Evidence for the direct involvement of βTrCP in Gli3 protein processing

Abstract

Hedgehog-regulated processing of the transcription factor cubitus interruptus (Ci) in Drosophila depends on phosphorylation of the C-terminal region of Ci by cAMP-dependent protein kinase and subsequently by casein kinase 1 and glycogen synthase kinase 3. Ci processing also requires Slimb, an F-box protein of SCF (Skp1͞ Cullin͞F-box proteins) complex, and the proteasome, but the interplay between phosphorylation and the activity of Slimb and the proteasome remains unclear. Here we show that processing of the Gli3 protein, a homolog of Ci, also depends on phosphorylation of a set of four cAMP-dependent protein kinase sites that primes subsequent phosphorylation of adjacent casein kinase 1 and glycogen synthase kinase 3. Our gain-and loss-of-function analyses in cultured cells further reveal that ␤TrCP, the vertebrate homolog of Slimb, is required for Gli3 processing, and we demonstrate that ␤TrCP can bind phosphorylated Gli3 both in vitro and in vivo. We also find that the Gli3 protein is polyubiquitinated in the cell and that its processing depends on proteasome activity. Our findings provide evidence for a direct link between phosphorylation of Gli3͞Ci proteins and ␤TrCP͞Slimb action, thus supporting the hypothesis that the processing of Gli3͞Ci is affected by the proteasome.casein kinase 1 ͉ Gli3 ͉ hedgehog ͉ cAMP-dependent protein kinase

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

“…A similar but opposite mechanism of regulation is operated downstream of Sufu at the levels of Gli2 and Gli3 by the SCF βTrcp ubiquitin ligase. In this case, the concerted action of PKA and GSK3β generates a phosphorylation‐dependent signal for ubiquitylation by βTrcp (Bhatia et al , 2006; Wang & Li, 2006). The ubiquitylation of Gli2 and Gli3 generates transcriptional repressors to avoid pathway activation.…”

Holoprosencephaly and preaxial polydactyly associated with a 1.24 Mb duplication encompassing FBXW11 at 5q35.1

“…Interestingly, lossof-function mutations in human GLI2 are associated with pituitary anomalies and HPE-like features (Roessler et al 2003), whereas mutations in GLI3 can cause three distinct congenital syndromes: Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, and postaxial polydactyly type A1 (Ming et al 1998). Recently, it has been demonstrated that bTRCP is directly involved in proteolytic processing of GLI3 in vertebrates (Wang and Li 2006). Thus, duplication of the FBXW11 gene is likely to result in increased bTRCP activity and consequently enhanced processing of GLI3 (Wang and Li 2006).…”

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…For example, protein kinase A is shown to retain Gli1 proteins in the cytoplasm (through a PKA site in the nuclear localization signal peptide) whereas active Ras signaling promotes Gli nuclear localization (Stecca et al, 2007). Second, ubiquitination and protein degradation of Gli molecules is also regulated by several distinct mechanisms, including b-TRCP-, cul3/BTBand numb/Itch-mediated Gli ubiquitination (Di Marcotullio et al, 2006;Huntzicker et al, 2006;Jiang, 2006;Pan et al, 2006;Wang and Li, 2006). In addition to protein degradation, Gli3 and (Gli2 to a less extent) can be processed into transcriptional repressors, which may be mediated by the b-TRCP E3 ligase (Wang and Li, 2006).…”

“…Second, ubiquitination and protein degradation of Gli molecules is also regulated by several distinct mechanisms, including b-TRCP-, cul3/BTBand numb/Itch-mediated Gli ubiquitination (Di Marcotullio et al, 2006;Huntzicker et al, 2006;Jiang, 2006;Pan et al, 2006;Wang and Li, 2006). In addition to protein degradation, Gli3 and (Gli2 to a less extent) can be processed into transcriptional repressors, which may be mediated by the b-TRCP E3 ligase (Wang and Li, 2006). Defects in the retrograde motor for IFT are also shown to affect Gli3 processing (Huangfu and Anderson, 2006).…”