Structural basis of βTrCP1-associated GLI3 processing

Abstract

Controlled ubiquitin-mediated protein degradation is essential for various cellular processes. GLI family regulates the transcriptional events of the sonic hedgehog pathway genes that are implicated in almost one fourth of human tumors. GLI3 phosphorylation by Ser/Thr kinases is a primary factor for their transcriptional activity that incurs the formation of both GLI3 repressor and activator forms. GLI3 processing is triggered in an ubiquitin-dependent manner via SCF βTrCP1 complex; however, structural characterization, mode of action based on sequence of phosphorylation signatures and induced conformational readjustments remain elusive. Here, through structural analysis and molecular dynamics simulation assays, we explored comparative binding pattern of GLI3 phosphopeptides against βTrCP1. A comprehensive and thorough analysis demarcated GLI3 presence in the binding cleft shared by inter-bladed binding grooves of β-propeller. Our results revealed the involvement of all seven WD40 repeats of βTrCP1 in GLI3 interaction. Conversely, GLI3 phosphorylation pattern at primary protein kinase A (PKA) sites and secondary casein kinase 1 (CK1) or glycogen synthase kinase 3 (GSK3) sites was carefully evaluated. Our results indicated that GLI3 processing depends on the 19 phosphorylation sites (849, 852, 855, 856, 860, 861, 864, 865, 868, 872, 873, 876, 877, 880, 899, 903, 906, 907 and 910 positions) by a cascade of PKA, GSK3β and CSKI kinases. The presence of a sequential phosphorylation in the binding induction of GLI3 and βTrCP1 may be a hallmark to authenticate GLI3 processing. We speculate that mechanistic information of the individual residual contributions through structure-guided approaches may be pivotal for the rational design of specific and more potent inhibitors against activated GLI3 with a special emphasis on the anticancer activity.