Phosphorylation of α/β Tubulin is a 'Toggle Switch' in Human Breast Cancer

“Phosphorylation of α/β-Tubulin is a ‘Toggle Switch’ in Human Breast Cancer The α/β-tubulin heterodimer is the building block of microtubules (MTs) which comprise an essential component of the cell cytoskeleton. MTs undergo growth/shrinkage episodes as dictated by whether GTP is bound to β-tubulin (β:GTP) (assembly) or is hydrolyzed to GDP (disassembly). Protein kinase C (PKC) is a key signaling enzyme that controls these MT events in human breast cells. In this regard, PKC phosphorylates α-tubulin at Ser165 (α:Ser165) resulting in persistent MT elongation and breast cell motility while proliferation is suppressed. Molecular dynamics simulations and validating experiments showed that phosphorylation of α:Ser165 misaligns the catalytic residue Glu254 in α-tubulin (α:Glu254) with its substrate β:GTP, leading to diminished hydrolysis of β:GTP, thereby increasing GTP caps and causing persistent MT elongation. β-tubulin undergoes phosphorylation at Ser172 by cyclin-dependent kinase-1 (cdk-1) which promotes proliferation, a process that requires α/β-tubulin to form spindle fibers during cell division. Thus, PKC and cdk1 produce opposing effects on microtubule structure, GTP binding/hydrolysis, and cell proliferation. By alternating the phosphorylation states of α− and β- tubulin in microtubules using phospho-mimetic mutants, a “toggle switch” model will be explored as a novel decision point that governs cancer-related phenotypes. This model will be rigorously tested by co-expression of site-directed mutants of α-tubulin (S165D or control S165N) and β-tubulin (S172D or control S172N) in human breast cells. By use of these constructs, proliferation and motility as well as EMT markers (E- vs. N-cadherin) will be evaluated. Molecular dynamics simulations will be used to screen the alignment of α:Glu254 with β:GTP both in the α/β-tubulin mutant constructs and in those mutants to be identified in tumor genomic databases; their significance will be verified experimentally by the presence of GTP caps and cell phenotypes (proliferation, motility). In vivo support for the α/β-tubulin toggle switch will be sought in an orthotopic animal model with highly aggressive breast cells to evaluate: 1) the impact of phospho-mimetic mutant constructs of α/β-tubulin, and 2) the efficacy of small molecule inhibitors of PKC and cdk1. The purpose of these studies is to establish a novel framework for assessing metastatic potential and to explore improved strategies for breast cancer chemotherapy.