Selenoprotein K and Promotion of FC-Gamma Receptor Mediated Phagocytosis

Norton, Robert
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[Honolulu] : [University of Hawaii at Manoa], [May 2016]
Fc gamma receptor (Fcү-R) mediated phagocytosis is a nutrient-dependent innate process whereby monocytes and macrophages ingest or phagocytose immunoglobulin G (IgG)- opsonized pathogens or antigens. Selenoprotein K (Selk) is a small mass (~12kDa) protein that is activated during immune cell stimulation that has been found to regulate several immune cell functions that rely on protein palmitoylation such as calcium flux. ASAP2 is a multi-domain protein with ARF-GAP activity that acts on ARF6 during FcүR mediated phagocytosis. Our previous work showed that Selk deficient macrophages stimulated through Fcү-R secreted lower levels of inflammatory mediators, but the role of Selk during the process of phagocytosis remains unclear. When we measured the phagocytic index of Selk KO macrophages for their uptake of IgG-opsonized microspheres through Fcү-R, it was found that their phagocytic capacity was significantly reduced compared to WT. Hence, to explore this novel role of Selk, we performed a screen for Selk binding partners and identified interactions between Selk and ASAP2. We proceeded to elucidate the functional significance of this interaction. We report that Selk is required for the palmitoylation of ASAP2 on cysteine-86 and this leads to subsequent cleavage of ASAP2 by calpain-2. Interestingly, in lysates from Selk KO BMDMs, ASAP2 is consistently detected as a high molecular weight form of 112kDa. Whereas, in WT BMDMs ASAP2 is detected as both 112kDa as well as a low MW form of 97kDa. The low MW form of ASAP2 can be generated in vitro by the addition of recombinant calpain-2. Moreover, this cleavage can be prevented in macrophages by treatment with 2-bromopalmitate, which is a chemical inhibitor of palmitoylation. Furthermore this cleavage leads to FAK2 phosphorylation of ASAP2, and our data suggests that the calin-2 cleavage occurs within the BAR domain that allows release of ASAP2 from the phagocytic cup. In support of this notion, ASAP2 was retained in the phagocytic cup in Selk KO macrophages compared to WT during Fc mediated phagocytosis. Finally, the retention of ASAP2 was reversed in Selk KO by rescuing with a plasmid encoding Selk but not with a control plasmid or a plasmid encoding a palmitoylation defective version (SH3-BD) of Selk. These data suggest that Selk affects FcγR mediated phagocytosis through a novel mechanism involving the regulation of ASAP2.
Ph.D. University of Hawaii at Manoa 2016.
Includes bibliographical references.
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