrane. Formation of these invaginations is dependent on the expression of Caveolin-1 or -3 and proteins of the cavin family. In response to stress, caveolae disassemble and cavins are released from caveolae, allowing cavins to potentially interact with intracellular targets. Here, we describe the intracellular (non-plasma membrane) cavin interactome using biotin affinity proteomics and mass spectrometry. We validate 47 potential cavin-interactor proteins using a cell-free expression system and protein-protein binding assays. These data, together with pathway analyses, reveal unknown roles for cavin proteins in metabolism and stress signaling. We validated the interaction between one candidate interactor protein, protein phosphatase 1 alpha (PP1α), and Cavin-1 and -3 and show that UV treatment causes release of Cavin3 from caveolae allowing interaction with, and inhibition of, PP1α. This interaction increases H2AX phosphorylation to stimulate apoptosis, identifying a pro-apoptotic signaling pathway from surface caveolae to the nucleus.
Assuntos
Apoptose/fisiologia , Cavéolas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Apoptose/efeitos da radiação , Cavéolas/efeitos da radiação , Núcleo Celular/metabolismo , Histonas/metabolismo , Humanos , Espectrometria de Massas/métodos , Fosforilação/efeitos da radiação , Ligação Proteica/efeitos da radiação , Transporte Proteico/efeitos da radiação , Proteômica/métodos , Raios UltravioletaRESUMO
Emerging evidence suggests the existence of a new mode of epidermal growth factor receptor (EGFR) signaling in which activated EGFR undergoes nuclear translocation following treatment with ionizing radiation. The authors provide evidence that the nuclear EGFR transport is a stress-specific cellular reaction, which is linked to src-dependent EGFR internalization into caveolae. These flask-shaped pits can fuse with endoplasmic reticulum and the EGFR is sorted into a perinuclear localization. This compartment may serve as a reservoir for nuclear EGFR transport which is regulated by PKCepsilon (protein kinase Cepsilon). Nuclear EGFR is able to induce transcription of genes essential for cell proliferation and cell-cycle regulation. Moreover, nuclear EGFR has physical contact with compounds of the DNA repair machinery and is involved in removal of DNA damage. Anti-EGFR strategies target radiation-associated EGFR nuclear translocation in different manners. EGFR-inhibitory antibodies, i.e., cetuximab (Erbitux((R))), can block nuclear translocation by EGFR immobilization within the cytosol in responder cell lines, whereas tyrosine kinase inhibitors rather target nuclear kinase activity of EGFR linked with cytosolic or nuclear functions. However, both strategies can inhibit DNA repair following irradiation.
Assuntos
Núcleo Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Receptores ErbB/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Translocação Genética/efeitos da radiação , Células Tumorais Cultivadas/efeitos da radiação , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Cavéolas/efeitos da radiação , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Divisão Celular/genética , Divisão Celular/efeitos da radiação , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cetuximab , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Genes src/efeitos da radiação , Humanos , Proteína Quinase C-épsilon/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Gênica/genética , Transcrição Gênica/efeitos da radiação , Translocação Genética/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Primary brain tumors, particularly glioblastomas (GB), remain a challenge for oncology. An element of the malignant brain tumors' aggressive behavior is the fact that GB are among the most densely vascularized tumors. To determine some of the molecular regulations occuring at the brain tumor endothelium level during tumoral progression would be an asset in understanding brain tumor biology. Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling, oncogenesis, and angiogenesis. In this work we investigated regulation of caveolin-1 expression in brain endothelial cells (ECs) under angiogenic conditions. In vitro, brain EC caveolin-1 is down-regulated by angiogenic factors treament and by hypoxia. Coculture of brain ECs with tumoral cells induced a similar down-regulation. In addition, activation of the p42/44 MAP kinase is demonstrated. By using an in vivo brain tumor model, we purified ECs from gliomas as well as from normal brain to investigate possible regulation of caveolin-1 expression in tumoral brain vasculature. We show that caveolin-1 expression is strikingly down-regulated in glioma ECs, whereas an increase of phosphorylated caveolin-1 is observed. Whole-brain radiation treatment, a classical way in which GB is currently being treated, resulted in increased caveolin-1 expression in tumor isolated ECs. The level of tumor cells spreading around newly formed blood vessels was also elevated. The regulation of caveolin-1 expression in tumoral ECs may reflect the tumoral vasculature state and correlates with angiogenesis kinetics.
