RESUMO
The CATS protein was recently identified as a novel CALM interacting protein. CATS increases the nuclear and specifically the nucleolar localization of the leukemogenic CALM/AF10 fusion protein. We cloned and characterized the murine Cats gene. Detailed analysis of murine Cats expression during mouse embryogenesis showed an association with rapidly proliferating tissues. Interestingly, the Cats transcript is highly expressed in murine hematopoietic cells transformed by CALM/AF10. The CATS protein is highly expressed in leukemia, lymphoma and tumor cell lines but not in non-proliferating T-cells or human peripheral blood lymphocytes. CATS protein levels are cell cycle dependent and it is induced by mitogens, suggesting a role of CATS in the control of cell proliferation and possibly CALM/AF10-mediated leukemogenesis.
Assuntos
Proteínas de Transporte/genética , Proliferação de Células , Desenvolvimento Embrionário/genética , Proteínas Monoméricas de Montagem de Clatrina/genética , Animais , Biomarcadores Tumorais , Proteínas de Transporte/análise , Ciclo Celular , Transformação Celular Neoplásica , Genes Essenciais/genética , Células-Tronco Hematopoéticas/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia/etiologia , Camundongos , Proteínas Monoméricas de Montagem de Clatrina/análise , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas de Fusão Oncogênica , RNA Neoplásico/análiseRESUMO
Epsin and AP180 are essential components of the endocytotic machinery, which controls internalization of protein receptors and other macromolecules at the cell surface. Epsin and AP180 are recruited to the plasma membrane by their structurally and functionally related N-terminal ENTH and ANTH domains that specifically recognize PtdIns(4,5)P2. Here, we show that membrane anchoring of the ENTH and ANTH domains is regulated by the acidic environment. Lowering the pH enhances PtdIns(4,5)P2 affinity of the ENTH and ANTH domains reinforcing their association with lipid vesicles and monolayers. The pH dependency is due to the conserved histidine residues of the ENTH and ANTH domains, protonation of which is necessary for the strong PtdIns(4,5)P2 recognition, as revealed by liposome binding, surface plasmon resonance, NMR, monolayer surface tension and mutagenesis experiments. The pH sensitivity of the ENTH and ANTH domains is reminiscent to the pH dependency of the FYVE domain suggesting a common regulatory mechanism of membrane anchoring by a subset of the PI-binding domains.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Bicamadas Lipídicas/química , Proteínas Monoméricas de Montagem de Clatrina/química , Fosfatidilinositol 4,5-Difosfato/química , Proteínas Adaptadoras de Transporte Vesicular/análise , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Montagem de Clatrina/análise , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estrutura Terciária de Proteína , Ratos , Alinhamento de SequênciaRESUMO
Clathrin assembly protein, AP180, was originally identified as a brain-specific protein localized to the presynaptic junction. AP180 acts to limit vesicle size and maintain a pool of releasable synaptic vesicles during rapid recycling. In this study, we show that polarized epithelial Madin-Darby canine kidney (MDCK) cells express two AP180-related proteins: the ubiquitously expressed 62-kDa clathrin assembly lymphoid myeloid leukemia (CALM, AP180-2) protein and a novel high-molecular-weight homolog that we have named AP180-3. Sequence analysis of AP180-3 expressed in MDCK cells shows high homology to AP180 from rat brain. AP180-3 contains conserved motifs found in brain-specific AP180, including the epsin NH2-terminal homology (ENTH) domain, the binding site for the alpha-subunit of AP-2, and DLL repeats. Our studies show that AP180-3 from MDCK cells forms complexes with AP-2 and clathrin and that membrane recruitment of these complexes is modulated by phosphorylation. We demonstrate by immunohistochemistry that AP180-3 is localized to cytoplasmic vesicles in MDCK cells and is also present in tubule epithelial cells from mouse kidney. We observed by immunodetection that a high-molecular-weight AP180-related protein is expressed in numerous cells in addition to MDCK cells.
Assuntos
Endocitose/fisiologia , Proteínas Monoméricas de Montagem de Clatrina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Cães , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Monoméricas de Montagem de Clatrina/análise , Proteínas Monoméricas de Montagem de Clatrina/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Ratos , Homologia de Sequência de AminoácidosRESUMO
Clathrin adaptor protein (AP)180 is a synaptic protein that regulates the assembly of clathrin-coated vesicles. Several endocytic proteins including AP2, CALM, and epsin 1 have functions or molecular structures similar to AP180. We determined if AP180 associates with functional synapses in cultured hippocampal neurons. We also compared the expression pattern of AP180 with the other endocytic proteins. The distribution of AP180 corresponded with the synaptic vesicle-associated protein synapsin I, and with functional presynaptic terminals labeled with the styryl dye FM1-43. Synaptic AP2 colocalized with AP180, but the distribution of AP2 was not limited to synapses of neurons and it was also expressed in glia. CLAM and epsin 1 immunoreactivities were also detected in both neurons and glia. Unlike AP180, the neuronal immunoreactivity of CALM was not intense in the synaptic puncta. Epsin 1 immunoreactivity was found in both synaptic and extrasynaptic sites, and its synaptic distribution only partially overlapped with that of AP180. These results support roles for AP180 in synaptic function in neurons. The findings also provide information on the distribution of AP2, CALM, and epsin 1 in cells of the nervous system that suggest different roles for these endocytic proteins in the biology of these cells.
