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. 2021 Oct 5;81(2):243–254. doi: 10.1136/annrheumdis-2021-221010

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.

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GLK (A410T) or GLK (K650R) mutant is resistant to MKRN4-induced GLK ubiquitination. (A) MKRN4-induced GLK ubiquitination. Flag-tagged GLK proteins were immunoprecipitated from lysates of HEK293T cells cotransfected with Flag-GLK plus Myc-MKRN4 plasmids, followed by immunoblotting with anti-Lys48-linked ubiquitination or anti-FLAG antibody. Cells were treated with 25 µM MG132 for 2 hours before being harvested. (B) Reduced GLK ubiquitination by GLK (A410T) or GLK (K650R) variant. Flag-tagged GLK proteins were immunoprecipitated from lysates of HEK293T cells cotransfected with Myc-MKRN4 plus Flag-GLK WT), A410T mutant, or K650R mutant, followed by immunoblotting with anti-Lys48-linked ubiquitination or anti-FLAG antibody. Cells were treated with 25 µM MG132 for 2 hours before being harvested. (C) Mass spectrometry analysis of the GLK peptides from the MKRN4 immunocomplex. The GLK peptide sequences containing Ub-Lys650 residue of GLK proteins that were detected in the MKRN4 immunocomplex are shown. (D) The structural domains of human MAP4K3 (GLK). Asterisks indicate the locations of A410T, K650R and A579T variants on GLK. (E) Coimmunoprecipitation of Flag-tagged GLKΔN with Myc-tagged MKRN4 proteins from lysates of HEK293T cells cotransfected with Myc-MKRN4 plus either Flag-GLKΔN (deletion of amino acids 1–272) wild-type or Flag-GLKΔN (A410T) mutant plasmids. (F) Coimmunoprecipitation of Flag-tagged GLK with Myc-tagged MKRN4 proteins from lysates of HEK293T cells transfected with Myc-MKRN4 plus either Flag-GLKΔN wild-type or Flag-GLKΔN (K650R) mutant plasmids. CNH, citron-homology domain; KD, kinase domain; WT, wild-type.