Anti-Gsk3b (N-termianl) polyclonal antibody

Background: Canonical Wnt signals, transduced by stabilized beta-catenin, play similar roles across animals in maintaining stem cell pluripotency, regulating cell differentiation, and instructing normal embryonic development. Dysregulated Wnt/beta-catenin signaling causes diseases and birth defects, and a variety of regulatory processes control this pathway to ensure its proper function and integration with other signaling systems. We previously identified GTP-binding protein 2 (Gtpbp2) as a novel regulator of BMP signaling, however further exploration revealed that Gtpbp2 can also affect Wnt signaling, which is a novel finding reported here. Results: Knockdown of Gtpbp2 in Xenopus embryos causes severe axial defects and reduces expression of Spemann-Mangold organizer genes. Gtpbp2 knockdown blocks responses to ectopic Wnt8 ligand, such as organizer gene induction in ectodermal tissue explants and induction of secondary axes in whole embryos. However, organizer gene induction by ectopic Nodal2 is unaffected by Gtpbp2 knockdown. Epistasis tests, conducted by activating Wnt signal transduction at sequential points in the canonical pathway, demonstrate that Gtpbp2 is required downstream of Dishevelled and Gsk3 beta but upstream of beta-catenin, which is similar to the previously reported effects of Axin1 overexpression in Xenopus embryos. Focusing on Axin in Xenopus embryos, we find that knockdown of Gtpbp2 elevates endogenous or exogenous Axin protein levels. Furthermore, Gtpbp2 fusion proteins co-localize with Dishevelled and co-immunoprecipitate with Axin and Gsk3b. Conclusions: We conclude that Gtpbp2 is required for canonical Wnt/beta-catenin signaling in Xenopus embryos. Our data suggest a model in which Gtpbp2 suppresses the accumulation of Axin protein, a rate-limiting component of the beta-catenin destruction complex, such that Axin protein levels negatively correlate with Gtpbp2 levels. This model is supported by the similarity of our Gtpbp2-Wnt epistasis results and previously reported effects of Axin overexpression, the physical interactions of Gtpbp2 with Axin, and the correlation between elevated Axin protein levels and lost Wnt responsiveness upon Gtpbp2 knockdown. A wide variety of cancer-causing Wnt pathway mutations require low Axin levels, so development of Gtpbp2 inhibitors may provide a new therapeutic strategy to elevate Axin and suppress aberrant beta-catenin signaling in cancer and other Wnt-related diseases.

Multiple myeloma (MM) patients with the t(14; 16) translocation have a poor prognosis, and unlike other molecular subgroups, their outcome has not improved with the introduction of bortezomib (Bzb). The mechanism underlying innate resistance of MM to Bzb is unknown. In the present study, we have investigated how MAF overexpression impacts resistance to proteasomeinhibitor (PI) therapy (Bzb and carfilzomib). High levels of MAF protein were found in t(14; 16) cell lines; cell lines from the t(4; 14) subgroup had intermediate levels, whereas cell lines from the other subgroups had low levels. High expression of MAF protein in t(14; 16) was associated with significantly higher PI halfmaximum inhibitory concentration values compared with other molecular subgroups. PI exposure abrogated glycogen synthase kinase 3b(GSK3b)-mediated degradation ofMAF protein, resulting in increased MAF protein stability and PI resistance. Subsequent studies using loss-of-function and gain-of-function models showed that silencing MAF led to increased sensitivity to PIs, enhanced apoptosis, and activation of caspase-3, -7, -8, -9, poly (ADP-ribose) polymerase, and lamin A/C. In contrast, overexpression of MAF resulted in increased resistance to PIs and reduced apoptosis. These results define the role of MAF and GSK3 in the resistance of t(14; 16) MM to PIs and identifies a novel mechanism by whichMAFprotein levels are regulated by PIs, which in turn confers resistance to PIs.