Selenoprotein K regulation of palmitoylation and calpain cleavage of ASAP2 is required for efficient FcγR-mediated phagocytosis

Abstract

Effective activation of macrophages through phagocytic Fcγ receptors (FcγR) has been shown to require selenoprotein K (Selk). We set out to determine whether the FcγR-mediated uptake process itself also requires Selk and potential underlying mechanisms. Macrophages from Selk knockout (KO) mice were less efficient compared with wild-type (WT) controls in engulfing IgG-coated fluorescent beads. Using LC-MS/MS to screen for Selk-binding partners involved in FcγR-mediated phagocytosis, we identified Arf-GAP with SH3 domain, ANK repeat, and PH domain-containing protein 2 (ASAP2). Coimmunoprecipitation assays confirmed interactions between Selk and ASAP2. Selk was required for ASAP2 to be cleaved by calpain-2 within the Bin/Amphiphysin/Rvs (BAR) domain of ASAP2. BAR domains promote membrane association, which was consistent with our data showing that Selk deficiency led to retention of ASAP2 within the phagocytic cup. Because Selk was recently identified as a cofactor for the palmitoylation of certain proteins, we investigated whether ASAP2 was palmitoylated and whether this was related to its cleavage by calpain-2. Acyl/biotin exchange assays and MALDI-TOF analysis showed that cysteine-86 in ASAP2 was palmitoylated in WT, but to a much lesser extent in KO, mouse macrophages. Inhibitors of either palmitoylation or calpain-2 cleavage and rescue experiments with different versions of Selk demonstrated that Selk-dependent palmitoylation of ASAP2 leads to cleavage by calpain-2 within the BAR domain, which releases this protein from the maturing phagocytic cup. Overall, these findings identify ASAP2 as a new target of Selk-dependent palmitoylation and reveal a new mechanism regulating the efficiency of FcγR-mediated phagocytosis.

Selenium, Selenoproteins, and Immunity

“…SELENOK itself does not function as an enzyme but instead binds to DHHC6 to stabilize the acylated intermediate of this enzyme so that it is not hydrolyzed by water, which does not hydrolyze the thioester bond between the acyl group and the cysteine residue in DHHC6 before it can transfer the acyl group to target proteins like IP3R [ 177 ]. Other proteins involved in immune functions also depend on SELENOK/DHHC6 for palmitoylation to carry out their activities including CD36 and Arf-GAP with SH3 domain, ANK repeat and PH domain-containing protein 2 (ASAP2) [ 178 , 179 ]. Overall, SELENOK plays an important, non-enzymatic role in regulating immunity by functioning as a cofactor for an enzyme involved in critical post-translational modifications of proteins.…”

Complement Receptor-Mediated Phagocytosis Induces Proinflammatory Cytokine Production in Murine Macrophages

“…Importantly, IκBα is another substrate of calpain (42), implicating a potential involvement in phagocytosis-induced cytokine production. Calpain has been shown to indirectly play a role in FcγR-mediated phagocytosis as the cleavage of ASAP2 by calpain reduced particle uptake during FcγRmediated phagocytosis (43). We employed the calpain inhibitor, the PD150606 peptide, which inhibits the protease core domains of both calpain-1 and calpain-2 (44) and examined gene expression and cytokine and chemokine protein expression during phagocytosis in BMDMs.…”

The molecular era of protein S-acylation: spotlight on structure, mechanisms, and dynamics

“…Human zDHHC6 was reported to interact with selenoprotein K (also known as SelK or SelenoK) via its SH3_2 domain and this interaction to be required for activity (Fredericks et al 2014). SelK was first found as necessary for the palmitoylation of CD36 in macrophages (Meiler et al 2013) and later on proposed to be a zDHHC6 cofactor for the S-acylation of the inositol 1,4,5-triphosphate (IP 3 ) receptor type 1 (IP3R1) and of ASAP2 (Fredericks et al 2014;Fredericks and Hoffmann 2015;Norton et al 2017).…”