Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Boggon, Titus

Abstract

The most common forms of cerebrovascular malformations are brain arteriovenous malformations (AVM), often caused by genetic mutations in the EphB4-p120RasGAP signaling pathway, and cerebral cavernous malformations (CCM), often caused by genetic mutations in the CCM signaling pathway. These pathways are well studied, but there are clear deficiencies in the molecular level understanding of how signals are transduced. Relevant to AVM, the GTPase activating protein (GAP) for Ras, p120RasGAP, is necessary for downregulating Ras signaling during vascular development, and loss of its activity is associated with impaired cerebrovascular development. In my thesis, I investigate how p120RasGAP uses its Src-homology 2 (SH2) domains to accommodate a wide variety of phosphotyrosine-containing binding partners and investigate the consequences of this on p120RasGAP signaling. I find that the dual SH2 domains function as a selectivity filter that may allow upstream signaling to differentially impact signaling downstream of p120RasGAP, a mechanism that may be used by other dual SH2 domain proteins to tune signaling. Relevant to CCM, the hub of proteins associated with CCM diseases regulates kinase activity of a MAP kinase kinase kinase, MEKK3, and in CCM disorder MEKK3 activity is significantly increased. MEKK3 and its paralog MEKK2 are the only MAP kinase kinase kinases (MAP3K) known to activate the ERK5 pathway, which is essential for vascular development. Nonetheless, despite their importance, the regulation and activation mechanisms of MEKK2 and MEKK3 remain unknown. I determined the crystal structure of the catalytic domain of MEKK2 bound to a clinically relevant inhibitor ponatinib. This is the first crystal structure of either MEKK2 or MEKK3. The structure reveals a dimerization surface that mediates MEKK2 and MEKK3 autophosphorylation. Furthermore, I demonstrate this surface is also necessary for MEKK2 to phosphorylate its downstream MAP kinase kinase (MAP2K) substrates MEK5 and MKK6. Importantly, I show this surface is important for recruitment of MKK6 but not MEK5, illustrating a difference in signaling decisions downstream of MEKK2 activation, thus providing potentially more generalizable insights into MAP kinase signaling pathways. My thesis therefore provides molecular level insights into signaling pathways relevant to cerebrovascular disorders, and are generalizable to wider signaling pathways.

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