mTORC1 phosphorylates LARP6 to stimulate type I collagen expression

Excessive deposition of type I collagen causes fibrotic diseases. Binding of La ribonucleoprotein domain family, member 6 (LARP6) to collagen mRNAs regulates their translation and is necessary for high type I collagen expression. Here we show that mTORC1 phosphorylates LARP6 on S348 and S409. The S348A/S409A mutant of LARP6 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 raptor. The S348A/S409A mutant weakly interacts with the accessory protein STRAP, needed for coordinated translation of collagen mRNAs. The interaction of wt LARP6 and STRAP is also attenuated by rapamycin and by raptor knockdown. Additionally, in the absence of S348/S409 phosphorylation LARP6 is sequestered in increasing amounts at the ER membrane. We postulate that phosphorylation of S348/S409 by mTORC1 stimulates the interaction of LARP6 and STRAP to coordinate translation of collagen mRNAs and to release LARP6 from the ER for new round of translation. These mechanisms contribute to high level of collagen expression in fibrosis.

mTORC1 phosphorylates LARP6 at S348/S409 and that lack of these phosphorylations has a dominant negative effect on type I collagen biosynthesis. We also provide evidence that mTORC1-dependent phosphorylation of LARP6 is required for recruitment of STRAP and for proper subcellular trafficking of LARP6.

Results
Inhibitors of mTOR pathway alter phosphorylation of LARP6. We have reported that LARP6 is phosphorylated at eight serines and that AKT is required for S451 phosphorylation 31 . For full understanding of the role of LARP6 in regulating collagen expression it was important to characterize the other phosphorylation sites. Among the eight sites, five resemble mTOR consensus sequence, which prefers a proline, a hydrophobic or an aromatic residue at the + 1 position 32 . To assess if these sites are mTOR targets, human lung fibroblasts (HLFs) were treated with mTORC1 and mTORC1/2 inhibitors, rapamycin and Torin 1 33 . In one dimensional SDS-PAGE (1DGE), endogenous LARP6 migrated as two bands (Fig. 1a, arrows), representing differently phosphorylated LARP6 isoforms 10 . The slower migrating band was markedly reduced by mTOR inhibitors (Fig. 1a, lanes 1 and 2), indicating that some phosphorylations of LARP6 may be mTOR-dependent. To verify this, we employed 2DGE, where endogenous LARP6 was resolved as series of isoforms with isoelectric point (pI) ranging from 6.0-7.0. However, upon rapamycin treatment LARP6 showed a pI shift towards the more basic region, from 6.2-7.2 ( Fig. 1b).
To verify that the signals on 1DGE and 2DGE represent LARP6 molecules carrying different phosphorylations, we treated the immunoprecipitated HA-LARP6 with calf intestinal alkaline phosphatase (CIP). In 1DGE HA-LARP6 was resolved as two bands and the upper band disappeared with CIP treatment (Fig. 1c). In 2DGE, LARP6 was resolved as multiple molecular species with pI ranging from 6.4-7.2. After CIP treatment only two molecular species remained, with pI of 7.0 and 7.2 (Fig. 1d). The drastic pI change by CIP suggests that most signals resolved on 2DGE represent differentially phosphorylated LARP6. We concluded that mTOR inhibitors can alter the phosphorylation status of LARP6.

Inhibitor of mTORC1, rapamycin, decreases secretion of type I collagen into cellular medium.
Having shown that mTOR is involved in phosphorylating LARP6, it was important to assess the effects of mTOR inhibition on collagen expression. The intracellular collagen was not affected by rapamycin (Fig. 2a), however, secretion of both collagen polypeptides was drastically reduced (Fig. 2b). Rapamycin did not change the expression of collagen α 1(I) or α 2(I) mRNAs (Fig. 2c), suggesting that the primary effect of mTORC1 inhibition is reduced collagen excretion.
Type I collagen is produced by folding of two α 1(I) and one α 2(I) polypeptides into a trimer after they are posttranslationally modified. Posttranslational modifications include hydroxylations of prolines/lysines and glycosylations 1 . Delayed folding of the triple helix results in excessive modification of collagen polypeptides, primarily due to excessive hydroxylations of prolines and lysines 1,34,35 . Although hydroxylations do not contribute to the charge of a protein, hydroxylations of lysines partially neutralize positive charge, resulting in more acidic pI of the polypeptides. This pI shift has been observed in patients with type I collagen folding defect 36,37 , therefore, it can be used as a readout of perturbed collagen synthesis. To assess if reduced secretion of collagen is associated with its hyper-modifications we analyzed the pI of collagen α 2(I) polypeptide. The α 1(I) polypeptide was not amenable to analysis, because the antibody could not recognize this polypeptide after isoelectric focusing. In  control cells α 2(I) polypeptide had the pI around 9.0 (arrow, Fig. 2d), but the pI was shifted to more acidic region by rapamycin, suggesting that hyper-modified polypeptides had been produced.
In the above experiments rapamycin treatment was 24 h, it was necessary to show if similar effects can be achieved with shorter time, when kinase inhibitors usually show an effect. While rapamycin did not alter intracellular collagen at 0.5, 1, and 2 h (Fig. 2e), the accumulation of collagen polypeptides into the cellular medium was lower at 0.5 h for the treated cells and did not increase further (Fig. 2f, RAPA). Control cells showed higher accumulation after 0.5 h, with further increase by 2 h (Fig. 2f, CON). These results suggest that secretion of collagen is compromised 30 min after mTORC1 inhibition.
Serines 348/409 of LARP6 are targets of mTORC1 phosphorylation. All serines which are candidates for mTOR phosphorylation are within the C-terminus of LARP6 (CTER). To identify which serines are mTOR targets we individually mutated S348, S396, S409, S421 and S451 into alanines and expressed the CTER mutants in the presence or absence of rapamycin. We surmised that if a serine is mTOR target, its mutation into alanine will abolish rapamycin-induced pI alteration. Wt CTER showed a broad range of pI (Fig. 3a, panel 1) and several signals were abolished by rapamycin (Fig. 3a, panel 2), suggesting that some phosphorylations are rapamycin sensitive. Phosphorylation of S451 is a prerequisite for other phosphorylations 31 , so this mutant does not show the multiple phosphorylated isoforms and does not respond to rapamycin (panels 3 and 4). When S348A, S396A, S409A, and S421A were analyzed, they all showed a pI alteration by rapamycin (Fig. 3a, panels 5-12), indicating that they are still rapamycin sensitive and that, perhaps, multiple serines need to be mutated to observe the effect. We found a double mutant S348A/S409A was resistant to rapamycin, because its pI did not change with rapamycin (Fig. 3a, panels 13 and 14). This suggests that mTORC1-dependent phosphorylation cannot be observed if S348/S409 are changed into alanines.
We also generated S348A/409A in the FL HA-LARP6 and tested its response to rapamycin. As shown in Fig. 3b, a similar result was observed for FL HA-LARP6 as CTER. This corroborated the finding that S348/S409 are the targets of mTOR.
LARP6 phosphorylation changes in activation of collagen-nonproducing cells into collagenproducing cells. HLFs produce high level of collagen constitutively and LARP6 phosphorylation in these cells reflects its status in terminally differentiated fibroblasts. To provide insight if LARP6 phosphorylation is a dynamic process in differentiation of collagen-nonproducing cells into collagen-producing cells we used culture activation of hepatic stellate cells (HSCs). HSCs are cells which produce type I collagen in liver fibrosis 40 . Freshly isolated HSCs from normal rat liver are in quiescent state and synthesize trace amount of collagen. However, HSCs spontaneously activate into myofibroblasts by culturing in vitro and increase collagen expression 50-100 fold [40][41][42] , mimicking the changes which HSC undergo in liver fibrosis. The activation starts after 3 days in culture and is well advanced by day 5. Therefore, we analyzed HSCs after 3 days and after 5 days in culture to investigate whether there is a change in LARP6 phosphorylation.
In day 3 HSCs, LARP6 had the pI from 6.5-7.0 ( Fig. 4a, panel 1), but by day 5 the pI shifted toward the more acidic range of 6.1-6.8 (Fig. 4a, panel 2). This alteration suggested that LARP6 undergoes additional phosphorylations in transition of HSCs from quiescent into activated state.
When rapamycin treated HSCs were analyzed at day 3, LARP6 exhibited more basic pI (6.8-7.9) (Fig. 4a, compare panel 1 and 3). This indicates that some phosphorylations of LARP6 are rapamycin sensitive even in quiescent HSCs. However, when day 5 HSCs were analyzed, a more dramatic pI shift with rapamycin was observed; from 6.1-6.8 to 6.8-7.9 (Fig. 4a, compare panels 2 and 4). This suggests that the additional phosphorylations observed in day 5 HSCs are also affected by inhibiting mTORC1.
Mapping of the mTORC1-dependent phosphorylation sites to S348/S409 allowed us to test if this mutant can undergo the dynamic phosphorylation changes during HSCs activation. The pI of S348A/S409A did not reveal an increase in phosphorylation at day 5 ( Fig. 4a, panels 5 and 6). This suggests that mTORC1-dependent S348/S409 phosphorylation is responsible for the increased phosphorylation of LARP6 during in vitro activation of HSCs. These indicate a correlation between S348/S409 phosphorylation and the ability to upregulate collagen.
To assess if there is an increased activity of AKT and mTORC1 signaling in HSCs between day 3 and day 5 in culture we analyzed expression of phosphorylated AKT and phosphorylated S6K. Increased phosphorylation of AKT and S6K was observed at day 5 compared to day 3, although the total AKT and S6K expression was comparable (Fig. 4b). These results indicate that there is an activation of AKT and mTOR signaling during HSCs activation, correlating with increased LARP6 phosphorylation.

Dominant negative effect of S348A/S409A on type I collagen expression. The experiments in
HSCs suggested that mTOR-dependent phosphorylation of LARP6 correlates with activation of collagen expression. To assess the functional significance of LARP6 phosphorylation we overexpressed S348A/S409A in HSCs and assessed its effect on collagen expression. We could not analyze α 2(I) polypeptide, because our antibody could not recognize the rodent polypeptide. At day 5, HSCs expressing wt HA-LARP6 showed high expression of α 1(I) polypeptide intracellularly and in the medium (Fig. 4c, top panel, lanes 1 and 3). The cellular level and the secretion of α 1(I) polypeptide were undetectable in HSCs overexpressing S348A/S409A (lanes 2 and 4). Thus, lack of phosphorylaitons on S348/S409 in HSCs shuts down type I collagen synthesis.
Since S348/S409 phosphorylation is dependent on mTOR, we expected that rapamycin treatment of HSCs will have a similar effect as S348A/S409A. Rapamycin reduced both, the cellular level and the secretion of collagen into the cellular medium (Fig. 4d), suggesting a similar effect on collagen expression as the S348A/S409A.
To extend the finding that S348A/S409A is dominant negative for collagen production we repeated the experiment in HLFs. To obtain an idea how much S348A/S409A is overexpressed in the dominant negative experiments we compared the expression of endogenous LARP6 and transduced wt LARP6 and S348A/S409A. The dominant negative effect was analyzed 48 h after overexpression of S348A/S409A and at this time point its expression was about 10-fold higher than that of endogenous LARP6 (Fig. 5a). Wt HA-LARP6 expressions at 48 h and 4 h are also shown. The 4 h expression was comparable to the endogenous LARP6 and this time point was utilized for analyzing the subcellular localization of LARP6 shown later in the manuscript.
S348A/S409A overexpression in HLFs slightly reduced the intracellular levels of both polypeptides. However, polypeptides secretion was dramatically reduced (Fig. 5b, top panels). The steady-state levels of both collagen mRNAs were similar in HLFs expressing wt LARP6 and S348A/S409A (Fig. 5c). The reduced secretion of collagen polypeptides by S348A/S409A resembles the result obtained with rapamycin ( Fig. 2). This suggests that phosphorylation of S348/S409 by mTORC1 regulates collagen production by HLFs. S348A/S409A decreases the rate of secretion of type I collagen. The decreased level of collagen found in the cellular medium by S348A/S409A or by rapamycin, suggests either the inefficient secretion of the protein or its accelerated degradation in the medium. To assess the secretion rate of collagen, we analyzed the accumulation of collagen α 1(I) and α 2(I) polypeptides in the medium after 1, 2, and 3 h. In cells overexpressing wt LARP6, a continuous increase in accumulation of collagen polypeptides in the cellular medium was observed within 3 h (Fig. 5d, lanes 1-3). However, their accumulation in the medium of cells overexpressing S348A/S409A was greatly retarded (Fig. 5d, lanes 4-6), suggesting that the mutant inhibited the rate of secretion of type I collagen.
To exclude the possibility that collagen polypeptides are subjected to more rapid degradation in cells overexpressing S348A/S409A, we collected the cellular medium at 3 h time point and incubated this medium for additional 6 h. The level of collagen polypeptides remained unchanged after the prolonged incubation (Fig. 5e), suggesting that medium did not contain proteolytic enzymes that would degrade collagen. We also measured the intracellular level of collagen polypeptides and it remained constant (Fig. 5f), indicating that collagen peptides were made in the cell. The slow excretion of collagen from S348A/S409A expressing cells was not accompanied by the increased intracellular retention, probably because the excessive polypeptides were degraded inside the cells. These results suggest that lack of mTORC1-dependent LARP6 phosphorylation results in inefficient secretion of collagen.
Next, we assessed if the slow secretion of collagen polypeptides by S348A/S409A is associated with their hyper-modifications. Hyper-modifications are indicative of inefficient folding as the reason for slow secretion. The pI of collagen α 2(I) polypeptide in control cells was around 9.5 (Fig. 5g, upper panel, arrow). S348A/S409A resulted in pI shift to 8.8, indicating hyper-modifications (Fig. 5g, lower panel). A similar shift was observed with rapamycin ( Fig. 2d). Therefore, we concluded that inability to phosphorylate LARP6 by mTORC1 impairs proper folding of collagen, resulting in hyper-modifications of polypeptides and their inefficient secretion.
The S451D mutation of LARP6 results in constitutive phosphorylations at multiple other sites, so using this mutant provides an opportunity to study the effects of constitutively hyperphosphorylated LARP6 in cells. We have described this mutant in our previous work 31 . We surmised that the activity of S451D may be independent on mTOR, because the protein is already hyperphosphorylated. We overexpressed S451D and analyzed if it could  overcome the inhibitory effect of rapamycin on collagen expression. We found that while rapamycin reduced secretion of collagen polypeptides from cells overexpressing WT LARP6, it did not alter intracellular collagen levels or the amount secreted into the medium of cells overexpressing S451D (Fig. 5h, lanes 3, 4, 7 and 8).
Phosphorylation of S348/S409 is required for effective interaction with essential cofactor for translation of collagen mRNAs, STRAP. To elucidate the underlying mechanism of poor secretion of collagen caused by S348A/S409A, we analyzed if this mutation affects the interaction of LARP6 with factors involved in collagen biosynthesis; STRAP and RHA [12][13][14] . STRAP is the cofactor which coordinates translation of collagen α 1(I) and α 2(I) mRNAs and in the absence of STRAP cells hyper-modify collagen polypeptides and inefficiently secrete collagen. We found that wt HA-LARP6 efficiently pulled down STRAP, while S348A/S409A showed less interaction with STRAP (Fig. 6a, left panel, compare lanes 2 and 3). The relative interaction of STRAP and LARP6 is shown at the bottom of Fig. 6a and indicates that about 50% less STRAP was pulled down with S348A/S409A.
We also analyzed if the interaction between LARP6 and STRAP is weaken by rapamycin or raptor knockdown. Rapamycin decreased the interaction between wt HA-LARP6 and STRAP (Fig. 6b, left panel, compare lanes 2 and 3); quantification of the interaction suggested that only 1/3 of STRAP was pulled down with wt HA-LARP6 after the rapamycin treatment (bottom panel, Fig. 6b). However, rapamycin did not change the already weak interaction of S348A/S409A with STRAP (Fig. 6c, left panel, compare lanes 2 and 3). Similar results were observed with raptor knockdown (Fig. 6d and e). Taken together, these results suggest that mTORC1-dependent S348/S409 phosphorylation is required for more efficient recruitment of STRAP.

Phosphorylation of S348/S409 regulates subcellular distribution of LARP6. The endogenous
LARP6 is found in both the nucleus and cytosol 11 . We assessed if S348A/S409A and mTORC1 inhibition alters LARP6 distribution. Wt HA-LARP6 accumulated throughout the cytoplasm and nucleus, but after rapamycin or raptor knockdown it was depleted in the nucleus and found predominantly in the cytosol, with more intense staining in the perinuclear regions ( Fig. 7a and b, upper panels). In contrast, S348A/S409A preferentially accumulated in the perinuclear region of the cytosol, while this distribution was not altered by rapamycin or by raptor knockdown ( Fig. 7a and b, lower panels). These indicate that phosphorylation of S348/S409 is required for nuclear accumulation of LARP6.
Similarly, endogenous LARP6 was found in the cytoplasm and nucleus of control cells (Fig. 7c, left panels). However, LARP6 was found predominantly in the cytoplasm after rapamycin treatment or after raptor knockdown (Fig. 7c, right panels). Taken together, these results are indicative that mTORC1-dependent phosphorylation of S348/S409 contributes to LARP6 nuclear distribution.
The perinuclear localization of LARP6 is suggestive of increased association with the ER. We have reported that LARP6 targets collagen mRNAs to the ER membrane 8 . To investigate this possibility we isolated the microsomal fraction, which represents ribosome-enriched ER membranes, and compared the relative levels of S348A/ S409A and wt HA-LARP6 in this fraction. Calnexin (CNX), a 67 kDa integral protein of ER membrane, which migrates as 90 kDa band in SDS-PAGE, was used as the ER marker 43,44 . The expression of both proteins in total cell lysate was similar, however, the relative abundance of S348A/S409A in the microsomal fraction was greater than that of wt LARP6 (Fig. 7d, top panel). This result is consistent with the notion that S348A/S409A localization around the perinuclear region is due to its accumulation at the ER membrane.
We also analyzed the microsomal distribution of wt HA-LARP6 after rapamycin treatment. Fig. 7e, top left panel, shows that in rapamycin treated cells twice as much of wt HA-LARP6 was found in microsome. The microsomal accumulation of S348A/S409A was high and was not further increased by rapamycin (Fig. 7f, top  left panel). Similar results were obtained with raptor knockdown (Fig. 7g and h). Taken together, these results strongly suggest that phosphorylation of S348/S409 by mTORC1 is required for dissociation of LARP6 from the ER membrane and re-entry into the nucleus. Presence of raptor in microsome ( Fig. 7g and h) has been indicated before 45,46 and suggests that phosphorylation of LARP6 by mTOR can occur on the ER membrane.
Sec61 translocon is a protein-conducting channel composed of Sec61α /β /γ subunits that mediates co-translational insertion of most secretory and membrane proteins [47][48][49] . We have previously reported that LARP6 tethers collagen mRNAs to the ER membrane, where it interacts with Sec61 8, 10 . Therefore, we assessed if the interaction of wt HA-LARP6 and S348A/S409A with Sec61 correlates with their accumulation in the microsomal fraction. Because greater amount of S348A/S409A accumulates in microsome, we adjusted the expression of wt HA-LARP6 and S348A/S409A so that similar amounts were found in the microsomal fraction (input in Fig. 7i). Under these conditions, more of S348A/S409A was pulled down with Sec61 than wt HA-LARP6, suggesting stronger interaction between the mutant and Sec61 (Fig. 7i, left panel, compare lanes 2 and 3). We concluded that LARP6 phosphorylation reduces its affinity for Sec61, allowing its release from the ER and shuttle into the nucleus.

Discussion
LARP6 is a RNA binding protein which specifically binds collagen mRNA. The binding serves to target collagen mRNA to the ER membrane and tether RHA and STRAP 8,13,14 . These factors increase translational competitiveness of collagen mRNAs (RHA) and couple translation of collagen α 1(I) and α 2(I) mRNAs (STRAP). Coupled  production of collagen polypeptides synchronizes the rate of modification to the rate of folding 8,12,14,50 . In contrast to the specific role of LARP6 in collagen mRNAs translation, mTOR pathway is involved in regulation of general translation by phosphorylating 4E-BP1 and S6K 22,24 . Our results are the first demonstration that mTOR signaling pathway regulates production of type I collagen via LARP6 phosphorylation, providing an example how translation of specific mRNAs is coupled to the general translation.
We recently identified eight phosphorylation sites on LARP6 and characterized the phosphorylation of S451 by AKT 31 . In this study we characterized the role of additional phosphorylations of LARP6. We show that (i) LARP6 is phosphorylated by mTORC1 on S348/S409, (ii) increased phosphorylation of LARP6 is observed during HSCs activation and is concomitant with increased collagen expression, (iii) S348A/S409A does not show the activation dependent phosphorylation in HSCs and has a dominant negative effect on collagen biosynthesis, (iv) the dominant negative effect is due to diminished rate of collagen secretion, (v) the interaction of S348A/S409A with STRAP, which couples translation of α 1(I) and α 2(I) mRNAs, is weakened, providing an explanation for its dominant negative effect, (vi) S348A/S409A predominantly accumulates at the ER membrane, while wt LARP6 shows such accumulation after mTORC1 inhibition, suggesting that phosphorylation of LARP6 is also required for its subcellular localization. Together, these results prompted us to postulate that LARP6 phosphorylation by mTORC1 has two functions. First, the phosphorylation increases the affinity of LARP6 for STRAP, coordinating the translation of collagen α 1(I) and α 2(I) mRNAs. Second, the phosphorylation reduces the accumulation of LARP6 at the ER membrane, allowing the protein to redistribute into the nucleus for new round of collagen mRNAs regulation. The results also provide novel rationale for the anti-fibrotic effect of rapamycin demonstrated in animal models of fibrosis.
Few reports implicated mTOR pathway in collagen biosynthesis. Leucine stimulated collagen α 1(I) mRNA translation in HSCs via mTOR activation 51 . mTOR contributes to HSCs activation by regulating expression of TGF-β 1 mRNA; TGF-β is the most potent profibrotic cytokine 27,52 . Our work shows that mTORC1 phosphorylates LARP6 at S348/S409 during HSCs activation and that these changes are important for upregulation of collagen. The first hint that LARP6 can be phosphorylated by mTOR was obtained when LARP6 phosphorylation sites were identified; five of the eight identified sites conformed to the mTOR consensus sequence 31,32 . This was further corroborated by altered electrophoretic mobility and pI of LARP6 after mTORC1 inhibition (Fig. 1). We have identified two mTORC1-dependent phosphorylation sites as the substitutions (S348A/S409A) which abolish rapamycin-induced pI change of the protein. Knockdown of raptor also could not change the phosphorylation status of S348A/S409A, while it had a profound effect on wt LARP6, further indicating that S348/S409 are the targets of mTORC1 (Fig. 3a,b and c). However, these results could not confirm that mTORC1 directly phosphorylates S348/S409 and we only concluded that mTORC1 is involved in LARP6 phosphorylation.
Type I collagen biosynthesis is unique among the synthesis of secretory proteins, because it involves coordinated translation, modifications and folding of collagen polypeptides into a triple helix 1,53,54 . Slow folding of collagen polypeptides results in hyper-modifications of the polypeptides and is seen in patients with osteogenesis imperfecta who have mutations affecting polypeptides folding 34,35 . S348A/S409A acts as a dominant negative protein in collagen biosynthesis by affecting excretion of collagen (Fig. 5). The slow rate of collage secretion by S348A/S409A is accompanied by hyper-modifications of collagen polypeptides, indicating their inefficient folding into the collagen trimer. Our previous work has shown that STRAP is essential for coordinating translation of collagen mRNAs and that it is tethered to collagen mRNAs by interaction with the CTER of LARP6 14 . STRAP knockout cells poorly secrete collagen and produce hyper-modified α 2(I) polypeptides. The interaction between LARP6 and STRAP is less efficient when phosphorylation on S348/S409 is prevented either by inhibiting mTORC1 or by mutation into alanines (Fig. 6), and the cells poorly secrete collagen and produce hyper-modified collagen polypeptides. This suggests that mTORC1-dependent LARP6 phosphorylation is an essential mechanism which activates productive collagen synthesis. mTORC1-mediated LARP6 phosphorylation increases its nuclear presence. S348A/S409A accumulates in increasing amounts at the ER membrane and interacts more strongly with Sec61 translocon. Similar effect is observed with wt LARP6 when mTORC1 is inhibited (Fig. 7). Based on our previous finding that LARP6 interacts with Sec61 8,10 , we postulate that in the absence of S348/S409 phosphorylation LARP6 has a diminished ability to dissociate from the ER membrane. This prevents its shuttling into the nucleus and participation in a new round of collagen mRNA metabolism 11,13,14 . Phosphorylation of LARP6 at the CTER does not change the binding affinity to collagen mRNAs, because this domain is dispensable for binding 5′ SL. As the S348A/S409A also weakly interacts with STRAP, combination of these events results in dysregulated translation of collagen α 1(I) and α 2(I) mRNAs, the phenotype evident by hyper-modifications of polypeptides and inefficient secretion of type I collagen.
Substantial evidence has been presented that rapamycin exerts anti-fibrotic effect in fibrosis of multiple organs, including skin/lung/kidney/heart/liver 27 effect; reduced infiltration of inflammatory cells and decreased expression of TGF-β 1 29 , decreased platelet growth factor-induced proliferation of HSCs 27 , and destabilization of collagen mRNA 57 . This report gives another rationale for anti-fibrotic effects of rapamycin; inhibition of LARP6 phosphorylation by mTOR, resulting in inactivation of LARP6 in collagen biosynthesis.
In conclusion, we propose a mechanism of LARP6 activation in collagen biosynthesis. Involvement of mTORC1 in LARP6 phosphorylation at S348/S409 increases the affinity of LARP6 for STRAP. By recruiting STRAP, LARP6 couples translation of collagen α 1(I) and α 2(I) mRNAs, facilitating folding of the polypeptides into triple helix and rapid secretion. This phosphorylation also helps recycling of LARP6 from the ER membrane for another round of binding, partitioning and translation initiation of collagen mRNAs. The mTORC1 involvement of LARP6 phosphorylation takes place during the critical period of activation of HSCs, suggesting that it is one of the central events in fibrosis.

Methods
Plasmids and adenovirus construction. The CTER LARP6 was made as described previously 11 . Site directed mutagenesis of the single amino acids was done by QuickChange mutagenesis kit (Stratagene, 200523-5). The identity of all constructs was verified by sequencing.
Adenoviruses were generated by re-cloning of full-length LARP6 and mutants from pcDNA3 vectors into pAd-CMV-Track vector, followed by recombination in BJ5183 E.coli cells 58 . Adenoviruses were amplified in HEK293 cells and purified by cesium chloride density gradient centrifugation. All adenoviral vectors expressed the protein of interest and GFP, the later was encoded by an independent transcription unit 58 . GFP was used as a marker to estimate the efficiency of cell transduction. Plasmid expressing raptor specific shRNA was co-transfected with pCMV-dR8.2 DVPR vector and pCMV-VSV-G vector at the ratio of 4:3:1 using LipoD293 Transfection Reagent (SignaGen Laboratories, SL100668) into HEK293T cells to allow packaging of the lentivirus. Virus containing supernatants were collected at 24 h intervals for three consecutive days and were used as source of virus. HLFs were plated in 6-well plates and the lentivirus transduction was carried out in media containing 8 μ g/ml hexadimethrine bromide (polybrene). Transduction efficiency was determined by monitoring the viral marker, RFP. The knockdown of raptor was confirmed by Western blot.

Chemicals and antibodies. Rapamycin was from
Rat hepatic stellate cell isolation and culture. Rat HSCs were isolated by perfusion of rat liver with 0.5 mg of pronase and 0.04 mg of collagenase per gram of animal weight, followed by centrifugation of the cell suspension over 20% Nykodenz gradient, as described 62 . All animal procedures were done according to the NIH guidelines and guidelines of Florida State University Animal Care and Use Committee (ACUC). The experimental protocol for isolation of HSCs was approved by the ACUC committee of Florida State University as the protocol #1428 on 7/30/14 and is valid for 3 years. The isolation of HSCs was performed strictly according to this protocol. After isolation, the cells were cultured in uncoated plastic dishes. HSCs Cell extracts were made at day 3 and day 5 and analyzed for AKT and mTOR expression by Western blot. For analysis of LARP6 phosphorylation, HSCs were transduced by adenovirus expressing wt LARP6 or mutants on day 2 or day 4 in culture and the cells and were harvested on day 3 or on day 5 for analysis. Treatment of HSCs with rapamycin (100 nM) was at day 5 for 2 h, followed by analysis of collagen levels by Western blot.
Real-time PCR analysis. RNA was extracted from HLFs by using Trizol (Invitrogen). 1 μ g of total RNA was used to prepare cDNA using the Superscript First Strand Synthesis System for RT-PCR (Invitrogen), according to the manufacturer's instructions. 5 μ l of 10-fold-diluted cDNA was used as in a SYBR Green qPCR assay (Applied Biosystems). The primers used for PCR amplification are shown in Table 1. Expression of collagen mRNAs was normalized to that of β -actin mRNA and statistical significance was determined using Student's t test. P values of < 0.05 were considered significant and the results are presented as means ± standard deviation (SD) (n = 3).
Preparation of microsomal fraction. Microsomal fraction was prepared as reported before 63  Immunoprecipitations. HLFs were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM Dithiothreitol, 170 μ g/ml PMSF, 1 × protease inhibitors and cleared lysate was incubated with 1 μ g of antibody for 3 h at 4 °C. 30 μ l of equilibrated protein A/G-agarose beads was added, and incubation continued for 1 h. The beads were washed 3 times and analyzed by Western blot.
For CIP treatment, HA-LARP6 was overexpressed in HLFs, immunoprecipitated and treated with or without CIP at 37 °C for 3 h. The beads were then washed 4 times and analyzed by Western blot and 2DGE.
For immunoprecipitation experiments with microsomal fraction, 1 μ g anti-Sec61β antibody was added and incubated for 30 min at 4 °C. 30 μ l of equilibrated protein A/G beads were then added and incubation continued for an additional 30 min. The beads were washed 4 times and the samples analyzed by Western blot.
Western blot. Cells were lysed in 50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1 mM DTT, protease inhibitors and protein concentrations were estimated with the Bradford assay (Biorad, 500-0006), with bovine serum albumin (BSA) as standard. 40 μ g of total cellular proteins was typically used for Western blot. For analysis of the medium proteins, equal numbers of cells were seeded and after 48 h the cells were washed 3 times with serum free medium. 600 μ l of serum free medium was added per well and collagen accumulation was allowed to proceed for 1, 2, and 3 h. Serum free medium was collected and analyzed by Western blot.
For analysis of collagen protein stability in the medium samples, after collection, the serum free medium samples were incubated for additional 6 h at 37 °C. Two-dimensional gel electrophoresis. Cells were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40 with protease inhibitors, and phosphatase inhibitors when necessary. The protein pellet or the immunoprecipitated HA-LARP6 was solubilized in 120 μ l of rehydration buffer (7 M Urea, 2 M Thiourea, 2% CHAPS, 0.8% Ampholytes, 65 mM Dithiothreitol, and trace amount of Bromophenol blue) for 1 h at room temperature, and loaded onto Immobiline Dry Strip strips (7 cm, pH 3 to 10, GE Healthcare, 17-6001-11). An Ettan IPGphor 3 instrument (GE Healthcare) was used for the isoelectric focusing, according to the recommended protocol 64 . After focusing, the strips were equilibrated in Equilibration buffer A (0.375 M Tris-HCl, pH 8.8, 6 M Urea, 20% Glycerol, 2% SDS, 2% Dithiothreitol, bromophenol blue) for 10 min, followed by Equilibration buffer B (0.375 M Tris-HCl, pH 8.8, 6 M Urea, 20% Glycerol, 2% SDS, 2.5% Iodoacetamide, bromophenol blue) for 2 × 10 min. The second-dimension separation was done by laying strips onto 7.5% SDS PAGE, followed by Western blot. Immobilized strips showed slight batch to batch variations in the ampholyte distribution, so only the samples run on the same batch of strips were directly compared.  Immunostaining of cells. HLFs were seeded onto glass coverslips. After treatment, cells were fixed in 4% paraformaldehyde in PBS for 10 min, followed by 3 washes with PBS. Cells were permeabilized with PBST (PBS containing 0.1% Triton X-100) for 10 min and blocked with PBTG (PBS containing 0.1% Triton X-100, 10% normal goat serum and 1% bovine serum albumin (BSA)) at room temperature for 2 h. Coverslips were incubated with primary antibody at 4 °C overnight. After 4 washes with PBS, secondary antibody diluted at 1:500 was added and incubated at room temperature for 1 h. Cells were washed 4 times with PBS and mounted with Prolong mounting solution containing DAPI. Images were taken by the EVOS FL Color fluorescence imaging system with 60 × objective.