title: Biochemical characterization of the phosphatase domain of the tumor suppressor PH domain leucine-rich repeat protein phosphatase.
abstract: PH domain leucine-rich repeat protein phosphatase (PHLPP) directly dephosphorylates and inactivates Akt and protein kinase C and is therefore a prime target for pharmacological intervention of two key signaling pathways, the phosphatidylinositol 3-kinase and diacylglycerol signaling pathways. Here we report on the first biochemical characterization of the phosphatase domain of a PHLPP family member. The human PHLPP1 and PHLPP2 phosphatase domains were expressed and purified from bacteria or insect cells and their activities compared to that of full-length proteins immunoprecipitated from mammalian cells. Biochemical analyses reveal that the PHLPP phosphatase domain effectively dephosphorylates synthetic and peptidic substrates, that its activity is modulated by metals and lipophilic compounds, and that it has relatively high thermal stability. Mutational analysis of PHLPP2 reveals an unusual active site architecture compared to the canonical architecture of PP2C phosphatases and identifies key acidic residues (Asp 806, Glu 989, and Asp 1024) and bulky aromatic residues (Phe 783 and Phe 808) whose mutation impairs activity. Consistent with a unique active site architecture, we identify inhibitors that discriminate between PHLPP2 and PP2Cα. These data establish PHLPP as a member of the PP2C family of phosphatases with a unique active site architecture.
authors.affiliation: Department of Pharmacology, University of California San Diego , La Jolla, California 92093, United States.
authors.forename: Emma
authors.initials: E
authors.lastname: Sierecki
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D006801:Humans; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D000071636:Protein Phosphatase 2C; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; C400601:PHLPP1 protein, human; C541572:PHLPP2 protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C