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Susan A. Rotenberg

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Protein kinase C (PKC), an enzyme important in signaling pathways that give rise to various cell phenotypes, has been closely associated with metastatic phenotypes of breast cancer. Tissues from patients of varying degrees of tumorgenicity have shown an elevated expression of the PKCα isoform as well as other diacylglycerol (DAG)-sensitive isoforms. Moreover, independent studies with highly invasive breast cell lines, such as MDA-MB-231 cells, have also shown an elevated level of PKCα. Therefore, PKCα has long been a therapeutic target for breast cancer. Among its known substrates (recently identified by our laboratory), α6-tubulin will, upon phosphorylation by PKC, enhance migration in breast epithelial cells. The mechanistic pathway as to how a phosphorylated state of α-tubulin can confer motility in breast epithelial cells was addressed in the present work. Different approaches were undertaken to dissect the role(s) played by phospho-α6-tubulin in enhancing cell migration: i) extensive live cell imaging to quantitate microtubule dynamics, ii) immunofluorescence of microtubules in fixed cells, iii) partitioning of phospho-α6-tubulin between particulate and soluble phases, and iv) pull-down assays to determine effects on the small GTPases. For these studies, two mutants of α6-tubulin namely, S165D (pseudo-phosphorylated) and S165N (phosphorylation-resistant), were used extensively. In addition, cells expressing wildtype (WT)-α6-tubulin were treated with either the PKC-activating DAG-lactone or the pan-PKC inhibitor bis-indoleylmaleimide (BIM). Experiments were conducted in non-motile MCF-10A cells that have a low expression level of PKC and therefore offer an ideal platform for studying PKC-mediated changes. Some experiments were also conducted in MDA-MB-231 metastatic breast epithelial cells to re-affirm the role of phospho-α-tubulin in motility and related phenotypes. Functionality of phospho-α-tubulin was also explored by examining its interaction with the microtubule cargo protein MARCKS (a PKC-substrate), and membrane proteins like IQGAP1 and Rac1 having known involvement in cell migration. Each α6-tubulin mutant was studied for its effects on the distinct morphological glandular structures that it produced in a 3D growth environment, in order to simulate an in vivo condition. This study is the first of its kind to establish a detailed mechanism of cell movement driven by the PKC substrate α-tubulin in human breast cells. The novel role of phospho-α-tubulin in promoting motility will establish it as an important predictive marker for metastatic breast cancer.

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