Role of A-Kinase Anchoring Protein Phosphorylation in Alcohol-Induced Liver Injury and Hepatic Stellate Cell Activation

Abstract

Alcoholic liver injury is associated with hepatic stellate cell (HSC) activation. A-kinase anchoring protein 12 (AKAP12) scaffolds protein kinase C and cyclin-D1, which is regulated by its phosphorylation, and spatiotemporally controls cell proliferation, invasiveness, and chemotaxis. HSC activation induces AKAP12 expression, but the role of AKAP12's scaffolding activity in liver function is unknown. Because AKAP12 phosphorylation is enhanced in ethanol-treated HSCs, we examined AKAP12's scaffolding functions in alcohol-mediated HSC activation and liver injury. AKAP12 expression, interaction, and phosphorylation were assayed in in vitro and in vivo ethanol models and human subjects by real-time PCR, coimmunoprecipitation, immunoblotting, and phosphorylated proteomics/Phos-tag. Ethanol induced AKAP12 phosphorylation in the liver and in primary HSCs, but not in hepatocytes. AKAP12's scaffolding activity for protein kinase C/cyclin-D1 decreased in ethanol-treated HSCs but not hepatocytes. AKAP12 negatively regulated HSC activation, which was reversed by ethanol-mediated AKAP12 phosphorylation. AKAP12 interacted with heat shock protein 47 (HSP47), which chaperones collagen and induces its secretion. Ethanol inhibited AKAP12-HSP47 and induced HSP47-collagen interaction. Ethanol-induced phosphorylated AKAP12 was unable to bind to HSP47 compared with its unphosphorylated counterpart, thereby proving that ethanol-mediated phosphorylation of AKAP12 inhibited the HSP47-AKAP12 scaffold. Silencing AKAP12 facilitated the chaperoning of collagen by HSP47. Hence, AKAP12 scaffolds HSP47 and regulates collagen-HSP47 interaction. Ethanol quenches AKAP12's scaffolding activity through phosphorylation and facilitates HSC activation.

S‐adenosylmethionine inhibits la ribonucleoprotein domain family member 1 in murine liver and human liver cancer cells

“…Coimmunoprecipitation of phospho‐LARP1 from Huh7 cells with anti‐phospho‐serine (PSer) antibodies was performed according to established protocols. [ 22 ]…”

“…Total RNA was subjected to real-time RT-PCR using a published protocol. [22] See the Supporting Information Supplemental Methods for details.…”

SUMOylation and phosphorylation cross-talk in hepatocellular carcinoma

“…However, a specific phosphorylation event at serine 505/serine 517 caused by PKC inhibits the scaffolding activity of AKAP12 for CCND1, leading to nuclear translocation of CCND1 and promotion of G1 to S transition (65). We have recently described that phosphorylation of AKAP12 in hepatic stellate cells alters its ability to scaffold key fibrogenic proteins, heat shock protein 47 (HSP47) and this may be associated with increased collagen expression during hepatic stellate cell activation (66).…”

Targeting A-Kinase Anchoring Protein 12 Phosphorylation in Hepatic Stellate Cells Regulates Liver Injury and Fibrosis in Mouse Models

“…Total RNA from cells or tissues was reverse transcribed to cDNA using M-MLV reverse transcriptase (Nxgen). CDNA was subjected to quantitative RT-PCR using TaqMan probes for mouse Akap12 and the housekeeping gene, Gapdh (mouse) (Life technologies) (24). The PCR profile was: initial denaturation: 95°C for 3 minutes, 45 cycles: 95°C, 3 seconds; 60°C, 30 seconds.…”

“…Total protein extract was processed for immunoprecipitation by incubating 200 µg of pre-cleared protein with with 2 µg of antibody as we described previously (24). Immunoprecipitated protein was processed for western blotting as previously published (24) and developed with Clean-blot™ IP detection reagent (HRP) (Thermo Scientific, IL). Antibodies used for western blotting are listed in supplementary table S7.…”