GSK3β?mediated Ser156 phosphorylation modulates a BH3?like domain in BCL2L12 during TMZ?induced apoptosis and autophagy in glioma cells

摘要

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 anti?apoptosis 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.