Apoptosis plays a critical role in organismal development and cellular homeostasis. When a cell is subjected to stimuli such as growth factor withdrawal, UV damage, or aberrant proliferative cues, it typically undergoes intrinsic or mitochondrial-associated apoptosis, which culminates in mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, and caspase activation. The Bcl-2 family of proteins serves as a crucial regulator of this pathway, with the balance between pro- and anti-apoptotic members determining whether a cell lives or dies.
The pro-apoptotic Bcl-2 protein Bim is upregulated in response to many cellular cues, and functions as a potent activator of the multi-domain effector proteins that permeabilize the mitochondria, and trigger MOMP. Because of its apoptogenic properties, Bim is extensively regulated at the transcriptional, posttranscriptional, and posttranslational levels. Notably, Bim is frequently phosphorylated, resulting in its proteasomal degradation and other alterations to its function. This occurs frequently in the context of cancer cells, where it is often necessary to suppress Bim function to facilitate oncogenic transformation. While several studies have examined Bim phosphorylation events that arise in response to various stimuli, little is currently known about the constitutive Bim phosphorylation state.
While studying the Bim phosphorylation events triggered by IL-6 stimulation of myeloma cells, we discovered that Bim was constitutively phosphorylated within these cells. Moreover, Bim was differentially phosphorylated across several myeloma cell lines and patient samples, with cells expressing anywhere from one to three or more phosphorylated forms. While we were unable to successfully utilize phospho-proteomics to identify specific sites, our use of small molecule inhibitors allowed us to partially elucidate the signaling pathways regulating Bim.
Additionally, while characterizing Bim phosphorylation within the Waldenström's macroglobulinemia cell line RPCI-WM1, we determined that the introduction of Bim phosphorylation site mutations impacted the ability of Bim to stabilize and prime Mcl-1. Our data provide evidence for the role of phosphorylation in determining the anti-apoptotic protein that Bim is bound to within a cell.
The implications of our research for cancer treatment are significant. Our data suggest that combining drugs that mobilize Bim with compounds that affect its phosphorylation state is a promising therapeutic strategy.
Table of Contents
I. INTRODUCTION.. 1
A. Intrinsic Apoptosis and the Bcl-2 Family of Proteins. 2
B. Bim.. 6
1. Transcriptional control of Bim.. 6
2. Posttranscriptional regulation of Bim.. 8
3. Posttranslational regulation of Bim.. 9
C. Dysregulation of the Bcl-2 Family in Cancer and Mitochondrial Priming. 13
D. Bcl-2 Family Antagonist Therapies. 16
E. Statement of Problem.. 19
II. CHARACTERIZING THE DIFFERENTIAL PHOSPHORYLATION OF PLASMA CELL DYSCRASIAS. 22
Materials and Methods. 22
III. DETERMINING THE IMPACT OF BIM PHOSPHORYLATION ON BINDING TO MCL-1. 37
Materials and Methods. 53
Author Contributions. 58
IV. DISCUSSION.. 71
A. Implications from the characterization of constitutive Bim phosphorylation. 71
B. Implications from studying the impact of Bim phosphorylation on Mcl-1 stability and priming. 77
V. REFERENCES 87
About this Dissertation
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|File download under embargo until 21 May 2019||2018-04-17||File download under embargo until 21 May 2019|