ABSTRACT
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.
Subject(s)
Apoptosis/physiology , Caveolae/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Phosphatase 1/metabolism , RNA-Binding Proteins/metabolism , Apoptosis/radiation effects , Caveolae/radiation effects , Cell Nucleus/metabolism , Histones/metabolism , Humans , Mass Spectrometry/methods , Phosphorylation/radiation effects , Protein Binding/radiation effects , Protein Transport/radiation effects , Proteomics/methods , Ultraviolet RaysABSTRACT
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.
Subject(s)
Cell Nucleus/radiation effects , Cell Survival/radiation effects , ErbB Receptors/radiation effects , Signal Transduction/radiation effects , Translocation, Genetic/radiation effects , Tumor Cells, Cultured/radiation effects , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/pharmacology , Caveolae/radiation effects , Cell Cycle/genetics , Cell Cycle/radiation effects , Cell Division/genetics , Cell Division/radiation effects , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cetuximab , DNA Damage/genetics , DNA Damage/radiation effects , DNA Repair/drug effects , DNA Repair/genetics , DNA Repair/radiation effects , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic/radiation effects , Genes, src/radiation effects , Humans , Protein Kinase C-epsilon/physiology , Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription, Genetic/genetics , Transcription, Genetic/radiation effects , Translocation, Genetic/drug effects , Tumor Cells, Cultured/drug effectsABSTRACT
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.
