Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

First Advisor

Calderwood, David

Abstract

Cerebral cavernous malformations (CCMs) are neurovascular abnormalities characterized by thin, leaky blood vessels resulting in lesions that predispose to hemorrhages, stroke, epilepsy, and focal neurological deficits. Familial CCMs arise due to loss-of-function mutations in genes encoding one of three CCM complex proteins, KRIT1, CCM2, or CCM3. These widely-expressed, multi-functional adaptor proteins can assemble into a CCM protein complex and either alone, or in complex, modulate signaling pathways that influence cell adhesion, cell contractility, cytoskeletal reorganization and gene expression. While recent advances, including analysis of the structures and interactions of CCM proteins, have allowed substantial progress towards understanding the molecular bases for CCM protein function and how their disruption leads to disease, key unanswered questions remain, including: 1) what biochemical signals regulate the localization of the CCM proteins and their subsequent function?, and 2) how do the various molecular pathways interplay and result in CCM pathogenesis when perturbed? My dissertation studies aim to tackle these questions. In this body of work, I first review CCM protein signaling with a focus on three pathways which have generated the most interest – the RhoA-ROCK, the MEKK3-MEK5-ERK5-KLF2/4, and cell junctional signaling pathways – but also consider ICAP1-β1 integrin and Cdc42 signaling. I discuss emerging links between these pathways and the processes that drive disease pathology and highlight open areas – among them, the role of subcellular localization in the control of CCM protein activity. In Chapter 2, I identify serine phosphorylation as a key factor in preventing nuclear accumulation of ICAP1 and the ICAP1/KRIT1 complex. Very few biochemical triggers that govern localization of proteins involved in CCM signaling have been identified, and much remains unknown including how these triggers occur mechanistically. My studies add to this field of knowledge by identifying a mechanism that regulates localization of ICAP1 and the ICAP1:KRIT1 complex, presumably impacting their as yet unidentified nuclear functions. I use quantitative microscopy to demonstrate that phosphorylation-mimicking mutations at Ser10, or to a lesser extent at Ser25, within ICAP1’s N-terminal region inhibits ICAP1 nuclear accumulation. I further demonstrate that p21-activated kinase 4 (PAK4) can phosphorylate ICAP1 at Ser10 in vitro and in cultured cells, and that active PAK4 inhibits ICAP1 nuclear accumulation in a Ser10-dependent manner. Finally, I show that ICAP1 phosphorylation controls nuclear localization of the ICAP1/KRIT1 complex. This work is the first to identify a biochemical mechanism to regulate localization of ICAP1 and the ICAP1:KRIT1 complex, potentially influencing their functions in CCM signaling and vascular development. In the appendix, I investigate the KRIT1:CCM2 binding stoichiometry and interaction, and how these impact relevant phenotypes including: MEKK3-MEK5-ERK5-KLF2/4 signaling, cell growth, endothelial network formation, and KRIT1:CCM2 protein stability. My preliminary studies indicate that KRIT1 or CCM2 knockdown in EA.hy926 human endothelial-like cells upregulates KFL2 and KLF4 mRNA levels, impairs cell growth, and perturbs network formation. Further work is needed to test whether wildtype or binding defective KRIT1 or CCM2 rescue these phenotypes. Ultimately, this work will reveal the signaling interplay between key CCM proteins and partners and may reveal how abnormal signaling results in CCM pathogenesis.

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