Abstract 4307: Characterizing the differential phosphorylation of Bim in plasma cell disorders
Authors: Conage-Pough Jason E. , Gupta Vikas A. , Matulis Shannon M. , Boise Lawrence H. 
Published: July 2017

Abstract
The pro-apoptotic Bcl-2 family protein Bim is known to be phosphorylated in response to many stimuli. These phosphorylation events have been linked to proteasomal degradation, release from dynein light chain, and impacting the ability of Bim to initiate apoptosis. We’ve recently shown that IL-6 stimulation of myeloma cells results in phosphorylation of Bim at S69 and S77. IL-6 treatment additionally led to increased association with Mcl-1 at the expense of Bcl-2/xL. This data, coupled with our longstanding interest in factors that influence Bim binding to anti-apoptotic proteins, led us to study the baseline phosphorylation state of Bim in plasma cell malignancies.
To study the global phosphorylation of Bim, we utilized PhosTag gel electrophoresis, which allows for detection of phosphorylated forms of a protein. We’ve shown that Bim is constitutively phosphorylated in myeloma cell lines and patient samples. Importantly, these cell lines and samples display differences in their distribution of Bim binding to anti-apoptotic proteins. The pattern of Bim phosphorylation varies significantly across cell lines, with expression of anywhere from 1 to 4 phosphorylated forms. Using the Bax/Bak-deficient Waldenström Macroglobulinemia cell line RPCI-WM1, we were able to recapitulate constitutive phosphorylation in stable cell lines overexpressing Bim.
In order to identify and characterize constitutive Bim phosphorylation sites, we created phospho-mimetic (E) and unphosphorylateable (A) versions with individual and combination of mutations of seven potential sites—S59, S77, S87, S94, S104, T116 and S118. PhosTag allowed us to identify individual phosphorylation sites—by mutating individual sites to A, we could determine if we eliminated phosphorylated Bim bands. These experiments showed that S118 and S59 are constitutively phosphorylated in RPCI-WM1. Lastly, we tested whether A and E mutations were affecting the distribution of Bim among anti-apoptotic proteins and dynein light chain. We observed that mutation of the T116 residue to either A or E resulted in increased binding to Mcl-1, suggesting this amino acid is critical for binding to dynein light chain, regardless of phosphorylation status.
Our results support the presence of multiple constitutive Bim phosphorylation events, differential signaling and regulation of Bim across a spectrum of plasma cell disorders, and a potential mechanism underlying the preferential binding of Bim. Characterization of the signaling cascades regulating these events may provide novel insights into improving therapies for cancer. The efficacy of drugs such as Navitoclax and Venetoclax can be improved by coupling them with kinase inhibitors that can either mobilize Bim from dynein light chain or alter its affinity for anti-apoptotic proteins.
Citation Format: Jason E. Conage-Pough, Vikas A. Gupta, Shannon M. Matulis, Lawrence H. Boise. Characterizing the differential phosphorylation of Bim in plasma cell disorders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4307. doi:10.1158/1538-7445.AM2017-4307