GSK3β interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3β and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3β interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3β/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3β binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3β in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3β binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3β compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3β activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3β interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3β away from the β-catenin destruction complex and as a competitor to compete for GSK3β binding, resulting in accumulation of S675 phosphorylated β-catenin.

Commitment of differentiating embryonic stem cells (ESCs) toward the various lineages is influenced by many factors, including androgens. However, the mechanisms underlying proteotoxic stress conferred by androgen receptor (AR) actions on embryonic cell fate remains unclear. Here we show that mouse ESCs display stress-related cellular phenotypes in response to androgens during early phase of differentiation. Androgen induced a significant increase in the percentage of ESCs and embryoid bodies with the intranuclear and juxtanuclear AR inclusions, which were colocalized with the E3 ubiquitin ligase, C terminus of Hsc70-interacting protein. Caspase-3 activity corresponded with AR expression, was enhanced in cells engaged more differentiation phenotypes. Androgen-mediated accumulation of AR aggregates exacerbated endoplasmic reticulum (ER) stress and rendered ESCs susceptible to apoptosis. Increasing expression levels of the ER chaperones, GRP78/BiP and GRP94, as well as ER stress markers, such as ATF6, phosphorylated PERK, GADD153/CHOP and spliced XBP-1 mRNA, were dramatically elevated in ESCs overexpressing AR. We found that androgen induced GRP78/BiP to dissociate from ATF6, and act as an AR-interacting protein, which was recruited into AR inclusions in ESCs. GRP78/BiP was also colocalized with AR inclusions in the cells of spinal bulbar muscular atrophy transgenic mouse model. Overexpression of GRP78/BiP suppressed ubiquitination of AR aggregates and ameliorated the misfolded AR-mediated cytopathology in ESCs, whereas knockdown of GRP78/BiP increased the accumulation of AR aggregates and significantly higher levels of caspase-3 activity and cell apoptosis. These results generate novel insight into how ESCs respond to stress induced by misfolded AR proteins and identify GRP78/BiP as a novel regulator of the AR protein quality control.

BH3 domains, classified initially as BCL2 homology domains, participate in both apoptosis and autophagy. Beclin-1 contains a BH3 domain, which is required for binding to antiapoptotic BCL2 homologs and BCL2-mediated inhibition of autophagy. BCL2-like 12 (BCL2L12) also harbors a BH3-like domain, which is 12 residues long and contains a LXXXAE/D motif. In a yeast two-hybrid system performed in the present study, BCL2L12 shared similar binding partnerships to antiapoptotic BCL2 homologs, such as Beclin-1. In addition, this BH3-like domain was involved in antiapoptosis and drug-induced autophagy in glioma cell lines. Mutations in S156 and hydrophobic L213 to alanine counteracted the antiapoptotic properties of BCL2L12 and downregulated the activation of microtubule associated protein 1 light chain 3B (LC3B), autophagy-related (ATG)12-ATG5 conjugates and Beclin-1, compared with a BCL2L12 wild-type group. Molecular dynamics simulations revealed that phosphorylation at Ser156 of BCL2L12 (within α-6 and α-7 helices) influenced the BH3-like domain conformation (α-9 helix), indicating that glycogen synthase kinase (GSK) 3β-mediated Ser156 phosphorylation modulated a BH3-like domain in BCL2L12. Altogether, the present findings indicated that BCL2L12 may participate in anti-apoptosis and autophagy via a BH3-like domain and GSK3β-mediated phosphorylation at Ser156. Furthermore, blockade of temozolomide (TMZ)-induced autophagy by 3-methyladenine (3-MA) resulted in enhanced activation of apoptotic markers, as well as tumor suppresor protein p53 (p53) expression in U87MG cells. The present results suggested that p53 and O6-methylguanine DNA methyltransferase activation, and BCL2, BCL-extra large, Beclin-1 and BCL2L12 expression may be used as a detection panel to determine which patients can benefit from TMZ and ABT-737 combination treatment.

Introduction The human JC polyomavirus (JCPyV) has been detected in colorectal cancer (CRC) tissues and is suggested to contribute to CRC tumorigenesis. The rearrangement of the JCPyV regulatory region is supposedly associated with CRC development. The progression of CRC involves the stepwise accumulation of mutations. The large tumor antigen (LT) of JCPyV can trigger uncontrolled cell cycle progression by targeting oncogenes, and tumor suppressor genes, and causing chromosome instability. Few studies have focused on the presence of JCPyV DNA in the higher grade of CRC tissues. Methods We collected 95 tissue blocks from samples of stages I, II, III, and IV CRC. Nested PCR targeting the regulatory region of the viral genome was performed to determine the presence of JCPyV DNA in the various stages of colorectal cancer tissues. Results The nested PCR results showed that the positive rate of JCPyV DNA increased with the progression of CRC stages. The archetypal-like, non-rearrangement genotype of JCPyV with subtle mutations was the major genotype found in CRC samples. Conclusions This finding in our study suggests that there may be an association between JCPyV and CRC progression.