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 SCFβTrCP1 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.
Highlights
Protein modification by ubiquitination plays a central role in multiple cellular processes including signal transduction, cell cycle progression, and metabolic pathways[1]
GL3 processing entails its extensive phosphorylation by three Ser/Thr kinases i.e. protein kinase A (PKA), glycogen synthase kinase 3 (GSK3), and casein kinase I (CKI) and component of SCF (SKP1, Cullin, F-box containing complex) E3 ligase i.e. βTrCP1, CUL1 (Cullin1) and RBX1 (RING-box protein 1)[7,8,18]
In the absence of Hh signaling, GLI3 processing is provoked by PKA-dependent phosphorylation, which is prerequisite for the later phosphorylation events by GSK3 and casein kinase 1 (CK1) leading to direct binding and ubiquitination by SCFβTrCP1 16,17
Summary
Protein modification by ubiquitination plays a central role in multiple cellular processes including signal transduction, cell cycle progression, and metabolic pathways[1]. In the absence of Hh signaling, GLI3 processing is provoked by PKA-dependent phosphorylation, which is prerequisite for the later phosphorylation events by GSK3 and CK1 leading to direct binding and ubiquitination by SCFβTrCP1 16,17. It has been reported that GLI3 processing is mediated by sequential phosphorylation of βTrCP1-binding sites through PKA, GSK3β and CK1 enzymes[16,17]. Unlike other substrates of E3 ligases, GLI3 requires PKA, GSK3 and CKI accessory proteins for phosphorylation and ubiquitination by βTrCP1. By exploring the association of βTrCP1 and GLI3, our study may provide invaluable insight in understanding the Hh relationship with cancer pathogenesis
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