The WD repeat protein FAN regulates lysosome size independent from abnormal downregulation/membrane recruitment of protein kinase C |
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Authors: | Möhlig Heike Mathieu Sabine Thon Lutz Frederiksen Marie-Catherine Ward Diane M Kaplan Jerry Schütze Stefan Kabelitz Dieter Adam Dieter |
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Affiliation: | Institut für Immunologie, Universit?tsklinikum Schleswig-Holstein Campus Kiel, Michaelisstr. 5, 24105 Kiel, Germany. |
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Abstract: | FAN (factor associated with neutral sphingomyelinase [N-SMase] activation) exhibits striking structural homologies to Lyst (lysosomal trafficking regulator), a BEACH protein whose inactivation causes formation of giant lysosomes/Chediak-Higashi syndrome. Here, we show that cells lacking FAN show a statistically significant increase in lysosome size (although less pronounced as Lyst), pointing to previously unrecognized functions of FAN in regulation of the lysosomal compartment. Since FAN regulates activation of N-SMase in complex with receptor for activated C-kinase (RACK)1, a scaffolding protein that recruits and stabilizes activated protein kinase C (PKC) isotypes at cellular membranes, and since an abnormal (calpain-mediated) downregulation/membrane recruitment of PKC has been linked to the defects observed in Lyst-deficient cells, we assessed whether PKC is also of relevance in FAN signaling. Our results demonstrate that activation of PKC is not required for regulation of N-SMase by FAN/RACK1. Conversely, activation of PKC and recruitment/stabilization by RACK1 occurs uniformly in the presence or absence of FAN (and equally, Lyst). Furthermore, regulation of lysosome size by FAN is not coupled to an abnormal downregulation/membrane recruitment of PKC by calpain. Identical results were obtained for Lyst, questioning the previously reported relevance of PKC for formation of giant lysosomes and in Chediak-Higashi syndrome. In summary, FAN mediates activation of N-SMase as well as regulation of lysosome size by signaling pathways that operate independent from activation/membrane recruitment of PKC. |
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Keywords: | amc, 7-amino-4methylcoumarin BEACH, beige and CHS E-64d, (2S, 3S)-trans-epoxyscuccinyl- smallcaps" >l-leucylamido-3-methylbutane ethyl ester EF, embryonic fibroblasts FAN, factor associated with neutral sphingomyelinase activation hTNF, human recombinant TNF Lyst, lysosomal trafficking regulator N-SMase, neutral sphingomyelinase PH, pleckstrin-homology PKC, protein kinase C PMA, phorbol 12-myristate 13-acetate RACK1, receptor for activated C-kinase 1 TNF, tumor necrosis factor TNF-R55, 55 kDa TNF receptor YAC, yeast artificial chromosome |
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