The Wnt/β-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated β-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, β-catenin phosphorylation by GSK3 is inhibited and β-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of β-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent β-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit β-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of β-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of b-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/β-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of β-catenin. This model provides a possible mechanism to account, in part, for inhibition of β-catenin phosphorylation by Wnt-activated LRP6.

To investigate the flavin utilization by dibenzothiophene monooxygenase (DszC), DszC of a desulfurizing bacterium Mycobacterium goodii X7B was purified from the recombinant Escherichia coli. It was shown to be able to utilize either FMNH2 or FADH2 when coupled with a flavin reductase that reduces either FMN or FAD. Sequence analysis indicated that DszC was similar to the C2 component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, which can use both FADH2 and FMNH2 as substrates. Both flavins at high concentrations could inhibit the activity of DszC due to autocatalytic oxidation of reduced flavins. The results suggest that DszC should be reclassified as an FMNH2 and FADH2 both-utilizing monooxygenase component and the flavins should be controlled at properly reduced levels to obtain optimal biodesulfurization results.

β-Catenin phosphorylation at serine 45 (Ser45), threonine 41 (Thr41), Ser37, and Ser33 is critical for β-catenin degradation, and regulation of β-catenin phosphorylation is a central part of the canonical Wnt signaling pathway. β-Catenin mutations at Ser45, Thr41, Ser37, and Ser33 perturb β-catenin degradation and are frequently found in cancers. It is established that Ser45 phosphorylation by casein kinase I (CKI) initiates phosphorylation at Thr41, Ser37, and Ser33 by glycogen synthase kinase 3 (GSK3) and that phosphorylated Ser37 and Ser33 are recognized by the F-box protein β-TrCP, a component of a ubiquitin ligase complex that mediates β-catenin degradation. While the roles of Ser45, Ser37, and Ser33 are well documented, the function of Thr41 remains less defined. Here we show that Thr41 strictly acts as a phosphorylation relay residue and that the Ser-X-X-X-Ser (X is any amino acid) motif is obligatory for β-catenin phosphorylation by GSK3. β-Catenin phosphorylation/degradation and its regulation by Wnt can occur normally in the absence of Thr41 as long as the Ser-X-X-X-Ser motif/spacing is preserved. These results suggest that Thr41 functions to bridge sequential phosphorylation from Ser45 to Ser37 and provide further insights into the discrete steps and logic in β-catenin phosphorylation-degradation.

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27Kip1 plays a central role in cell cycle progression, and enhanced degradation of p27Kip1 is associated with many common cancers. Proteolysis of p27Kip1 is triggered by Thr187 phosphorylation, which leads to the binding of the SCFSkp2 (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27Kip1 ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27Kip1 phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27Kip1 binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27Kip1 is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27Kip1 to the SCFSkp2-Cks1 complex.

In the February issue of Developmental Cell, Nastasi et al. describe Ozz, a muscle-specific ubiquitin ligase adaptor that regulates myofibril organization. Ozz appears to function in ubiquitination and degradation of membrane-bound, but not eytosolie, β-eatenin,whose turnover may be required for alignment and growth of the sareomere, the basic contractile unit of myofibers.