Abstract
The protein factor Glomulin (Glmn) is a regulator of the SCF (Skp1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex. Mutations of Glmn lead to glomuvenous malformations. Glmn has been reported to be associated with FK506-binding proteins (FKBP). Here we present in vitro binding analyses of the FKBP—Glmn interaction. Interestingly, the previously described interaction of Glmn and FKBP12 was found to be comparatively weak. Instead, the closely related FKBP12.6 and FKBP51 emerged as novel binding partners. We show different binding affinities of full length and truncated FKBP51 and FKBP52 mutants. Using FKBP51 as a model system, we show that two amino acids lining the FK506-binding site are essential for binding Glmn and that the FKBP51-Glmn interaction is blocked by FKBP ligands. This data suggest FKBP inhibition as a pharmacological approach to regulate Glmn and Glmn-controlled processes.
Highlights
Glmn regulates the Skp1-Cullin-F-box complex, an E3-Ligase, which primes proteins for proteasomal degradation [1]
To quantify the Glmn-FK506-binding proteins (FKBP) interactions we developed a protein-protein interaction assay with purified proteins
We generated labelled mutants of FKBP51FK1 and FKBP12 containing a single cysteine at their respective C-terminus with Fluorescein-5-Maleimide
Summary
Glmn regulates the Skp1-Cullin-F-box complex, an E3-Ligase, which primes proteins for proteasomal degradation [1]. One of the most studied examples of this E3 ligase family is CRL1Fbw7 [2] Glmn binds to this complex by intercalating between Cul and Rbx. Glmn is thought to be a regulator of ligase function and to prevent an overshooting ubiquitinylation reaction, which is mediated by Rbx1-binding cullins. This was further supported by a crystal structure of the Glmn-Cullin complex [3]. Glomulin was initially described as FKBP Associated Protein (called FAP48 or FAP68) [5,9] In these studies, the FK506-binding proteins FKBP12 and 52 were identified as interaction partners of Glmn in a yeast-2-hybrid system [9,10].
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