Phospholipid-binding Sites of Phosphatase and Tensin Homolog (PTEN): EXPLORING THE MECHANISM OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE ACTIVATION* |
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Authors: | Yang Wei Boguslaw Stec Alfred G. Redfield Eranthie Weerapana Mary F. Roberts |
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Affiliation: | From the ‡Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467 and ;the §Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454 |
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Abstract: | The lipid phosphatase activity of the tumor suppressor phosphatase and tensin homolog (PTEN) is enhanced by the presence of its biological product, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This enhancement is suggested to occur via the product binding to the N-terminal region of the protein. PTEN effects on short-chain phosphoinositide 31P linewidths and on the full field dependence of the spin-lattice relaxation rate (measured by high resolution field cycling 31P NMR using spin-labeled protein) are combined with enzyme kinetics with the same short-chain phospholipids to characterize where PI(4,5)P2 binds on the protein. The results are used to model a discrete site for a PI(4,5)P2 molecule close to, but distinct from, the active site of PTEN. This PI(4,5)P2 site uses Arg-47 and Lys-13 as phosphate ligands, explaining why PTEN R47G and K13E can no longer be activated by that phosphoinositide. Placing a PI(4,5)P2 near the substrate site allows for proper orientation of the enzyme on interfaces and should facilitate processive catalysis. |
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Keywords: | Enzyme Kinetics Nuclear Magnetic Resonance (NMR) Phosphatase and Tensin Homolog (PTEN) Phosphatidylinositol Phosphatase Phosphoinositide 31P Field Cycling Micelles Phospholipid-binding Site Spin-labeled Protein |
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