PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding : LIVER BIOLOGY/PATHOBIOLOGY

Abstract

<p class="first" id="P1">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. The high throughput screening showed that PKC412, a multi-kinase 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 non-muscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. NMHC-IIA association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent and occurs with K8, the obligate K18 partner, and is enhanced by PKC412 in cells and mouse liver and blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments. </p><div class="section"> <a class="named-anchor" id="S1"> <!-- named anchor --> </a> <h5 class="section-title" id="d14443165e219">Conclusion</h5> <p id="P2">PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NMHC-IIA in a myosin dephosphorylation regulated manner. Targeting of intermediate filament disorganization by compounds that alters keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other IF-associated diseases. </p> </div>