Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1

Abstract

The Down syndrome-associated DYRK1A kinase has been reported as a stimulator of the developmentally important Hedgehog (Hh) pathway, but cells from Down syndrome patients paradoxically display reduced Hh signaling activity. Here, we find that DYRK1A stimulates GLI transcription factor activity through phosphorylation of general nuclear localization clusters. In contrast, in vivo and in vitro experiments reveal that DYRK1A kinase can also function as an inhibitor of endogenous Hh signaling by negatively regulating ABLIM proteins, the actin cytoskeleton and the transcriptional co-activator MKL1 (MAL). As a final effector of the DYRK1A–ABLIM-Actin-MKL1 sequence, we identify the MKL1-interactor Jumonji-domain demethylase 1A (JMJD1A) as a novel Hh pathway component stabilizing the GLI1 protein in a demethylase-independent manner. Furthermore, a Jumonji-specific small molecule antagonist represents a novel and powerful inhibitor of Hh signal transduction by inducing GLI1 protein degradation in vitro and in vivo.

Identification of a DYRK1A-mediated phosphorylation site within the nuclear localization sequence of the hedgehog transcription factor GLI1

“…Three sites for AMPK phosphorylation of GLI1 were identified in the medulloblastoma study; Ser 102, Ser 408 and Thr 1074. Interestingly, two of these sites were those identified by us herein (Ser408) or by others (Ser102; [40]) as DYRK1A-mediated sites on GLI1. In an independent study, phosphorylation of GLI1 by AMPK at Ser408 was shown to be inhibitory leading to GLI1 ubiquitination and proteasomal degradation [45].…”

“…DYRK1A has also been shown to phosphorylate GLI1 in the N-terminal domain at two SPS clusters (S102/104 and S130/32) and that this phosphorylation promotes nuclear transport of GLI1 [40]. Further, when both SPS clusters were mutated, GLI transcriptional activity was not enhanced by DYRK1A [40]. Direct phosphorylation of Ser102 was shown by mass spectrometry [40].…”

“…Further, when both SPS clusters were mutated, GLI transcriptional activity was not enhanced by DYRK1A [40]. Direct phosphorylation of Ser102 was shown by mass spectrometry [40]. We also identified a tryptic phospho-peptide spanning this SPS (TsPSSLVAFINSR; see Table 2 in [18]).…”

“…SUFU binds GLI directly within residues 116-125 of GLI1 [44]. The SUFU binding site falls between the two SPS clusters identified by Schnieder et al [40] but interestingly, although DYRK1A was shown to promote GLI1/SUFU dissociation, it was independent of the SPS clusters [40]. Direct phosphorylation of GLI1 by the AMP-activated protein kinase (AMPK) in medulloblastoma [45] has been shown to lead to GLI1 destabilization [46].…”

The Nuclear Translocation of Mitogen-Activated Protein Kinases: Molecular Mechanisms and Use as Novel Therapeutic Target

“…Only when calcium concentrations decrease, ERK1/2 translocate to the nucleus to regulate transcription [63]. Finally, recent studies indicate that the selectivity of the ERK1/2-NTS mechanism of stimulated nuclear import is quite specific and is used only by a few other proteins, such as MEK1/2 [32], SMAD3 [32], EGR-1 [65], GLI1 [66], Notch1 [67], and probably also Pyk2 [68] and Drosha [69]. …”