2. Academic Validation
  3. PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding

PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding

  • Hepatology. 2015 Dec;62(6):1858-69. doi: 10.1002/hep.27965.
Raymond Kwan 1 2,  Lu Chen 1 3,  Koksun Looi 1,  Guo-Zhong Tao 4,  Sujith V Weerasinghe 1,  Natasha T Snider 1,  Mary Anne Conti 5,  Robert S Adelstein 5,  Qing Xie 3,  M Bishr Omary 1 2
Affiliations

Affiliations

  • 1 Departments of Molecular & Integrative Physiology and of Medicine, University of Michigan, Ann Arbor, MI.
  • 2 VA Ann Arbor Healthcare System, Ann Arbor, MI.
  • 3 Infectious Diseases Department, Ruijin Hospital, Shanghai Jiao Tong University Medical School, Shanghai, People's Republic of China.
  • 4 Department of Surgery, Stanford University, Palo Alto, CA.
  • 5 The Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
PMID: 26126491 DOI: 10.1002/hep.27965
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.

Conclusion: 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.

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