Chemical genetic dissection of aurora a kinase in breast cancer
Abstract
Aurora A kinase is a mitotic protein which is overexpressed in a variety of cancer and plays a significant role in tumorigenesis. Our goal is to identify Aurora A kinase substrates in metastatic breast cancer cell lines. These substrates will be used as cues to unravel the molecular mechanism by which Aurora A promotes breast malignancy. We employed a chemical genetic technique to identify direct substrates of Aurora A kinase in breast cancer. It involves a space-creating mutation at the kinase ATP binding pocket, which renders it sensitive to orthogonal ATP analogs. The chemical genetic technique which has been applied to over 50 different kinases, did not work for the Aurora A kinase. Using modeling, we identified a new residue, which was less than 5 angstroms away from the N-6 group of the adenine ring of ATP. This single mutation along with the mutation at the gate keeper residue made the kinase susceptible to N6(phenethyl)-ATP. Using the engineered Aurora A kinase and mass spectrometry we have identified several direct substrates in breast cancer cells. Two of the identified substrates were chosen for further validation: Pleckstrin homology-like domain family A member 1 (PHLDA1) and Lim kinase 2 (LIMK2). PHLDA1 encodes a protein of 262 amino acids and has recently been suggested to be a tumor suppressor. Loss of PHLDA1 mRNA and protein has been correlated with breast adenocarcinoma and melanoma progression in clinical samples. The exact mechanism as to how PHLDA1 mediates its role as a tumor suppressor is still not known. Our data shows Aurora A kinase directly phosphorylates and interacts with PHLDA1. Phosphorylation of PHLDA1 by Aurora A on residue S98 facilitates its degradation. On the other hand, overexpression of PHLDA1 downregulates Aurora A mediated breast cancer cell invasion, proliferation and migration to suppress it oncogenic activity. Taken together, our data indicate that deregulation of the functional balance between Aurora A and PHLDA1 could be another mechanism in Aurora A mediated tumorigenesis and metastasis. In contrast to PHLDA1, LIMK2 a serine/threonine kinase have recently been shown to have an essential role in the migration and metastatic behavior of pancreatic cancer cells. However, LIMK2 has not been analyzed in breast cancer. We report for the first time, the importance of LIMK2 in Aurora A mediated transformation and tumorigenesis in breast cancer. Aurora A positively regulates LIMK2 protein levels by phosphorylating at S283, T494 and T505. We further observed that LIMK2 also positively regulates Aurora A levels. Ablation of LIMK2 abrogate Aurora A mediated cell migration and colony formation in soft agar. Most importantly, in nude mouse xenografts, ablation of LIMK2 completely abrogates tumor formation, suggesting that LIMK2 is an important therapeutic target in breast cancer.
Degree
Ph.D.
Advisors
Shah, Purdue University.
Subject Area
Molecular biology|Biochemistry|Organic chemistry
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