Abstract
Engineered overexpression of protein kinase Cα (PKCα) is known to phosphorylate Ser165 in α-tubulin resulting in stimulated microtubule dynamics and cell motility, and activation of an epithelial-mesenchymal transition (EMT) in non-transformed human breast cells. Here it is shown that endogenous phosphorylation of native α-tubulin in two metastatic breast cell lines, MDA-MB-231-LM2-4175 and MDA-MB-468 is detected at PKC phosphorylation sites. α-Tubulin mutants that simulated phosphorylated (S165D) or non-phosphorylated (S165 N) states were stably expressed in MDA-MB-231-LM2-4175 cells. The S165D-α-tubulin mutant engendered expression of the EMT biomarker N-cadherin, whereas S165 N-α-tubulin suppressed N-cadherin and induced E-cadherin expression, revealing a 'cadherin switch'. S165 N-α-tubulin engendered more rapid passage through the cell cycle, induced shorter spindle fibers and exhibited more rapid proliferation. In nude mice injected with MDA-MB-231-LM2-4175 cells, cells expressing S165 N-α-tubulin (but not the S165D mutant) produced hyper-proliferative lung tumors with increased tumor incidence and higher Ki67 expression. These results implicate the phosphorylation state of Ser165 in α-tubulin as a PKC-regulated molecular switch that causes breast cells to exhibit either EMT characteristics or hyper-proliferation. Evaluation of genomic databases of human tumors strengthens the clinical significance of these findings.