RESUMO
Clathrin assembly lymphoid myeloid leukemia protein (CALM) is a clathrin assembly protein with a domain structure similar to the neuron-specific assembly protein AP180. We have previously found that CALM is expressed in neurons and present in synapses. We now report that CALM has a neuron-related function: it facilitates the endocytosis of the synaptic vesicle protein VAMP2 from the plasma membrane. Overexpression of CALM leads to the reduction of cell surface VAMP2, whereas knockdown of CALM by RNA interference results in the accumulation of surface VAMP2. The AP180 N-terminal homology (ANTH) domain of CALM is required for its effect on VAMP2 trafficking, and the ANTH domain itself acts as a dominant-negative mutant. Thus, our results reveal a role for CALM in directing VAMP2 trafficking during endocytosis.
Assuntos
Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Endocitose , Humanos , Proteínas Monoméricas de Montagem de Clatrina/antagonistas & inibidores , Proteínas Monoméricas de Montagem de Clatrina/química , Proteínas Monoméricas de Montagem de Clatrina/genética , Mutagênese Sítio-Dirigida , Células PC12 , Estrutura Terciária de Proteína , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Transferrina/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
Phosphoinositide 3-kinase (PI3K) is important in TCR signaling. PI3K generates phosphatidylinositol 3, 4, 5-trisphosphate (PI-3,4,5-P3), which regulates membrane localization and/or activity of multiple signaling proteins. PTEN (phosphatase and tensin homologue deleted on chromosome 10) opposes PI3K, reversing this reaction. Maintaining the balance between these two enzymes is important for normal T cell function. Here we use the PTEN-null Jurkat T cell line to address the role of PTEN in modulating proximal and distal TCR-signaling events. PTEN expression at levels that restored low basal Akt phosphorylation (an indicator of PI-3,4,5-P3 levels), but which were not themselves cytotoxic, had minimal effect on TCR-stimulated activation of phospholipase Cgamma1 and Ca2+ flux, but reduced the duration of extracellular signal-regulated kinase (Erk) activation. Distal signaling events, including nuclear factor of activated T cells (NFAT) activation, CD69 expression and IL-2 production, were all inhibited by PTEN expression. Notably, PTEN did not block TCR-stimulated PI-3,4,5-P3 accumulation. The effect of PTEN on distal TCR signaling events was strongly correlated with the loss of the constitutive Akt activation and glycogen synthase kinase-3 (GSK3) inhibition that is typical of Jurkat cells, and could be reversed by expression of activated Akt or pharmacologic inhibition of GSK3. These results suggest that PTEN acts in T cells primarily to control basal PI-3,4,5-P3 levels, rather than opposing PI3K acutely during TCR stimulation.