Abstract
Bcl-2 plays a central role in the regulation of apoptosis. Structural studies of Bcl-2 revealed the presence of a flexible and natively disordered loop that bridges the Bcl-2 homology motifs, BH3 and BH4. This loop is phosphorylated on multiple sites in response to a variety of external stimuli, including the microtubule-targeting drugs, paclitaxel and colchicine. Currently, the underlying molecular mechanism of Bcl-2 phosphorylation and its biological significance remain elusive. In this study, we investigated the molecular characteristics of this anti-apoptotic protein. To this end, we generated synthetic peptides derived from the Bcl-2 loop, and multiple Bcl-2 loop truncation mutants that include the phosphorylation sites. Our results demonstrate that S87 in the flexible loop of Bcl-2 is the primary phosphorylation site for JNK and ERK2, suggesting some sequence or structural specificity for the phosphorylation by these kinases. Our NMR studies and molecular dynamics simulation studies support indicate that phosphorylation of S87 induces a conformational change in the peptide. Finally, we show that the phosphorylated peptides of the Bcl-2 loop can bind Pin1, further substantiating the phosphorylation-mediated conformation change of Bcl-2.
Highlights
Apoptosis is an essential physiological process for the development and homeostasis of multi-cellular organisms [1]
The peptides were treated by jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 2 (ERK2), and the degree of phosphorylation was determined by analyzing the differences in the elution profiles between the peptides and phosphopeptides on a HPLC column
From the HPLC elution profiles, we showed that both JNK and ERK2 were able to phosphorylate the T56 peptide to a similar level (Fig. S1A & S1B)
Summary
Apoptosis is an essential physiological process for the development and homeostasis of multi-cellular organisms [1] It is a well-orchestrated and highly controlled cellular process, regulated by pro- and anti-apoptotic proteins [2]. Bcl-2 is phosphorylated at multiple sites in the flexible loop (T56, S70, T74, S87) in response to various external stimuli [7,8,9]. Kinases, such as c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 2 (ERK2), are involved in Bcl-2 phosphorylation [7,10,11,12], and the phosphorylation appears to regulate the activity of Bcl-2. Peptidylprolyl cis-trans isomerization of some proteins serves as a molecular switch and influences protein function [16]
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