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

> **NIH NIH R16** · QUEENS COLLEGE · 2024 · $170,349

## Abstract

“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.

## Key facts

- **NIH application ID:** 10850006
- **Project number:** 1R16GM153696-01
- **Recipient organization:** QUEENS COLLEGE
- **Principal Investigator:** Susan A. Rotenberg
- **Activity code:** R16 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $170,349
- **Award type:** 1
- **Project period:** 2024-08-05 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10850006

## Citation

> US National Institutes of Health, RePORTER application 10850006, Phosphorylation of α/β Tubulin is a 'Toggle Switch' in Human Breast Cancer (1R16GM153696-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10850006. Licensed CC0.

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