Characterization of Role of LARP6 Phosphorylation in Regulating Type I Collagen Biosynthesis in Liver Fibrosis

Liver fibrosis is the common end stage of all chronic liver diseases, such as chronic viral hepatitis, alcoholism, nonalcoholic fatty liver disease, autoimmune hepatitis, alpha 1 anti-trypsin deficiency and some rare metabolic diseases. Fibrosis it is a major cause of morbidity and mortality worldwide. However, the specific and efficient anti-fibrotic therapy is still lacking. Thus, better understanding the underlying mechanism of liver fibrosis is critical in order to find a cure. Liver fibrosis is histologically characterized by excessive deposition of extracellular matrix composed primarily of type I collagen. Type I collagen is a complex protein composed by folding by two α1(I) and one α2(I) polypeptides into triple helix. The production of collagen polypeptides is regulated by the cis-acting sequence of their respective mRNAs, the 5' stem loop (5'SL). In the 5' untranslated region of collagen α1(I) and α2(I) mRNAs, there is a secondary structure forming a stem loop (5'SL). This cis-acting element regulates type I collagen expression in fibrosis by binding an RNA binding protein, La ribonucleoprotein domain family, member 6 (LARP6). LARP6 specifically binds to 5'SL of collagen mRNAs with high affinity and sequence specificity. The binding recruits several effector proteins to stimulate type I collagen production in fibrosis. LARP6 is a phosphor-protein, however, how the phosphorylation of LARP6 is involved in the process of collagen biosynthesis has not been studied before. My dissertation focuses on the role of LARP6 phosphorylation in biosynthesis of type I collagen in fibrosis. I have identified eight serines of LARP6 that undergo phosphorylation and six of these serines have never been reported to be phosphorylated before. I have characterized the functional consequence of phosphorylation of these serines, identified the responsible kinases, and analyzed the role in collagen biosynthesis. These studies are presented in the dissertation as three logically connected chapters. In the chapter two provide evidence that phosphorylation of LARP6 follows a hierarchical order; namely, that phosphorylation of S451 is the initial event, which is required for phosphorylations of other serines. Phosphorylation of S451depends on the activity of PI3K/Akt signaling pathway. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production with EC50 of 150 nM. This effect is explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of xi various forms of fibrosis. The S451A mutant of LARP6 lacks phosphorylation, not only at 451 position, but also at several other serines. Its overexpression has a dominant negative effect on collagen biosynthesis; the S451A mutant drastically reduces secretion of type I collagen and induces synthesis of aberrant and over-modified collagen polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for activation of the protein in translation and folding of collagen polypeptides. In the chapter three I have characterized two other phosphorylations of LARP6, the phosphorylation of S348 and S409. These sites are phosphorylated by mTORC1 and are redundant. Mutation of both of these serines is required to inactivate LARP6. The double mutant, S348A/S409A, acts as a dominant negative protein in collagen biosynthesis, which retards secretion of type I collagen and causes excessive posttranslational modifications. Similar effects are seen using mTORC1 inhibitor rapamycin or by knocking down mTORC1 function by siRNA. The phosphorylation of S348A or S409A is needed for two processes: 1. To recruit an accessory protein STRAP to collagen mRNAs and 2. To enable normal subcellular trafficking of LARP6. STRAP is needed to coordinate translation of collagen α1(I) and α2(I) mRNAs, what becomes critical in fibrosis. In the absence of S348/S409 phosphorylation LARP6 is also sequestered in increasing amounts at the ER membrane. The mechanistic details and significance of the S348/S409 phosphorylation are described in chapter 2. The role of TGF-β1 in LARP6 phosphorylation is described in the fourth chapter. TGF-β is the most potent profibrotic cytokine and this discovery provides the link between the TGF-β activity and the LARP6 dependent mechanism of collagen synthesis. The phosphorylation of LARP6 at S396 is stimulated by TGF-β and it promotes the distribution of LARP6 into the nucleus. This is necessary for binding of the newly transcribed collagen mRNAs and their inclusion in the LARP6 dependent metabolic pathway, resulting in more efficient type I collagen expression. In conclusion, my dissertation work has characterized different phosphorylation events of LARP6 and how they are involved in regulating the function of LARP6 in type I collagen biosynthesis. These findings will contribute to better understanding of the underlying mechanism of overproduction of type I collagen in fibrosis, and provide the rationale of using kinase inhibitors for treating fibrotic disorders.