Gsk3 Beta-Mediated Ser156 Phosphorylation Modulates A Bh3-Like Domain In Bcl2l12 During Tmz-Induced Apoptosis And Autophagy In Glioma Cells

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 alpha-6 and -7 helices) influenced the BH3-like domain conformation (alpha-9 helix), indicating that glycogen synthase kinase (GSK) 3 beta-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 GSK3 beta-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.