FBW2 Targets GCMa to the Ubiquitin-Proteasome Degradation System

Abstract

The GCM proteins GCMa/1 and GCMb/2 are novel zinccontaining transcription factors critical for glial cell differentiation in fly and for placental as well as parathyroid gland development in mouse. Previous pulsechase experiments have demonstrated differential protein stabilities of GCM proteins with half-lives from ϳ30 min to 2 h (Tuerk, E. E., Schreiber, J., and Wegner, M. (2000) J. Biol. Chem. 275, 4774 -4782). However, little is known about the machinery that controls GCM protein degradation. Here, we report the identification of an SCF complex as the GCM ubiquitin-protein isopeptide ligase (E3) that regulates human GCMa (hGCMa) degradation. We found that SKP1 and CUL1, two key components of the SCF complex, associate with hGCMa in vivo. We further identify the human F-box protein FBW2 (hFBW2) as the substrate recognition subunit in the SCF E3 complex for hGCMa. We show that hFBW2 interacts with hGCMa in a phosphorylation-dependent manner and promotes hGCMa ubiquitination. Supporting a critical role for hFBW2 in hGCMa degradation, knockdown of hFBW2 expression by RNA interference leads to a reduction in hGCMa ubiquitination and a concomitant increase in hGCMa protein stability. Our study identifies the SCF hFBW2 E3 complex as the key machinery that targets hGCMa to the ubiquitin-proteasome degradation system.

Small Ubiquitin-like Modifier Modification Regulates the DNA Binding Activity of Glial Cell Missing Drosophila Homolog a

“…As shown in Fig. 4A, the luciferase activity directed by p(pGBS) 4 E1bLUC was activated by GCMa. Moreover, the observed GCMa-mediated transcriptional activation was repressed when pEGFP-SUMO1 was cotransfected.…”

“…HeLa cells were transfected with different combinations of p(pGBS) 4 E1bLUC, pHAGCMa, pEGFP-SUMO1, pSENP1-FLAG, and pUbc9-FLAGdn. As shown in Fig.…”

“…The proximal GCMa-binding site (pGBS, 28026 -28033) in p(27950/28314)LUC was deleted to generate p(27950/28314)LUC-⌬GBS. The reporter construct, p(pGBS) 4 E1bLUC, has been described previously (2). All constructs were verified by DNA sequencing using the dideoxy chain termination method.…”

“…On the other hand, in vitro labeling experiments have also revealed that GCMa is a phosphoprotein. Being phosphorylated, GCMa can be targeted by the F-box protein, FBW2, and subjected to protein degradation by the ubiquitin-proteasome degradation system (4). Therefore, regulation of GCMa activity is achieved at multiple levels by phosphorylation, acetylation, deacetylation, and ubiquitination.…”

Gcm protein degradation suppresses proliferation of glial progenitors

“…The ubiquitination and degradation of GCMa is mediated by the SCF FBW2 E3 ubiquitin ligase complex in which the F-box protein FBW2 is the substrate adaptor (23). Sequence comparison indicates that FBW2 shares limited identity (Ϸ18%) to all 3 Drosophila FBWs (16).…”