title: The phosphoinositide-dependent protein kinase 1 inhibitor, UCN-01, induces fragmentation: possible role of metalloproteinases.
abstract: Phosphoinositide-dependent protein kinase 1 (PDK1) is a key enzyme, master regulator of cellular proliferation and metabolism; it is considered a key target for pharmacological intervention. Using membranes obtained from DDT1 MF-2 cells, phospho-PDK1 was identified by Western blotting, as two major protein bands of Mr 58-68 kDa. Cell incubation with the PDK1 inhibitor, UCN-01, induced a time- and concentration-dependent decrease in the amount of phospho-PDK1 with a concomitant appearance of a ≈42 kDa phosphorylated fragment. Knocking down PDK1 diminished the amount of phospho-PDK1 detected in membranes, accompanied by similarly decreased fragment generation. UCN-01-induced fragment generation was also observed in membranes from cells stably expressing a myc-tagged PDK1 construct. Other PDK1 inhibitors were also tested: OSU-03012 induced a clear decrease in phospho-PDK1 and increased the presence of the phosphorylated fragment in membrane preparations; in contrast, GSK2334470 and staurosporine induced only marginal increases in the amount of PDK1 fragment. Galardin and batimastat, two metalloproteinase inhibitors, markedly attenuated inhibitor-induced PDK1 fragment generation. Metalloproteinases 2, 3, and 9 co-immunoprecipitated with myc-PDK1 under baseline conditions and this interaction was stimulated by UCN-01; batimastat also markedly diminished this effect of the PDK1 inhibitor. Our results indicate that a series of protein kinase inhibitors, namely UCN-01 and OSU-03012 and to a lesser extent GSK2334470 and staurosporine induce PDK1 fragmentation and suggest that metalloproteinases could participate in this effect.
authors.affiliation: Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México DF 04510, México.
authors.forename: Rocío
authors.initials: R
authors.lastname: Alcántara-Hernández
authors.affiliation: Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México DF 04510, México.
authors.forename: Aurelio
authors.initials: A
authors.lastname: Hernández-Méndez
authors.affiliation: Departamento de Biología Celular y Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México DF 04510, México. Electronic address: agarcia@ifc.unam.mx.
authors.forename: J Adolfo
authors.initials: JA
authors.lastname: García-Sáinz
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D006224:Cricetinae; D007191:Indazoles / Q000494:pharmacology; D045726:Metalloproteases / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; C555257:GSK 2334470; D007191:Indazoles; C500894:OSU 03012; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D013449:Sulfonamides; C054852:7-hydroxystaurosporine; D017346:Protein Serine-Threonine Kinases; D045726:Metalloproteases; D019311:Staurosporine
keywords: Batimastat (BB-94) (CID 5362422). Galardin (GM 6001) (PubChem CID 132519); GSK2334470, (3S,6R)-1-[6-(3-amino-1H-indazol-6-yl)-2-(methylamino)-4-pyrimidinyl]-N-cyclohexyl-6-methyl-3-piperidinecarboxamide. ) (PubChem CID 46215815); OSU-03012, (2-amino-N-[4-[5-(2-phenanthrenyl)-3-trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide) (PubChem CID 10027278); PDK1; Protein fragmentation; Protein kinase; Protein kinase inhibitor; Staurosporine (PubChem CID 44259); UCN-01; UCN-01, (7-hydroxystaurosporine (3R*,8S*, 9R*, 10R*,12R*)-2,3,9,10,11,12-hexahydro-3-hydroxy-9-methoxy-8-methyl-10-(methylamino)-8,12-epoxy-1H, 8H-2,7b,12a-triazadibenzo[a,g]-cyclonona[cde]triden-1-one) (PubChem CID 3078519)