title: Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis.
abstract: Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) plays an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs). Here we characterize the mechanism by which PILSAP regulates the vascular endothelial growth factor (VEGF)-stimulated proliferation of ECs. The specific elimination of PILSAP expression or its enzymatic activity inhibited VEGF-stimulated G1/S transition in ECs. This G1 arrest correlated with reduced cyclin dependent kinase 4/6 (CDK4/6) activity and retinoblastoma (Rb) protein phosphorylation. Analyses of signaling molecules upstream of CDK4/6 revealed that S6 kinase (S6K) activation was affected by PILSAP, whereas that of phosphatidylinositol-3 kinase (PI3K), Akt, and extracellular signal-related kinase 1/2 (ERK1/2) was not. We further demonstrated that PILSAP bound phosphatidylinositol-dependent kinase 1 (PDK1) and removed 9 amino acids from its N-terminus, which allowed S6K to associate with PDK1 and PILSAP upon VEGF stimulation. We constructed mutant PILSAP, which lacked the aminopeptidase activity but bound PDK1. Mutant PILSAP abrogated S6K activation upon VEGF stimulation in a dominant-negative manner. An N-terminal truncated form of PDK1 abolished the dominant-negative effect of mutant PILSAP. Finally, the introduction of a mutated PILSAP gene in ECs inhibited angiogenesis and retarded tumor growth in vivo. These results indicate that PILSAP plays a crucial role in the cell cycle progression of ECs and angiogenesis via the binding and modification of PDK1.
authors.affiliation: Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aobaku, Sendai 980-8575, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yamazaki
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Akada
authors.forename: Osamu
authors.initials: O
authors.lastname: Niizeki
authors.forename: Takahiro
authors.initials: T
authors.lastname: Suzuki
authors.forename: Hiroki
authors.initials: H
authors.lastname: Miyashita
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Sato
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002453:Cell Cycle; D002455:Cell Division; D002460:Cell Line; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000098:blood supply / Q000235:genetics / Q000473:pathology; D009389:Neovascularization, Pathologic; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D016160:Retinoblastoma Protein; D042461:Vascular Endothelial Growth Factor A; D004247:DNA; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; C578564:Pdpk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D018844:Cyclin-Dependent Kinases; D007931:Leucyl Aminopeptidase; C456799:puromycin-insensitive leucyl-specific aminopeptidase