Contribution of Bcl-2 Phosphorylation to Bak Binding and Drug Resistance

• Published in: Cancer research (Chicago, Ill.) Vol. 73; no. 23; pp. 6998 - 7008
• Main Authors: HAIMING DAI, HUSHENG DING, WEI MENG, X, LEE, Sun-Hee, SCHNEIDER, Paula A, KAUFMANN, Scott H
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 01-12-2013
• Subjects: Antineoplastic agents; Antineoplastic Agents - pharmacology; Apoptosis Regulatory Proteins - metabolism; bcl-2 Homologous Antagonist-Killer Protein - metabolism; Bcl-2-Like Protein 11; Biological and medical sciences; CDC2 Protein Kinase - metabolism; Cells, Cultured; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm - drug effects; Drug Resistance, Neoplasm - genetics; Humans; Jurkat Cells; K562 Cells; Medical sciences; Membrane Proteins - metabolism; Pharmacology. Drug treatments; Phosphorylation - drug effects; Phosphorylation - physiology; Protein Binding - drug effects; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-bcl-2 - antagonists & inhibitors; Proto-Oncogene Proteins c-bcl-2 - genetics; Proto-Oncogene Proteins c-bcl-2 - metabolism; RNA, Small Interfering - pharmacology; Tumors; Phosphorylation; Treatment resistance; C-Onc gene; Protooncogene; Protein Bak
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.CAN-13-0940

Bcl-2 is phosphorylated on Ser(70) after treatment of cells with spindle poisons. On the basis of effects observed in cells overexpressing Bcl-2 S70E or S70A mutants, various studies have concluded that Ser(70) phosphorylation either enhances or diminishes Bcl-2 function. In the present study, the ability of phosphorylated Bcl-2, as well as the S70E and S70A mutants, to bind and neutralize proapoptotic Bcl-2 family members under cell-free conditions and in intact cells was examined in an attempt to resolve this controversy. Surface plasmon resonance indicated that phosphorylated Bcl-2, Bcl-2 S70E, and Bcl-2 S70A exhibit enhanced binding to Bim and Bak compared with unmodified Bcl-2. This enhanced binding reflected a readily detectable conformation change in the loop domain of Bcl-2. Furthermore, Bcl-2 S70E and S70A bound more Bak and Bim than wild-type Bcl-2 in pull-downs and afforded greater protection against several chemotherapeutic agents. Importantly, binding of endogenous Bcl-2 to Bim also increased during mitosis, when Bcl-2 is endogenously phosphorylated, and disruption of this mitotic Bcl-2/Bim binding with navitoclax or ABT-199, like Bcl-2 downregulation, enhanced the cytotoxicity of paclitaxel. Collectively, these results provide not only a mechanistic basis for the enhanced antiapoptotic activity of phosphorylated Bcl-2, but also an explanation for the ability of BH3 mimetics to enhance taxane sensitivity.