In vitro reconstitution of Sgk3 activation by phosphatidylinositol 3-phosphate

Abstract

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Molecular basis for the recruitment of the Rab effector protein WDR44 by the GTPase Rab11

“…Sgk3 used to phosphorylate WDR44 was purified as previously described ( 40 ). The human Sgk3 gene was codon optimized for expression in insect cells (Genscript) and fused to the C terminus of His10/StrepII-tagged enhanced GFP (A206 K monomeric variant) to aid expression of soluble Sgk3.…”

Discovery of small molecule ligands for the von Hippel-Lindau (VHL) E3 ligase and their use as inhibitors and PROTAC degraders

“…166,167 Though overexpression of SGK3 has been linked to several solid cancers, the precise mechanism of SGK3 regulation has yet to be elucidated. 168 Starting from a pan-selective SGK inhibitor linked via an optimised ether-containing alkyl linker to VH032's N-terminus, the Ciulli and Alessi laboratories developed the SGK3 selective degrader SGK3-PROTAC1 (72, Fig. 28), inducing efficient SGK3 degradation with DC 50 o 100 nM and D max = 80%.…”

A critical evaluation of protein kinase regulation by activation loop autophosphorylation

“…Site-directed mutations and covalent modifications, including common post-translational modifications such as phosphorylation, can be deduced from the mass shifts of the unmodified protein, as well as the stoichiometry of modification. In a more general sense, intact mass spectrometry is an essential quality control step that provides confidence in the interpretation of biochemical results ( Elsner et al, 2019 ; Levina et al, 2022 ; Pokorny et al, 2021 ; Reinhardt et al, 2023 ; Truebestein et al, 2023 ; Truebestein et al, 2021 ; Truebestein et al, 2015 ; von Raußendorf et al, 2017 ). Intact mass spectrometry does not, however, provide site-specific information and is limited by the quality and complexity of the sample.…”