CK2 phosphorylates Sec31 and regulates ER-To-Golgi trafficking.
CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking Mayuko Koreishi1, Sidney Yu2, Mayumi Oda1, Yasuko Honjo3, Ayano Satoh1* 1 The Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan, 2 School of Biomedical Sciences and Epithelial Cell Biology Research Center, The Chinese University of Hong Kong, Hong Kong, People’s Republic of China, 3 The Research Core for Interdisciplinary Sciences (RCIS), Okayama University, Okayama, Japan Abstract Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking. Citation: Koreishi M, Yu S, Oda M, Honjo Y, Satoh A (2013) CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking. PLoS ONE 8(1): e54382. doi:10.1371/ journal.pone.0054382 Editor: Wanjin Hong, Institute of Molecular and Cell Biology, Singapore Received August 18, 2012; Accepted December 11, 2012; Published January 18, 2013 Copyright: ß 2013 Koreishi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded to AS by the National Institute of Aging (R03 AG030101), the special Coordination Fund for Promoting Science and Technology of MEXT (Ministry of Education, Culture, Sports, Science and Technology, Japan), Hayashi Memorial Foundation for Female Natural Scientists, Ryobi Teien Memorial Foundation, Naito Foundation, Uehara Memorial Foundation, Kurata Memorial Hitachi Science and technology foundation, and Kanae Foundation for the Promotion of Medical Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: email@example.com Introduction Molecular trafficking and secretion is initiated by the ratelimiting step of protein export from the endoplasmic reticulum (ER). This process is mediated by coat protein II (COPII)-coated vesicles, the formation of which is a crucial step. The COPII coat consists of small GTPase Sar1 and 6 Sec proteins. Coat assembly is initiated by the exchange of GDP for GTP on Sar1 [1,2], which is catalyzed by the ER-localized guanine nucleotide exchange factor Sec12 [3,4]. Sar1–GTP recruits the heterodimeric complex Sec23–Sec24 to ER membranes [5,6,7]. Sec23 is a GTPaseactivating protein (GAP) for Sar1 . Sec24 is thought to bind directly to and sort cargo [9,10,11]. The Sec23–Sec24 heterodimers recruited to ER membranes by Sar1–GTP in turn recruit Sec13–Sec31. The Sec13–Sec31 heterodimers form a clathrin-like cage lattice to promote budding of COPII vesicles from ER membranes [12,13]. Other molecules not included in the COPII coat also modulate COPII vesicle formation . A transmembrane protein, Sec16, has been shown to be necessary for COPII vesicle formation by binding to multiple components of the COPII coat [15,16,17,18,19,20]. It is well established that COPII vesicle formation is initiated by the activation of Sar1. However, the regulation of the subsequent process has not been elucidated completely. One way of achieving such a regulation could be Sec31 phosphorylation. Sec31 is phosphorylated in yeast, and its phosphorylation–dephosphorylation cycle is implicated in the budding of COPII vesicles . In addition, mammalian Sec31 has been isolated as a phosphoprotein . However, the molecular nature underlying Sec31 phosphorylation including the responsible kinase(s), phosphorylation sites, and the functional significance in COPII vesicle formation have not been well characterized. Casein Kinase II (CK2) is a constitutively active serine/ threonine kinase that regulates cellular events such as cell cycle and transcriptional regulation, cell survival, virus infection and tumor growth . CK2 is also known as a master kinase that links these cellular events to other kinases . Importantly, the role of CK2 in the regulation of the secretory pathway has been documented by its effect on the trafficking of cystic fibrosis transmembrane conductance regulator (CFTR), mannose 6phosphate receptor (MPR), and taurine [25,26,27]. It is also speculated that CK2 phosphorylates p115, a vesicle tethering factor that is essential for ER-to-Golgi transport . Similar to the proteins described above Sec31 contains multiple CK2 consensus phosphorylation sites. Therefore, we hypothesized that CK2 is responsible for Sec31 phosphorylation, and attempted to determine the role of Sec31 phosphorylation in COPII vesicle formation. PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e54382 CK2 Phosphorylates Sec31 Results Sec31 Phosphorylation Reduces its Membrane Association To determine the role of Sec31 phosphorylation in COPIImediated ER-to-Golgi transport, we first examined whether Sec31 phosphorylation affects its membrane association by subcellular fractionation. Ultracentrifugation was used to separate the membranes from the cytosol. Sec31 was recovered from each fraction by immunoprecipitation with anti-Sec31 and its phosphostatus was determined by western blotting using anti-phosphoserine/threonine antibodies. As shown in Figure 1, phosphorylated Sec31 was only found in the supernatant (cytoplasmic fraction), whereas Sec31 was detected in both the supernatant and the pellet (membrane fraction; indicated by calnexin). These findings indicate that the phosphorylation of Sec31 reduces its membrane association. Sec31 is Phosphorylated at Serines 527, 799, and 1163 and at Threonine 1165 Since Sec31 phosphorylation appears to be important for its membrane dissociation, we determined its phosphorylation sites. Endogenous Sec31 was isolated from total cell lysates by immunoprecipitation and analyzed by mass spectrometry. Peptide fragments containing serines 527 (S527), 799 (S799), and 1163 (S1163) and threonine 1165 (T1165) were phosphorylated. These sites (indicated by the arrows in Figure 2A) are distributed in the linker region (S527 and S799) and C-terminal Sec23 binding site (S1163 and T1165) of Sec31. To confirm their phosphorylation, the 4 amino acids were mutated to alanines using QuikChange. FLAG-tagged wild-type (WT) and alanine mutant Sec31 (4SA) were expressed and immunoprecipitated from the total cell lysate. Their phosphorylation was analyzed by western blotting using anti-phosphoserine/threonine antibodies. As shown in Figure 2B, the phosphorylation of the 4SA mutant was reduced by approximately 60%. The 4SA mutant was still partially phosphorylated, which suggests that the 4SA mutant is able to form complexes with endogenous WT Sec31 that can be phosphorylated or that there are other phosphorylation sites that were not identified. The Non-phosphorylatable Mutant of Sec31 Increases its Membrane Association Membrane association of the WT Sec31 and the 4SA mutant was assessed by fluorescence recovery after photobleaching (FRAP). GFP-tagged WT Sec31 and the 4SA mutant were expressed transiently in HeLa cells. A GFP-positive dot (an ERES) was then photobleached, and the fluorescence recovery was monitored. As shown in Figure 3, the FRAP of the 4SA mutant was slower than that of the WT. Analysis by fitting the FRAP data to 2 equations (Materials and Methods) showed that the 4SA mutant appeared to have a larger immobile fraction and kon/koff (Table 1). To determine the cause of the difference in the FRAP of WT and 4SA, the FRAP was performed in the presence of cycloheximide. Cycloheximide inhibits protein synthesis resulting in less cargo loading into transport vesicles. Lowering cargo loading by cyclohexmide has been shown to change the turnover of COPII coat . The FRAP of 4SA in the presence or absence of cyclohexmide did not change, suggesting that the difference between WT and SA in Figure 3 may be due to the changes in the turnover rather than lateral diffusion (data not shown). These findings suggest that the 4SA mutant remains on the membranes longer than the WT, which is consistent with the decreased membrane association of phospho-Sec31 shown in Figure 1. Dephosphorylation of the Linker Region of Sec31 Increases its Binding to Sec23 Sec23 forms the inner layer of the COPII coat on ER membranes, whereas Sec31 is part of the outer layer. It is thought that Sec31 is recruited to the membrane through direct binding to Sec23 [15,22]. Therefore, the decreased association of Sec31 with membranes due to phosphorylation led us to examine whether Sec31 phosphorylation also affects its affinity for Sec23. For this purpose, we generated a series of Sec31 non-phosphorylatable alanine mutants. FLAG-tagged Sec31 (WT and alanine mutants) and GFP-tagged Sec23 were then co-expressed, and Sec23 was recovered from total cell lysates by immunoprecipitation with antiGFP antibodies. As shown in Figure 4A, double mutation of the serines at 527 and 799 (S527/S799) in the linker region led to a 3fold increase in Sec31 binding to Sec23, whereas there was no or a minimal effect with only a single mutation of S527 or S799, respectively. These effects were specific to Sec23. As shown in Figure 4B, the association of Sec31 with Sec13 was not affected by these mutations. These results indicate that Sec31 phosphorylation regulates its binding to Sec23 but not Sec13. Importantly, the Figure 1. Membrane associated Sec31 is not phosphorylated. HeLa cells were subjected to subcellular fractionations to prepare cytosol (Sup) and membranes (Ppt) by ultracentrifugation. The recovery of the transmembrane protein, calnexin in the membrane fraction (Ppt) indicates that the fractionation was performed properly. Subsequently, Sec31 from both fractions were immunoprecipitated and subjected to western blotting with anti-Sec31 and anti-phospho-serine/threonine antibodies. doi:10.1371/journal.pone.0054382.g001 PLOS ONE | www.plosone.org 2 January 2013 | Volume 8 | Issue 1 | e54382 CK2 Phosphorylates Sec31 Figure 2. Identification of the phosphorylation sites in Sec31. (A) The predicted domain structure of human Sec31 by SMART (http://smart. embl-heidelberg.de). The domain structure of Sec31 is: N-terminal WD repeats for Sec13 binding, a linker region, and a C-terminal extensin-like proline-rich domain for Sec23 binding. The phosphorylation sites identified by mass-spectrometry were indicated by arrows. (B) HEK293 cells were transfected with FLAG-tagged wild type (WT) Sec31 or its S527A/S799A/S1163A/T1165A mutant (4SA). Tagged proteins were immunoprecipitated with antibodies to the FLAG tag and subjected to western blotting with anti-FLAG and anti-phospho-serine/threonine antibodies. The immunoprecipitated (IP’ed) phospho-Sec31 levels were normalized with IP’ed Sec31 levels and expressed as the normalized ratio. doi:10.1371/journal.pone.0054382.g002 increased affinity of the Sec31 alanine mutants for Sec23 may explain the larger immobile fraction and slower FRAP of the 4SA mutant shown in Figure 3. CK2 Phosphorylates Sec31 Our preliminary data showed that Sec31 was phosphorylated using 32P-ATP as well as 32P-GTP as a phosphate source (data not shown), which is consistent with the fact that CK2 and CK2-like kinases can use both ATP and GTP as a phosphate source . Therefore, we tested whether CK2 could phosphorylate Sec31. FLAG-tagged Sec31 was expressed, immunoprecipitated from the total cell lysates, and incubated with recombinant CK2 protein (recCK2) before western blotting using anti-phosphoserine/ threonine antibodies. recCK2 treatment increased Sec31 phosphorylation more than 3-fold, indicating that CK2 can phosphor- Table 1. Kinetics of Sec31 turnover at single ERES. Mobile fraction* Wild type (WT) 0.22160.004 4SA mutant 0.11860.002 t1/2 maximum recovery (s)* 6.13 s 60.01 6.46 s 60.01 kon/koff** 8.4760.14 12.3260.20 *Calculated using the equation 1 in the text and . **Calculated using the equation 2, which is the reaction dominant model described in . doi:10.1371/journal.pone.0054382.t001 ylate Sec31 directly at least in vitro (Figure 5A). We did not observe phosphorylation of Sec13 treated with recCK2 in the same way suggesting that the phosphorylation of Sec31 by recCK2 is specific (Figure 5B). In addition, recCK2 treatment did not change the weak phosphorylation of 4SA suggesting that there might be additional unidentified phosphorylation sites in Sec31 (data not shown). To confirm whether CK2 is responsible for Sec31 phosphorylation in cells, we depleted CK2 by RNAi. Depletion or inhibition of CK2 reduced Sec31 phosphorylation (Figure 5C and 5D, respectively). These results suggest that CK2 is responsible for Sec31 phosphorylation. The protein levels of CK2 after depletion were 10%–15%, as determined by western blotting using anti-CK2 (Figure S1). CK2 Regulates Sec31–Sec23 Interactions Through Sec31phosphorylation Since Sec31 phosphorylation decreased its binding to membranes and the alanine mutations increased Sec31 binding to Sec23, we predicted that phospho-Sec31 may not bind to Sec23. To test the role of CK2 in Sec31–Sec23 interactions, we manipulated CK2 levels prior to Sec31–Sec23 co-immunoprecipitation in a process similar to that of the experiment shown in Figure 4. As shown in Figure 6, depletion of CK2 by siRNA1 increased Sec31 binding to Sec23 by approximately 3-fold. In contrast, CK2 overexpression decreased such binding (data not shown). We also noticed that CK2 was co-immunoprecipitated with Sec31 in the presence of 1 mM Ca++ (Figure S2). PLOS ONE | www.plosone.org 3 January 2013 | Volume 8 | Issue 1 | e54382 CK2 Phosphorylates Sec31 Figure 3. The dynamic of membrane association and dissociation of Sec31 is reduced with the non-phosphorylatable mutant. (A) Shown are the results of fluorescence recovery after photobleaching (FRAP) of Sec31 and its 4SA. GFP-tagged wild type Sec31 (WT) and the 4SA mutant were expressed transiently in HeLa cells. A GFP positive dot was photobleached and subsequent fluorescence recovery was monitored for 30 s with 1.2 s intervals. Eight dots were photobleached per cell and results were normalized. Represented are the average of ,7 experiments. Bars, SEM. (B) The representative images at the indicated time points of Figure 3A. Sizes, 1.85 (w) x 1.85 (h) mm. doi:10.1371/journal.pone.0054382.g003 CK2 Depletion Reduces Membrane Trafficking To investigate the function of CK2 in the secretory pathway, we tested whether depletion of CK2 by RNAi affects membrane trafficking. We first monitored the secretion of secretory alkaline phosphatase (SEAP) by measuring SEAP activity in the culture supernatants of mock or CK2 siRNA-treated cells. As shown in Figure 7A, the secretion of SEAP was reduced by approximately 50% in CK2-depleted cells. To confirm the phenotypes of CK2 knockdown by siRNAs, we used siRNA1 and siRNA2. The depletion of CK2 by siRNA1 was more efficient with just 10%– 15% of the original CK2 protein level remaining after depletion (Figure S1). The reduction of CK2 by siRNA2 (Figure S3A) was less efficient than that by siRNA1, but the trend in the SEAP assay was the same (Figure S3B). To further determine whether CK2 exerts its effect in ER-to-Golgi transport, we assessed the trafficking of a temperature-sensitive mutant of vesicular stomatitis virus G protein (VSVG). HeLa cells were transfected with VSVGGFP at 40uC and incubated overnight. After being shifted to 32uC for 15 min, the cells were processed for fluorescence microscopy. The GFP fluorescence intensity in the Golgi region was quantified using Image J software. As shown in Figure 7B, VSVG in the Golgi region was reduced by approximately 40% in CK2-depleted cells. The immunofluorescence images in Figure 7C indicated VSVG is retained in the ER and ERES in CK2-depleted cells. To examine the primary effect of CK2 inhibition in SEAP transport, a CK2 inhibitor was then used. As shown in Figure 7D, the CK2 inhibitor also inhibited SEAP transport similar to CK2 RNAi (Figure 7A). Furthermore, to test if the inhibition of CK2 is primarily caused by inhibition of Sec31 phosphorylation, the SEAP transport was measured in 4SA mutant expressing cells. As shown in Figure 7D, the expression of 4SA mutant reduced SEAP transport by 70%. The 4SD mutant did not affect the transport. These data suggest that CK2 is involved in the regulation of membrane trafficking particularly in ER-to-Golgi transport though Sec31 phosphorylation. Finally, we measured the colocalization of wild type Sec31 (WT) or 4SA mutant (SA) with Sec24, which is equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | www.ploscompbiol.org 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study . The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step , raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps ) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee , or a slightly open conformation as found in Jimenez-Useche et al. . Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | www.ploscompbiol.org 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. 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