The WD repeat protein FAN regulates lysosome size independent from abnormal downregulation/membrane recruitment of protein kinase C.
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.
Möhlig
Heike
H
Institut für Immunologie, Universitätsklinikum Schleswig-Holstein Campus Kiel, Michaelisstr. 5, 24105 Kiel, Germany.
Mathieu
Sabine
S
Thon
Lutz
L
Frederiksen
Marie-Catherine
MC
Ward
Diane M
DM
Kaplan
Jerry
J
Schütze
Stefan
S
Kabelitz
Dieter
D
Adam
Dieter
D
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D009479:Neuropeptides / Q000172:deficiency / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D020449:Repetitive Sequences, Amino Acid; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C491133:Lyst protein, mouse; D009479:Neuropeptides; C491112:Nsmaf protein, mouse; D011506:Proteins; C090294:RACK1 protein, mouse; D000075370:Receptors for Activated C Kinase; D014409:Tumor Necrosis Factor-alpha; D033921:Vesicular Transport Proteins; D011493:Protein Kinase C