Date of Graduation


Document Type

Dissertation (PhD)

Program Affiliation

Genetics and Epigenetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Xuetong Shen, PhD

Committee Member

Mark T. Bedford, PhD

Committee Member

Xiaodong Cheng, PhD

Committee Member

David G. Johnson, PhD

Committee Member

Dianna M. Milewicz, MD, PhD

Committee Member

Prabodh Kapoor, PhD


Actin is an abundant and evolutionarily conserved protein and a key component of the cytoskeleton. Post-translational modifications of actin are emerging as an important mechanism for regulating actin functions, and may form an ‘Actin Code’. In this work, I investigate the role of actin phosphorylation at tyrosine 53 (pY53), one of the most frequently detected actin PTMs, through identifying interaction partners, or ‘readers’, for this modification. Using an SH2 (Src Homology 2) protein domain array, we identify N-terminal SH2 domains of p85, regulatory subunits of Phosphatidylinositol 3-kinase (PI3K), and VAV2, a Rho GTPase guanine nucleotide exchange factor, as phosphorylation-dependent binding partners of an actin pY53 peptide. Through biochemical and structural biology approaches, I define the interaction mechanism of the actin pY53 peptide with p85α, p85β and VAV2. My work provides evidence for an interaction mechanism of the actin pY53 peptide to the p85 N-terminal SH2 domains that is partially distinct from the canonical mechanism as it lacks the common binding motif for this domain. Moreover, I present the first high-resolution crystal structure of the p85β N-terminal SH2 domain bound to a peptide ligand. I also performed functional analysis of the possible roles of the actin pY53 modification. To do so, I generated a human cell line with β-actin Y53F mutation to abolish phosphorylation of this residue, using CRISPR/Cas9-based gene editing. I find that this cell line exhibits slightly higher levels of AKT phosphorylation, as well as an altered gene and protein expression profile that includes components of the PI3K pathway. Taken together, my findings suggest that actin tyrosine 53 phosphorylation may play a role in cell signaling, possibly through the phosphorylation-dependent interactions with SH2 domains of p85 and/or VAV2 proteins, and highlight a largely unexplored, and potentially highly important area of actin biology through its post-translational modifications and their ‘reader’ proteins.


actin, PTM, tyrosine phosphorylation