Role Of RACK1 In Hepatocyte Autophagy And The Underlying Molecular Mechanisms

Background:Scaffold proteins play essential roles in regulating many cellular activities from growth control to apoptosis. Receptor for activated C kinase 1(RACK1, official gene name Gnb2l1) is such an adaptor protein. RACK1 was originally identified on the basis of its ability to anchor activated form of protein kinase C(PKC). RACK1 contains seven WD40 repeats. A large number of studies show that RACK1 plays an important role in promoting the malignant growth of hepatocellular carcinoma. RACK1 is highly expressed in normal liver, but its pathophysiological role in the liver is unknown.Autophagy is a highly conserved cell intrinsic life process, in this process, aggregated proteins, damaged/excess organelles and lipids are transported to the lysosomes for degradation and decomposition. Hepatocytes are a major cellular storehouse for neutral lipids. The loss of hepatocyte autophagy leads to lipid accumulation in vivo. VPS34, which generates PI3 P, is the only class III phosphatidylinositol 3-kinase(thereby also named as PIK3C3) in mammals. The ATG14L-Beclin1-VPS34-VPS15 complex functions in autophagosome formation. Thus it is named as the autophagy-initiation complex. Starvation-induced AMPK activation and mTOR inhibition results in Ulk1 activation. The formation of the autophagy-initiation complex depends on Beclin1 phosphorylation by Ulk1 and Bcl-2 dissociation from Beclin1 triggered by JNK-mediated Bcl-2 phosphorylation. VPS34 kinase activity increases upon the assembly of the autophagy-initiation complex. The generated PI3 P can recruit DFCP1 to form Omega body. And then ATG12-ATG5 conjugation leads to elongation and closure of the membranes and membrane-bound LC3 B II generation from cytosolic LC3 B I. But the formation mechanism of autophagy initiation complex is not clear.Objective:To investigate the role of RACK1 in hepatocyte autophagy and the underlying molecular mechanisms and to analyze the role of RACK1 in preventing lipid accumulation in the liver.Method:Autophagy was induced by starvation or mTOR inhibitor rapamycin treatment. Chloroquine(CQ) was used to inhibit lysosomal protease. Under these conditions, the correlation between the protein levels of RACK1 and autophagy markers such as LC3 B II and ATG12-ATG5 was analyzed by Western Blot; autophagosomes and lipid droplets were observed by electron microscopy; nile red staining was employed to examine hepatocyte lipid accumulation; DFCP1 puncta were observed by immunofluorescence; VPS34 kinase activity was investigated by in vitro immune complex kinase assays; RACK1-interacting proteins were explored by immunoprecipitation and mass spectrometry; the interaction of RACK1 with the autophagy-initiation complex was confirmed by coimmunoprecipitation analysis; the direct binding of in vitro translated Atg14 L, Beclin1, VPS34, and VPS15 to RACK1 was examined by GST-pull down.Results:1) RACK1 promotes autophagy and limits lipid accumulation in the liverWestern Blot revealed that RACK1 knockdown led to reduced levels of LC3 B II at the basal state and upon starvation. RACK1 overexpression showed opposite effects. Because CQ treatment failed to reverse the reduction of LC3 B II levels upon RACK1 knockdown, RACK1 promotes autophagy onset, but not autophagic protein degradation.Western Blot revealed hepatocyte-specific RACK1 deficiency in Gnb2l1?hep mice when compared with Gnb2l1F/F mice. Gnb2l1?hep mice began to show enlarged and discolored livers which weigh more than those from Gnb2l1F/F mice as early as 4 months of age. Ultrastructural analysis revealed increased number of cytosolic lipid droplets in fed 6-week-old Gnb2l1?hep livers. Furthermore, smaller mitochondria and the lack of glycogen deposition were also observed in fed Gnb2l1?hep livers. Notably, although a 48 h fasting induced autophagic vesicles in Gnb2l1F/F livers, Gnb2l1?hep livers exhibited virtually no autophagosomes, lipid droplets with increased number and size, and many swollen mitochondria. Nile red staining confirmed lipid accumulation in Gnb2l1?hep livers. Western Blot revealed Gnb2l1?hep livers showed diminished basal and starvation-induced LC3B-II generation. These data confirm that RACK1 promotes autophagy onset.2) RACK1 is required for the early stage of autophagosome formationGnb2l1?hep livers showed diminished ATG12-ATG5 conjugation at the basal state and upon 48 h fasting/chloroquine(CQ) treatment. RACK1 knockdown led to diminished numbers and sizes of GFP-DFCP1 puncta at the basal state and upon nutrient deprivation. Moreover, Gnb2l1?hep livers showed diminished ATG14L-linked VPS34 kinase activity at the basal state and after 48 h fasting.3) RACK1 promotes the assembly of the autophagy-initiation complexSilver staining and mass spectrometry identified VPS15 might be a binding partner of endogenous-like GFP-RACK1. Coimmunoprecipitation analysis revealed the interaction of RACK1 with the autophagy-initiation complex. Gnb2l1?hep livers showed diminished formation of the autophagy-initiation complex.4) RACK1 forms dimers and directly binds to ATG14 L, Beclin1, and VPS15Binding of GFP-RACK1 to FLAG-RACK1 was detected with coimmunoprecipitation analysis, which was augmented upon nutrient deprivation. GST-pull down assays demonstrate that RACK1 directly binds to ATG14 L, Beclin1, and VPS5.5) RACK1 does not affect the activation of AMPK, Ulk1, and JNKWestern Blot disclose that Gnb2l1?hep livers showed unchanged P-AMPK, P-Ulk1, and P-JNK levels at the basal state and after 48 h fasting.Conclusion:RACK1 directly interacts with ATG14 L, Beclin1, and VPS15, thereby promoting the assembly of the ATG14L-Beclin1-VPS34-VPS15 complex and ATG14L-linked VPS34 kinase activity. These observations provide a function for RACK1 in autophagy and hepatosteatosis.