Death receptor ligation triggers membrane scrambling between Golgi and mitochondria.

Subcellular organelles such as mitochondria, endoplasmic reticulum (ER) and the Golgi complex are involved in the progression of the cell death programme. We report here that soon after ligation of Fas (CD95/Apo1) in type II cells, elements of the Golgi complex intermix with mitochondria. This mixing follows centrifugal dispersal of secretory membranes and reflects a global alteration of membrane traffic. Activation of apical caspases is instrumental for promoting the dispersal of secretory organelles, since caspase inhibition blocks the outward movement of Golgi-related endomembranes and reduces their mixing with mitochondria. Caspase inhibition also blocks the FasL-induced secretion of intracellular proteases from lysosomal compartments, outlining a novel aspect of death receptor signalling via apical caspases. Thus, our work unveils that Fas ligand-mediated apoptosis induces scrambling of mitochondrial and secretory organelles via a global alteration of membrane traffic that is modulated by apical caspases.

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels.

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.