title: PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding.
abstract: Keratins, among other cytoskeletal intermediate filament proteins, are mutated at a highly conserved arginine with consequent severe disease phenotypes due to disruption of keratin filament organization. We screened a kinase inhibitor library, using A549 cells that are transduced with a lentivirus keratin 18 (K18) construct, to identify compounds that normalize filament disruption due to K18 Arg90Cys mutation at the conserved arginine. High-throughput screening showed that PKC412, a multikinase inhibitor, ameliorated K18 Arg90Cys-mediated keratin filament disruption in cells and in the livers of previously described transgenic mice that overexpress K18 Arg90Cys. Furthermore, PKC412 protected cultured A549 cells that express mutant or wild-type K18 and mouse livers of the K18 Arg90Cys-overexpressing transgenic mice from Fas-induced apoptosis. Proteomic analysis of proteins that associated with keratins after exposure of K18-expressing A549 cells to PKC412 showed that nonmuscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. The nonmuscle myosin-IIA (NM-IIA) association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent; occurs with K8, the obligate K18 partner; is enhanced by PKC412 in cells and mouse liver; and is blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments.
  
  
  CONCLUSIONS
  The inhibitor PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NM-IIA in a myosin dephosphorylation-regulated manner. Targeting of intermediate filament disorganization by compounds that alter keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other intermediate filament protein-associated diseases.
authors.affiliation: Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.|VA Ann Arbor Healthcare System, Ann Arbor, MI.
authors.forename: Raymond
authors.initials: R
authors.lastname: Kwan
authors.affiliation: Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.|Infectious Diseases Department, Ruijin Hospital, Shanghai Jiao Tong University Medical School, Shanghai, People's Republic of China.
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.affiliation: Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.
authors.forename: Koksun
authors.initials: K
authors.lastname: Looi
authors.affiliation: Department of Surgery, Stanford University, Palo Alto, CA.
authors.forename: Guo-Zhong
authors.initials: GZ
authors.lastname: Tao
authors.affiliation: Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.
authors.forename: Sujith V
authors.initials: SV
authors.lastname: Weerasinghe
authors.affiliation: Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.
authors.forename: Natasha T
authors.initials: NT
authors.lastname: Snider
authors.affiliation: The Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
authors.forename: Mary Anne
authors.initials: MA
authors.lastname: Conti
authors.affiliation: The Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Adelstein
authors.affiliation: Infectious Diseases Department, Ruijin Hospital, Shanghai Jiao Tong University Medical School, Shanghai, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.|VA Ann Arbor Healthcare System, Ann Arbor, MI.
authors.forename: M Bishr
authors.initials: MB
authors.lastname: Omary
mesh_terms: D000818:Animals; D007382:Intermediate Filaments / Q000235:genetics*; D007633:Keratins / Q000378:metabolism*; D008107:Liver Diseases / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000502:physiology
chemical_list: D007633:Keratins; D009218:Myosins; D019311:Staurosporine; C059539:midostaurin