AKAP10, regulates heme biosynthesis during erythropoiesis at the outer mitochondrial membrane. This integrated pathway culminates with the direct phosphorylation of the crucial heme biosynthetic enzyme, ferrochelatase (FECH) by protein kinase A (PKA). Biochemical, pharmacological, and genetic inhibition of this signaling pathway result in a block in hemoglobin production and concomitant intracellular accumulation of protoporphyrin intermediates. Broadly, our results implicate aberrant PKA signaling in the pathogenesis of hematologic diseases. We propose a unifying model in which the erythroid transcriptional program works in concert with post-translational mechanisms to regulate heme metabolism during normal development.Heme is required for survival of all cells, and in most eukaryotes, is produced through a series of eight enzymatic reactions. Although heme production is critical for many cellular processes, how it is coupled to cellular differentiation is unknown. Here, using zebrafish, murine, and human models, we show that erythropoietin (EPO) signaling, together with the GATA1 transcriptional target, DOI:http://dx.doi.org/10.7554/eLife.24767.001 Heme is an iron-containing compound that is important for all living things, from bacteria to humans. Our red blood cells use heme to carry oxygen and deliver it throughout the body. The amount of heme that is produced must be tightly regulated. Too little or too much heme in a person’s red blood cells can lead to blood-related diseases such as anemia and porphyria. Yet, while scientists knew the enzymes needed to make heme, they did not know how these enzymes were controlled.et al. show that an important signaling molecule called erythropoietin controls how much heme is produced when red blood cells are made. The experiments used a combination of red blood cells from humans and mice as well as zebrafish, which are useful model organisms because their blood develops in a similar way to humans. When Chung et al. inhibited components of erythropoietin signaling, heme production was blocked too and the red blood cells could not work properly.Now, Chung These new findings pave the way to look at human patients with blood-related disorders to determine if they have defects in the erythropoietin signaling cascade. In the future, this avenue of research might lead to better treatments for a variety of blood diseases in humans.DOI:http://dx.doi.org/10.7554/eLife.24767.002 FECH (EC 4.99.1.1), which encodes the terminal rate-limiting enzyme in heme production, is strongly associated with the disease erythropoietic protoporphyria (EPP)  .The dependence of RBC biology on heme metabolism makes erythropoiesis an excellent system to gain insight into this process. Previous genetic analyses using RBCs have identified several mechanisms regulating heme metabolism most of which are transcriptional networks controlling mRNA expression of heme metabolism genes . CurrentAkap10  cells were analyzed by quantitative mass spectrometry  Figure . MEL celAs expected, erythroid differentiation was associated with the elevated mitochondrial expression of a number of mitochondrial proteins known to have a role in erythropoiesis such as FECH, ATPIF1, and ABCB10 while the expression of housekeeping proteins such as components of the mitochondrial transport and protein translation machinery were relatively unchanged . StrikinWe wondered whether increased mitochondrial PKA was specific for a particular suborganellular compartment, and next, performed a series of experiments to determine their submitochondrial expression. First, intact mitochondria isolated from maturing erythroid cells were treated with proteinase K that would digest all proteins exposed on the outer mitochondrial membrane. Immunoblot analysis of untreated and treated mitochondria revealed that the majority of PKA subunits were sensitive to proteinase K activity similar to TOM20 while VDAC1, a mitochondrial outer membrane (OM) marker known to be resistant to proteinase K digestion, remained largely unaffected  Rapapor. BiochemA great deal of work has demonstrated that PKA is localized to subcellular compartments through interactions with a family of anchoring proteins called AKAPs (a kinase anchoring proteins) . The majAKAP10 expression increases in maturing erythroid cells and is a downstream target of the GATA1 erythroid lineage master transcription factor AKAP10], parts of exons 1 (Ex1) and 3 (Ex3) along with all of exon 2 including the ATG start codon were deleted by our targeting strategy (Cas9(184delTG)AKAP10] had a 2 base-pair deletion that resulted in a premature stop codon (Stop’)  . Neither (Stop’) .Total expression of PKA subunits in maturing KO cells was similar to wild-type (WT) cells . Howevero-dianisidine staining, which can be blocked by PKA antagonists H-89 or PKI (14-22) (2) (2) that has a physiologic role during multiple aspects of hematopoiesis (2 can be similarly inhibited by PKI (14-22)  . An incr-22) (2) , which iopoiesis . The eff (14-22) , undersc for PKA .10.7554/Although our results from pharmacologic experiments suggest that widespread modulation of PKA has an impact on heme production on maturing erythroid cells, they do not explicitly examine the role of mitochondrial OM PKA. The precise contributions of distinct mitochondrial pools of PKA have been a topic of controversy . EmerginIt is difficult to rely solely on pharmacologic data to unambiguously dissect the contributions of intracellular PKA pools since dose responses are known to vary form one cell type to another . Howeverin vivo significance of AKAP10 and mitochondrial OM PKA signaling by using morpholinos to block akap10 expression in zebrafish (Danio rerio) embryos (termed morphants) (akap10 morphants were anemic with decreased hemoglobinization (globin-LCR:eGFP)] (akap10 morphants had reduced RBC counts (in vivo.We next examined the rphants) . For overphants) . RemarkaR:eGFP)] . Flow-cyC counts . TogetheNext, we asked whether mitochondrial OM PKA signaling directly regulated heme biosynthesis by phosphorylating mitochondrial heme enzymes. Of the mitochondrial enzymes ALAS2, PPOX, CPOX, and FECH, the only enzyme with a predicted high-confidence PKA site  is FECH at Thr116 (human FECH and Thr115 for murine FECH) . This rein vitro kinase assay by mixing together purified His-tagged human FECH and PKAc, followed by western analysis. This experiment showed that FECH is directly phosphorylated by PKA in an ATP-dependent fashion  abolished this phosphorylation  that can be produced by unmodified and modified FECH. DP, as a more soluble analog of the naturally occurring heme precursor protoporphyrin-IX (PPIX), is frequently employed in such measurements and is similarly metalated by FECH to generate deuteroheme (55Fe measurements (max) but did not significantly change the Michaelis-Menten (Km) constant  . PKC-medteroheme . Signifiteroheme  and the urements . This inurements . More deconstant , suggested cells . In conted cells —instead reatment . Conversreatment  and 5D. FECH mutations retain residual FECH activity  analysis revealed that erythroid cells expressing only FECHT115A protein had reduced hemoglobinization as well as lower intracellular hemin levels  precursor  signaling through its cognate receptor tyrosine kinase (EPOR) regulates survival and proliferation of erythroid progenitors . HoweverSer133 phosphorylation but not STAT5Tyr694 phosphorylation was blocked by PKI (14-22) /signal transducer and activator of transcription 5 (STAT5), mitogen activated protein kinase (MAPK), and phosphatidyl 3’-inositol kinase (PI3K) pathways , leading (14-22) , indicatrylation , stronglrylation , which irylation .10.7554/Ser133 and FECHThr116 in human erythroleukemia (HEL) cells that express the constitutively active JAK2V617F mutant protein  treated with either EPO or vehicle. Indeed, EPO stimulation resulted in an increase in FECH phosphorylation . Further protein . TogetheThe role of PKA in erythropoiesis has remained enigmatic. This is because early studies failed to detect changes in cAMP levels despite its stimulatory effect on iron incorporation during RBC maturation that is likely mediated by the synergistic effects of CREB on STAT5 transcription . HoweverV617F signaling revealed that STAT5 formed a complex with PKAc  that we observed. It is also very possible that binding of the FECH substrate to the PKA kinase induces conformational changes that would render the target motif more favorable to modification. This mechanism has previously been proposed as a means to prevent ‘promiscuous’ phosphorylation  was kindly provided by Arthur Skoultchi . Parental human UT7 erythroid cells (RRID:CVCL_5202) were kindly provided by Meredith S. Irwin . Human erythroleukemia (HEL) cells (RRID:CVCL_2481) were kindly provided by Ann Mullally . All cells are mycoplasma negative and the International Cell Line Authentication Committee lists none of them as a commonly misidentified cell line. The identities of all cells were confirmed by their labs of origin since none of them are commercially available and have no standard authentication reference sample.The DS19 murine erythroleukemia (MEL) subclone  for 60 min and stained with o-dianisidine as described below. Inhibition with 20 µM H-98 , 100 nM PKI (14-22) (ThermoFisher Scientific), or 30 nM bis-indolylmaleimide II (Tocris Bioscience) were performed by pre-treating the cells for 30 min prior to PKA pharmacologic activation.DS19 MEL and primary murine erythroid progenitors from E13.5 fetal liver were cultured and differentiated as previously described . DiffereBulk murine erythroid progenitors from adult spleen (splenocytes) were prepared and EPO-stimulated as previously described . After tHuman UT7 erythroid cells were cultured in αMEM  supplemented with 20% heat-inactivated fetal bovine serum  and 10 ng/mL GM-CSF . For stimulation experiments, UT7 cells were starved overnight without GM-CSF. The next morning, rhEPO (2 U/mL) was added for the indicated times prior to harvesting.Human erythroleukemia cells (HEL) were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum and treated with 1 µM Ruxolitinib, which was a kind gift of Dr. Ann Mullally  and added to the cells for the indicated times.Akap10-targeting and NT9 control shRNAs in the pLKO.1-puro vector were purchased from Sigma-Aldrich. MEL cells were electroporated and monoclonal populations expressing these shRNAs were isolated as previously described . Samples were then dounce homogenized and pelleted by spinning at 1000 g for 10 min at 4°C. The supernatant was separated and the leftover pellet was resuspended in 200 µL of MSHE buffer. All samples were centrifuged again at 1000 g for 10 min at 4°C. All supernatants from each undifferentiated and differentiating samples were collected and combined and centrifuged again at 1000 g for 10 min at 4°C. This supernatant was then transferred to a new tube and centrifuged at 8000 g for 20 min at 4°C. The resulting supernatant containing cytosolic proteins was transferred to another tube and flash frozen. The pellet was resuspended and washed twice more in 200 µL of MSHE buffer. The final pellet, containing mitochondrially enriched membrane fractions, was flash frozen until subjected to mass spectrometry analysis. For western analyses, the pellet was lysed in NP-40 lysis buffer. For ferrochelatase activity assays, the Mitochondrial Isolation Kit for Mammalian Cells (ThermoScientific) was used according to manufacturer’s instructions. The mitochondrial pellet was resuspended in 200 µL of reaction buffer and immediately used.1 × 10Intact mitochondria were isolated as described above without protease inhibitors and resuspended in MSHE buffer containing 5 mg/mL digitonin that was prepared fresh at 4% in water immediately before each experiment. Samples were incubated on ice for 15 min with vortexing at maximum setting every few minutes for 10 s intervals. After 15 min, samples were centrifuged at 10,000 g for 10 min at 4°C. The supernatant was transferred to a second tube and the leftover pellet was re-extracted twice more with 80 µL of MSHE containing 5 mg/mL digitonin, leaving a final pellet enriched for MPs. The supernatant from the second and third extractions were discarded. The supernatant from the first extraction was then centrifuged at 144,000 g for 1 hr at 4°C. The supernatant containing the IMS was removed and kept in a separate tube. The pellet contained the OM fraction. Proteinase K protection assays were performed as previously described .Protein was extracted from purified mitochondria by dissolution in 8 M urea, 50 mM Tris pH 8.0, followed by probe sonication. Extracted protein was reduced and alkylated with dithiothreitol and iodoacetamide, respectively. Alkylated protein samples were first digested with endoproteinase LysC for 4 hr at ambient temperature with an enzyme to protein ratio of 200:1. Each sample was diluted with 50 mM Tris to 1.5 M urea and further digested with trypsin at an enzyme to protein ratio of 50:1, overnight and at ambient temperature. Peptides from each sample were desalted over a C18 solid phase extraction cartridge and dried down. Each sample was resuspended in 0.2 M triethylammonium bicarbonate pH 8.5, and labeled with tandem mass tag reagents (TMT) as previously described . Labeledm/z. Peptide precursors in the survey scan were sampled for ms/ms analysis by data dependent top 15 selection with dynamic exclusion turned on. Each peptide precursor selected for sampling was isolated in the ion trap, fragmented by higher energy collisional dissociation (HCD) at 35 NCE, followed by mass analysis of the fragments in the Orbitrap at resolving power 15,000 at 400 m/z.All nano UPLC separations were performed on a nanoAcquity system. From each fraction, approximately 2 µg of peptides was injected onto a 75 µm inner diameter, 30 cm long, nano column packed with 1.7 BEH C18 particles. The mobile phases were as follows: A) 0.2% formic acid and B) 100% acetonitrile with 0.2% formic acid. Peptide were eluted with a gradient of increasing B from 0%30% over the course of 100 min, followed by a wash with 100% B and re-equilibration at 0% B. Eluting peptides were electrospray ionized and analyzed with an Orbitrap Elite mass spectrometer. The mass spectrometry analysis cycle was as follows. First a survey scan was performed with Orbitrap analysis at 60,000 resolving power at 400 2 fold change was calculated.All data analysis was performed in the COMPASS software suite . SpectraAkap10 (NM_019921.3) was performed as previously described with modifications  wild-type (5’-CATCGCCAAACGCCGAACC-3’) and mutant (5’-CATTGCGAAGCGACGTGCG-3’) oligos as described elsewhere  primer, 5’-GAAGGGCTCGCGGACTCG-3’; reverse-1 (R1) primer, 5’-CCCTGACAAAACCCTTGC-3’; and reverse-2 (R2) primer, 5’-CACTTGCAGTGTTTTGGGGTTT-3’.Genomic DNA was isolated from DS19 MEL cells according to manufacturer’s instructions . For screening Site-directed mutagenesis was performed using the Agilent QuikChange Lightning Multi Site-Directed Mutagensis Kit  according to the manufacturer’s instructions. The following mutagenesis primers were used: T116A, 5’-CATCGCCAAACGCCGAGCCCCCAAGATTCAAG-3’ and 5’-CTTGAATCTTGGGGGCTCGGCGTTTGGCGATG-3’; K113L, 5’-GCACCATTCATCGCCTTACGCCGAACCCCCAAG-3’ and 5’-CTTGGGGGTTCGGCGTAAGGCGATGAATGGTGC-3’; and R114L, 5’-CCATTCATCGCCAAACTCCGAACCCCCAAGATTC-3’ and 5’-GAATCTTGGGGGTTCGGAGTTTGGCGATGAATGG-3’.Recombinant His-tagged human FECH proteins were expressed and purified as previously described .The 2.0 Å structure of human FECH (PDB 2QD4) was visualized by PyMOL .32P]-ATP , the reaction was performed with an incubation time of 30 min instead of 10 min and scaled down to where 0.2 pmol of purified FECH and PKAc were used. Four-fold excess [γ-32P]-ATP was added. After 30 min, each reaction was immunoprecipitated with anti-FECH antibodies (see below). The incubation time for [γ-32P]-ATP labelling was three-times longer than for other assays to ensure maximal phosphorylation in vitro. The amount of radioactivity was quantified in a scintillation counter . 20% of the kinase reaction was subjected to western blot analysis. The stoichiometry of the kinase reaction was kept at 1:1. For kinase assays using eLife. Your revised article has been evaluated by Ivan Dikic (Senior editor), David Ginsberg (Reviewing editor and Reviewer #1), and two additional reviewers (Susan Taylor and John Scott).Thank you for resubmitting your work entitled "Erythropoietin signaling regulates heme biosynthesis" for further consideration at eLife could be considered. The independent comments of all three reviewers are appended below. Though multiple comments/suggestions are included, the key issues that must be satisfactorily addressed in a revised submission are points 2, 4, 5 and 8 from reviewer #2 and comments #4 and 5 from reviewer #3.All three reviewers agreed that the manuscript has been significantly improved. However, several key issues remain, that must be addressed satisfactorily, before acceptance for publication in Reviewer #1:This revised manuscript is significantly improved and considerable new data have been added to address the initial critiques.Reviewer #2:Chung, et al. "Erythropoietin signaling regulates heme biosynthesis"The authors have introduced a significant amount of new information to this revised manuscript and as a result both its significance and its impact have been greatly improved. While I would now recommend publication there are still some questions that the authors could address. Some are beyond the scope of this work but others should be answered.1) The discovery that AKAP10 is the AKAP the mechanism by which PKA is targeted to the mitochondria is compelling and very interesting. In addition to the recent Geiger paper, we found early on that there was an advantageous mutation in AKAP10 when one compared older populations vs. younger populations . There were two mutations but the one in the AKAP binding site specifically reduced the affinity for RIa. We speculated at the time that this had a cardioprotective effect, but this could also be relevant for your findings.2) There are some concerns about the in vitro phosphorylation in 3) In spite of the modest phosphorylated bands, the mutants look good and it is good that you did both the Thr116Ala mutant as well as the K mutants. However, did you try the phosphomimetic mutants (D/E)? This is an important experiment and should give an opposite phenotype to the Ala mutant?4) The stoichiometry is also important for the kinetic assays. The Vmax effect seems to be rather modest and if the protein is not stoichiometrically phosphorylated then you would see a more modest effect. I suspect that the effect on activity would be even more pronounced if the protein were fully phosphorylated.5) One last question that relates to the kinase assays that you used. What was the amount of PKA that you used relative to the amount of FECH? If it was sub-stoichiometric then you may have a situation where there is a single turnover but the substrate stays bound until some other signal releases it. If this were the case you would need to add near stoichiometric amounts of kinase and substrate to see full phosphorylation. Just a thought.6) I do not think it is a concern that the motif is in a helix as this is a surface helix and it is likely that it is quite dynamic. I would guess that the helix propensity of this helix is not great especially because it has two prolines in it. This is unusual for a helix where Pro is typically a helix breaker. There was an earlier example of this with a structure of PRK tethered to its substrate eIG2alpha . In this case the helix in the substrate became disordered near the active site when the protein was tethered to the kinase. Sicheri suggests in a later paper a putative mechanism whereby the helix protects against phosphorylation until the substrate binds to the kinase.7) Although it would not be necessary for this paper, I think that the helix could actually provide a good mechanism for docking onto the C-subunit using the groove that the PKI peptide uses. A peptide array might indicate that there were recognition motifs beyond the immediate site of phosphorylation. I would guess that the FI motif could be important for docking onto this surface. You could test this with an FI/AA mutant.8) The pull down assays showing the C-subunit binding to Tyr phosphorylated STAT5 is intriguing but still preliminary. The mechanism is unclear. Is the RIIb subunit no longer associated with this complex? ie Does the binding of STAT5 induce the dissociation of the holoenzyme or does it just cause a conformational change that opens up the active site making is accessible to FECH? Does the entire complex pull down so that it is poised for binding to FECH? Are additional PKA substrates phosphorylated other than FECH or are you looking at the events in a specific signaling complex? Is the C-subunit now released into the cytoplasm and no longer tethered to the mitochondria or is it still tethered to the mitochondria? Do you trap a complex with STAT5 and FECH if you use the Ala mutant of FECH? As you do not fully dissociate the complex even in the presence of cAMP, which is consistent with the very early Johnson results, which you reference, and also with earlier mass spec data and Scott's recent papers. The high forskolin experiments would force the holoenzyme apart but this is not physiological.9) One last point. AKAP10 has a PDZ motif at its C-terminus that immediately follow the A Kinase Binding motif. In kidney this interacts with PDZK1 but I do not know if other binding partners have been discovered. It could just be something to look for in your various data sets.In conclusion, this is a very nice story and the biological and physiological relevance is obviously very high. If some of the above questions can be satisfactorily addressed, publication is recommended.Reviewer #3:eLife on how erythroid transcriptional programming regulates post-translational mechanisms that influence heme metabolism. The quality of the western blot characterization of the phenomena in This is an extensively revised version of a manuscript that was previously submitted to 1) AKAP10 has two locations that could be pertinent to this work. It is found at the plasma membrane where it is believed to have some RGS function and on or in the mitochondria. The membrane-associated fraction of AKAP10 may play an important role and needs to be considered by the authors. A few comments in the discussion would suffice.2) In subsection “Mitochondrial PKA is localized to the outer mitochondrial membrane via AKAP10” it states that AKAP10 has long been recognized as a downstream target for the GATA1 erythroid lineage transcription factor. This is not immediately apparent in the Fujiwara et al., 2009 paper (text or supplementary figures). Please clarify.3) On the basis of other studies it is quite possible that deletion of AKAP10 results in the up-regulation of other mitochondrial anchoring proteins. This is an easily performed set of experiments. Also, the authors should incorporate analysis of WAVE1 they cite the paper that identified it at the mitochondria but never follow up on this.4) The experiments in 5) The data linking PKA and Stat5 seems rather weak. The authors need to deftly describe how biochemical effects at the mitochondria with PKA relate to changes in nuclear CREB phosphorylation. Especially since Cam Kinases, Mist1 & Mist 2 and Akt are just as potent modulators of Ser 133 on CREB.eLife. Your revised article has been favorably evaluated by Ivan Dikic (Senior editor), a Reviewing editor, and two reviewers.Thank you for resubmitting your work entitled "Erythropoietin signaling regulates heme biosynthesis" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) All reviewers were concerned about the quality of 2) Given that the sample is only 10% phosphorylated, interpretation of the kinetic data in 3) If possible, the manuscript would be strengthened by including a native gel for the in vitro phosphorylated protein, looking for a gel shift to confirm the quantification reported. Also, would it be possible to separate the phospho protein by ion exchange chromatography? [Editors’ note: the author responses to the first round of peer review follow.]All three reviewers agreed that this is an intriguing and well written manuscript that links regulation of heme biosynthesis to Epo activation of JAK2 and PKA signaling in the mitochondria. Specifically the authors show first using an unbiased MS analysis of mitochondrial proteins that PKA isoforms are up-regulated in the mitochondria of differentiated MEL cells. They then go on to show that ferrochetalase (FECH) has a PKA phosphorylation site and that this site is selectively phosphorylated in response to high levels of forskolin and cAMP but down regulated in the presence of PKA inhibitors. They then go on to link the increased PKA signaling to EPO stimulation of JAK2. Because the mechanism for regulation of heme biosynthesis at the metabolic level in contrast to the gene transcriptional regulation is poorly understood, this is a potentially significant advance. However, the biochemical and mechanistic evidence, including the overall elucidation of PKA signaling, is rather superficial at this point, and would require considerable additional data to justify the sweeping conclusions that are made. Specific comments/suggestions are appended at the end of this letter.On behalf of all the authors, I want to thank you for your efforts in evaluating our work. We are very encouraged by the positive tone of the reviews and the in-depth analysis provided by everyone is reflective of the broad interest and provocative nature our work. Your insightful feedback has propelled us to explore new concepts. Based upon our new data, we have revised our original conclusions and developed a new unifying model where transcriptional and post-translational mechanisms work in concert to regulate heme metabolism. These new additional results have significantly strengthened our revised manuscript, which is now composed of 7 main figures instead of 4 in the original submission. In particular, our new results include:1) in vivo evidence using an animal model that this EPO-PKA signaling cascade is important in a physiologic setting.2) Biochemical and genetic evidence that FECH phosphorylation occurs on the outer mitochondrial membrane and not in the mitochondrial matrix.3) A mechanism of how PKA become localized, which links the erythroid transcriptional program with EPO signaling and also explains our observed PKA- isoform-specific effects.4) Extensive quantitative analysis including kinetic measurements of enzymatic activity of phosphorylated FECH.5) Direct evidence of how erythropoietin signaling regulates PKA activity.eLife, and respectfully, ask that the Editorial Office and the reviewers give our revised work consideration.Our manuscript uncovers a novel signal transduction pathway that couples cell differentiation with heme metabolism – two fundamental cellular processes that were, previously, thought to occur independently. All reviewers agreed that our work is of significant interest and addresses this fundamental biological problem that, to date, has been unresolved. We strongly believe that our revised manuscript would strongly appeal to the broad readership of Specific comments/suggestions from the reviewers:1) Forskolin vs. cAMP. The dose of forskolin is very high and low doses show no effect although this figure  is looking at the total cell extract vs. purified mitochondria. If the authors wish to promote the idea that there are localized pools of cAMP, then the requirement for high doses of forskolin, a general cyclase activator, are counterintuitive. Isoproterenol is a much more specific activator of β adrenergic signaling and should also be tested to see if there is enhanced specificity. The difference between high vs. low levels of forskolin is disconcerting and not explained or well justified. Also it is not at all established or well accepted that high doses vs. low doses of forskolin will activate mitochondrial PKA. Mitochondrial soluble cyclase is predicted to be activated by bicarbonate, not by forskolin.Indeed, the contribution of distinct pools of cAMP and their corresponding activating signals are two significant controversies in the field. They are interrelated issues because the roles of various intracellular PKA pools have typically been extrapolated from pharmacologic studies. We thank the reviewer for bringing these key points to our attention. Over the past several months, we have worked hard to experimentally address these issues and we would like the opportunity to discuss how they address the reviewer’s queries.A significant proportion of our new results involve the identification and characterization of AKAP10 as a mitochondrial scaffold that nucleates PKA at the mitochondrial outer membrane. Much of what we know of AKAP10 is from studies carried out by Dr. Susan Taylor (reviewer #3) over the past 20 years . Recently, AKAP10 has garnered a great deal of attention since its association with human cardiac function and hematologic parameters in genome-wide association studies (GWAS) Our new work demonstrates that the majority of mitochondrial PKA in maturing red cells is actually tethered onto the outer mitochondrial membrane, which is dependent upon AKAP10. In the absence of AKAP10, we found a significant depletion of PKA in this compartment and reduced FECH phosphorylation and function , and 5. As it specifically relates to the Reviewer’s comments, we would like to begin by clarifying that our conclusion is not that cAMP is the sole activator of PKA during erythropoiesis. In fact, as we note later , suggesting that discrepancies between independent studies may reflect technical differences. Second, the methods in which suborganellular PKA activity is monitored has been debated, making it even more difficult to reconcile differences that are observed between studies . Thus, not only do our new data uncover a novel physiologic pathway, it also sheds light into this controversy from a different scientific vantage point.The reviewer also astutely noted that we use total cell extract instead of purified mitochondria to perform our assays, implying that the latter approach would be superior. In this case, we, respectfully, disagree. The use of purified mitochondria to study signaling pathways overlooks the dynamic nature of signal transduction. As previous studies have demonstrated, proteins  can be modified prior to mitochondrial transport . Furthermore, mitochondrial outer membrane PKA is a compartment with distinct biochemical properties that can be activated by cytosolic PKA agonists such as forskolin while having vastly different kinetics . Our characterization of the role of AKAP10 is actually consistent with previous work in which case, treatment of purified mitochondria with PKA pharmacologic agents would only lead to ambiguous negative results.eLife is an excellent venue to publish our revised manuscript as it deserves a broad audience. We hope that the reviewer is satisfied with our responses.In addition to including in vivo genetic and biochemical data on the role of AKAP10 into our revised manuscript, we have also included a much more thorough discussion of these topics. We are very excited that our work has elicited such a fantastic discussion, which underscores our belief that 2) In the Results section the authors state "We independently confirmed these results using immunoblotting analysis where we also found increased total PKA expression in maturing erythroid cells  It is stated in the Results that “Thr116 (humanFECH) . This reThis is an excellent point raised by the reviewer. It is true that access to the consensus motif is crucial for kinase phosphorylation. Our new results demonstrating the involvement of AKAP10 at the outer mitochondrial membrane corroborates this paradigm since the import of proteins across this membrane requires that proteins remain in an unfolded state . This would increase accessibility of this site to PKA. Notably, this mechanism of regulation by PKA has been observed previously . Outer membrane PKA phosphorylates BAX prior to its integration within the mitochondrial membrane . These points are of significant interest to basic cell signaling and we have now included a discussion of this in our revised manuscript.4) Phosphorylation of FECH on the mitochondria matrix. The mitochondrial MS proteomic data shows a global enrichment of PKA signaling proteins. Specifically, the data shows an increase in RIa, RIIa, RIIb, Ca and Cb although only two of these proteins, Ca and RIIb, are actually shown in the protein blots shown in In our revised manuscript, we have addressed these concerns. As the reviewer has noted, PKA refers to an enzyme complex consisting of regulatory and catalytic subunits. In humans, there are three catalytic subunits  and four regulatory subunits  . Mice do not have PKA-catalytic-g  and, thus, was not detected in our analysis using a murine model. In addition, the expression of RIa is restricted to neurons  and is similarly not detected in our analysis. We apologize to the reviewer for omitting these critical details, which we have now included in our revised manuscript. We have also performed a more thorough expression analysis of all PKA isoforms and included it in The points brought-up by the reviewer made us realize that we were premature in our conclusions. We performed a significant amount of new experiments that now demonstrate that FECH is phosphorylated by PKA at the outer mitochondrial membrane. To avoid being repetitive, we kindly ask the reviewer refer to our responses to points #1 and #3 regarding our new data and their implications on the PKA signaling.5) Isoform specificity. To demonstrate specificity in PKA signaling the authors need to rigorously show that one isoform, and not all three, is responsible for the phosphorylation of FECH in the mitochondria. shRNA experiments should be done to demonstrate isoform specificity. Targeting experiments can also be done. Will disruption of targeting abolish the activation of FECH? Several peptides that disrupt targeting are available. If so, can the authors discriminate between RI and RII targeting? Tools are available to carry out these experiments.We thank the reviewer for bringing up this point. Isoform specificity is very difficult to address in PKA signaling because of functional redundancy and compensatory responses both of which have been well documented . In fact, in mouse knockouts of individual PKA regulatory subunits compensation of the other isoforms is frequently found . In addition, loss-of-function shRNA experiments do not adequately discriminate between total and mitochondrial PKA effects as noted by the reviewer in point #8 below. Thus, we, respectfully, do not believe that the approach recommended by the reviewer will yield satisfactory and unambiguous scientific answers.We agree that aberrant targeting of PKA away from mitochondria is critical to deciphering a mitochondrial role for PKA. However, instead of using peptides, we have uncovered an adaptor protein – AKAP10 – that is a transcriptional target of GATA-1, the master regulatory erythroid transcription factor . Strikingly, inhibition of AKAP10 in erythroid cells as well as a zebrafish model resulted in reduced heme production and anemia . MoreoveIn our revised manuscript, we have included a much more thorough discussion on the interplay of the various PKA isoforms as well as included our analysis of the role of AKAP10 as a dual-specificity AKAP in heme biosynthesis. We sincerely hope that we have satisfactorily addressed the reviewer’s concerns.6) Mutations in PKA that lead to hematopoetic dysfunction. What are the specific mutations in PKA signaling that lead to hematopoietic dysfunction and can these also recapitulate the effects of FECH phosphorylation? These mutations will certainly also support the isoform specificity of PKA signaling.We thank the reviewer for asking these two critical questions. As the reviewer has noted, point #6 is very much related to point #5 above and, to avoid being repetitive, we kindly ask the reviewer to refer to point #5 for our response to isoform specificity.The most prevalent PKA mutations associated with hematologic symptoms are deletions of PRKAR1A . This is not to say that other isoforms do not have a role. We are aware that different PKA isoforms have different biochemical properties . However, as we noted in our response to point #5, compensatory responses has made it very difficult to address isoform specificity using classical genetic loss-of-function approaches in vivo. In addition, as noted in reviewer point #8 below, global inhibition of PKA isoforms (with RNAi or CRISPR), would be highly ineffective at delineating cytosolic and mitochondrial effects.In contrast, our characterization of AKAP10 addresses the issue previously raised by the reviewer in an elegant and biologically relevant fashion. AKAP10-dependent recruitment of PKA requires the regulatory subunits . Thus, loss of AKAP10 essentially mimics loss of PKAr specifically from mitochondria while leaving all other PKA signaling components intact. Strikingly, we found that AKAP10 deletion in erythroid cells resulted in diminished FECH phosphorylation and activity. Interestingly, recent GWAS studies have found that AKAP10 variants are associated with hematologic blood traits . We have included all these details in the expanded Discussion section of our revised manuscript.7) Dissociation of R- and C-subunits. While high levels of cAMP can promote dissociation of the PKA subunits in cells, growing evidence suggests that unleashing of the catalytic activity of the C-subunit does not require dissociation of the R and C-subunits. These experiments with whole cells shed little light on the mechanism that is invoked here for the phosphorylation of FECH in the mitochondria. The authors show only the general dissociation of RIIb under conditions that are far from physiological. They do not address specific PKA signaling at the mitochondria, much less in the matrix of the mitochondria. PKA activity reporters are also available. Is PKA initially activated in the cytoplasm as a result of EPO binding to JAK2?not in the mitochondrial matrix .We thank for the reviewer for raising these concerns, which has propelled us to explore new possibilities to explain our observations. Our new experiments demonstrate that activated PKA responsible for phosphorylating FECH is With regards to how EPO signaling activates PKA, our new results argue that PKA is activated directly through phosphorylated STAT5 in a cAMP-independent fashion. As we have discussed, early studies in red blood cells failed to detect changes in cAMP levels throughout red cell maturation . This is not to say that PKA has no role in erythropoiesis but rather it may be cAMP-independent. In support, exogenous cAMP promotes iron uptake in red cells by phospho-CREB dependent synergism with STAT5 . Thus, there is clearly cross talk between these two pathways that does not involve endogenous cAMP.not matrix. Rather, it is cytosolic, which also activates mitochondrial outer membrane PKA as previously shown by other groups .In our new We appreciate the enthusiasm demonstrated by the reviewer in our work. However, we believe that unraveling the kinetics behind PKA activation and whether dissociation between the regulatory and catalytic subunits is absolutely required in red cells is beyond the scope of the current manuscript. The goal of our work here is not to test every nuance of PKA signaling that has been previously published; follow-up studies are more appropriate to address all these issues. Instead, the key conceptual advance is that PKA is activated by EPO and heme metabolism is regulated by this mechanism. Lastly, we would like to emphasize that, akin to EPO signaling, iron uptake induced by cAMP does not result in iron overload . This is a key point because it suggests that PKA activation can not only trigger iron import but also coordinate iron assimilation into heme. Our new data, model, and in-depth discussion are now included in our revised work.8) Total cell differences vs. mitochondrial differences. The authors need to rigorously distinguish between total cell effects vs mitochondrial effects.Please see our responses to points #5 and #6.9) In vitro phosphorylation of FECH. These studies carried out with recombinant expressed His-tagged FECH are also somewhat superficial. The modeling with space filling models is inadequate. The authors should at least show the individual residues and their interactions, and each residue should be clearly labeled. The phosphorylation in We thank the reviewer for noting these technical issues and we apologize for not providing sufficient detail. We have now performed extensive in vitro analysis to examine enzyme kinetics . In addi10) Phosphoproteomics. There should be a phosphoproteomic analysis of the mitochondrial fractionations and of the global proteins to see to what extent PKA substrates in general are upregulated.We absolutely agree with the reviewer that further confirmation of PKA activation would greatly strengthen our work. This is why we are excited to report that we have addressed this in two ways. Our response to this reviewer concern consists of unpublished and ongoing work and we would kindly request that our response be held in strict confidence and not be released for public knowledge.https://ash.confex.com/ash/2016/webprogram/Paper93991.html). They are in the process of publishing their work and, thus, while we are excited by an independent validation of our model, we are also eager to publish our own work to capitalize on its novelty.First, using phospho-antibody-specific flow cytometry analysis, a colleague  has independently found that PKA substrates are hyper-phosphorylated in response to EPO treatment of CD34+ human hematopoietic progenitor cells. This was presented at the recent 58th American Society of Hematology Annual Meeting in San Diego, CA  and superimposed it onto gene expression databases that have been well annotated according to stages of erythroid differentiation . In p < 0.05). We are in the process of performing careful analysis on the relevance of the PKA/CREB transcriptional program in erythropoiesis the results of which are beyond the scope of the current manuscript. However, taken together, the biochemical analysis in our revised manuscript along with the analysis performed by Dr. Sankaran in human CD34+ cells and the preliminary gene expression bioinformatics analysis, we believe that we have made a strong and convincing argument that PKA is important in red blood cell development.At every stage of erythroid development, we found that this “CREB gene signature” was indeed up-regulated in red blood cells (11) PKC phosphorylation. If PKC is known to be an activator of FECH, then the authors should use this as a control to make sure that PKC cannot phosphorylate Thr116, which actually looks like a good PKC site, and also to demonstrate the robustness of activation and phosphorylation by PKC. Is the effect comparable for PKA?bis-indolylmaleimide II  was unable to block PKA-dependent heme production suggesting that they function in two independent and parallel pathways. In addition, we have performed additional experiments using pharmacologic PKA agents on erythroid cells harboring the Thr115Ala FECH mutation (We thank the reviewer for suggesting using PKC as a specificity control. We are excited to say that we have now performed the suggested experiments . PKC inhmutation . When we12) Steps from JAK2 activation to FECH phosphorylation. There are many steps going from JAK2 activation at the plasma membrane to FECH phosphorylation in the matrix of the mitochondria, and many of these steps could be regulated by cAMP including not only phosphorylation of key metabolic enzymes such as FECH but also proteins involved in translocation from the plasma membrane to the matrix of the mitochondria. How does the signal from JAK2 activation by EPO get transmitted to the mitochondria? This mechanism is completely unclear. CREB phosphorylation at S133 is also very weak and not very convincing in the gels shown in We thank the reviewer for their interest in this mechanism. We agree that it is a major question and have addressed it in our revised manuscript. To avoid being repetitive, we kindly request that the reviewer visit our response to point #7 in which we go into great detail explaining our new data.With regards to our blots in 13) FECH disease mutations. What are the FECH mutations that cause EPP and do they correlate with the PKA regulation that is being proposed? Have any EPP mutations been identified at or near the FECH Thr116 phosphorylation site?We thank the reviewer for asking these astute questions and have now included an appropriate discussion in our manuscript. In short, mutation of the phosphorylation site has yet been documented in EPP. However, this does not indicate that this pathway is not important in this disease for a number of reasons.GATA1  to the first identification of humans with GATA1 mutations . To date, the only FECH mutation that is consistently found in EPP is a hypomorphic allele that is inherited in combination with other mutations . The search for these other genetic lesions is an active area of study. In fact, the characterization of an X-linked, dominant EPP-like disease that is associated with C-terminal mutations in ALAS2 highlights the emerging molecular and genetic heterogeneity of EPP .First, EPP is a rare disorder and because of its low prevalence, one of the major hurdles in understanding the genetics of rare diseases is finding a comprehensive genotype- phenotype correlation. For example, it took 11 years from the initial reports of FECH mutations must only occur in certain combinations in order for EPP to develop.Second, complete loss of FECH function has never been found in EPP and one very likely explanation is that severe FECH deficiencies are non-viable . This would further complicate any attempts at finding genotype- phenotype correlations since FECH gene is not mutated . We have included an appropriate discussion covering this topic in our revised work and hope that the reviewer finds it satisfactory.We are actively engaged in efforts to collect more clinical data from EPP patients to see if the FECH Thr116 site or the surrounding motif is mutated in EPP. In addition, we are also examining whether regulators of the EPO/PKA pathway can account for the 4% of EPP cases in which the [Editors' note: the author responses to the re-review follow.]Reviewer #2:Chung, et al. "Erythropoietin signaling regulates heme biosynthesis"The authors have introduced a significant amount of new information to this revised manuscript and as a result both its significance and its impact have been greatly improved. While I would now recommend publication there are still some questions that the authors could address. Some are beyond the scope of this work but others should be answered.1) The discovery that AKAP10 is the AKAP the mechanism by which PKA is targeted to the mitochondria is compelling and very interesting. In addition to the recent Geiger paper, we found early on that there was an advantageous mutation in AKAP10 when one compared older populations vs. younger populations . There were two mutations but the one in the AKAP binding site specifically reduced the affinity for RIa. We speculated at the time that this had a cardioprotective effect, but this could also be relevant for your findings.We thank the reviewer for bringing this study to our attention and apologize for having missed it throughout the course of preparing our manuscript. It would be interesting to see if AKAP10 mutations in older populations correlate with hematologic parameters. We are also actively searching GWAS databases to determine if there is a link between AKAP10 mutations and porphyrias (EPP in particular). We have included the Kammerer et al., 2003 reference into our discussion.2) There are some concerns about the in vitro phosphorylation in We thank the reviewer for suggesting this experiment. Indeed, we have used [γ-32P]-ATP labeling and determined that in vitro, the amount of phosphorylated FECH is 9.75 ± 3.18%. This result has now been included in the main text with an accompanying description of the protocol in the Materials and methods section.3) In spite of the modest phosphorylated bands, the mutants look good and it is good that you did both the Thr116Ala mutant as well as the K mutants. However, did you try the phosphomimetic mutants (D/E)? This is an important experiment and should give an opposite phenotype to the Ala mutant?This is an excellent suggestion. We have actually tried this experiment with the phosphomimetic mutant using in vitropurified proteins. Unfortunately, we were unable to detect any significant change in enzyme activity. This has lead us to hypothesize that the phospho-group somehow interacts with the porphyrin/iron substrates in a manner that we still do not fully understand and will require a great deal of additional structural biology experiments to decipher, which are beyond the time line for the revision.4) The stoichiometry is also important for the kinetic assays. The Vmax effect seems to be rather modest and if the protein is not stoichiometrically phosphorylated then you would see a more modest effect. I suspect that the effect on activity would be even more pronounced if the protein were fully phosphorylated.We agree that stoichiometry is an important consideration when interpreting the results of our kinetic assays. As we discussed in our responses to points 2 and 3, not all FECH protein is phosphorylated in vitroand, as a complementary approach, we have tried to examine the activity of His-tagged phospho-mimetic mutant FECH proteins without much success. Future work is needed to better understand the structural changes that occur with FECH upon modification that would provide important clues as to the relationship between stoichiometry and effect on catalysis.5) One last question that relates to the kinase assays that you used. What was the amount of PKA that you used relative to the amount of FECH? If it was sub-stoichiometric then you may have a situation where there is a single turnover but the substrate stays bound until some other signal releases it. If this were the case you would need to add near stoichiometric amounts of kinase and substrate to see full phosphorylation. Just a thought.For our in vitroassays, the ratio of PKA to FECH is approximately 1:1. We had never considered that PKA may remain bound to FECH following the modification and thank the reviewer for bringing this to our attention. We have also included a small note regarding the stoichiometry of the in vitrokinase reaction in our Materials and methods section as well as the corresponding Figure Legends.6) I do not think it is a concern that the motif is in a helix as this is a surface helix and it is likely that it is quite dynamic. I would guess that the helix propensity of this helix is not great especially because it has two prolines in it. This is unusual for a helix where Pro is typically a helix breaker. There was an earlier example of this with a structure of PRK tethered to its substrate eIG2alpha . In this case the helix in the substrate became disordered near the active site when the protein was tethered to the kinase. Sicheri suggests in a later paper a putative mechanism whereby the helix protects against phosphorylation until the substrate binds to the kinase.We thank the reviewer for the insightful feedback and providing us with their expertise. Indeed, we have considered that this phosphorylation event may be highly dynamic, which would explain its location within a helix. Furthermore, we have also wondered how stable this helical structure would remain throughout enzyme catalysis (ie. “helix propensity” as referred to by the reviewer). We have included these details and accompanying references  into our discussion to further strengthen our manuscript.7) Although it would not be necessary for this paper, I think that the helix could actually provide a good mechanism for docking onto the C-subunit using the groove that the PKI peptide uses. A peptide array might indicate that there were recognition motifs beyond the immediate site of phosphorylation. I would guess that the FI motif could be important for docking onto this surface. You could test this with an FI/AA mutant.We thank the reviewer for their interest in our work. The role of PKA signaling in hematopoiesis is an emerging concept , and we are actively exploring avenues to pursue for follow-up studies.8) The pull down assays showing the C-subunit binding to Tyr phosphorylated STAT5 is intriguing but still preliminary. The mechanism is unclear. Is the RIIb subunit no longer associated with this complex? ie Does the binding of STAT5 induce the dissociation of the holoenzyme or does it just cause a conformational change that opens up the active site making is accessible to FECH? Does the entire complex pull down so that it is poised for binding to FECH? Are additional PKA substrates phosphorylated other than FECH or are you looking at the events in a specific signaling complex? Is the C-subunit now released into the cytoplasm and no longer tethered to the mitochondria or is it still tethered to the mitochondria? Do you trap a complex with STAT5 and FECH if you use the Ala mutant of FECH? As you do not fully dissociate the complex even in the presence of cAMP, which is consistent with the very early Johnson results, which you reference, and also with earlier mass spec data and Scott's recent papers. The high forskolin experiments would force the holoenzyme apart but this is not physiological.We thank the reviewer for their enthusiasm and are excited to announce that we have performed additional experiments in an effort to further address the reviewer’s questions. Some of these concerns are also related to concerns raised by reviewer 3 point 5.First, we performed additional co-immunoprecipitation experiments using antibodies directed against PKAr subunits. The goal was to examine whether phosphorylated STAT5 formed a macromolecular complex with PKAc and PKAr. In contrast to The second experiment we performed is to use mitochondrial lysates for coimmunoprecipitations. This would help address the issue of whether the phospho-STAT5/PKAc complex is tethered to the mitochondria. Our results show that we could not detect a stable complex between phospho-STAT5 and PKAc using isolated mitochondria new . This reWhether dissociation of the PKAr/PKAc complex is absolutely required for kinase activity is a difficult issue to address in our system. We were never able to completely disrupt the PKAr/PKAc complex with EPO stimulation see . Thus, i9) One last point. AKAP10 has a PDZ motif at its C-terminus that immediately follow the A Kinase Binding motif. In kidney this interacts with PDZK1 but I do not know if other binding partners have been discovered. It could just be something to look for in your various data sets.We thank the reviewer for their enthusiasm for our proteomics screen. We are in the process of validating additional hits that would be of biological significance.Reviewer #3:This is an extensively revised version of a manuscript that was previously submitted to eLife on how erythroid transcriptional programming regulates post-translational mechanisms that influence heme metabolism. The quality of the western blot characterization of the phenomena in 1) AKAP10 has two locations that could be pertinent to this work. It is found at the plasma membrane where it is believed to have some RGS function and on or in the mitochondria. The membrane-associated fraction of AKAP10 may play an important role and needs to be considered by the authors. A few comments in the discussion would suffice.We thank the reviewer for this comment that raises an important issue regarding isoform specificity. When AKAP10 was first identified by Huang, LJ, et al., PNAS, 1997, they found multiple isoforms with different molecular weights. These isoforms may possess different N-terminal signal peptides which would direct AKAP10 to various subcellular compartments. This is a main reason why we designed our CRISPR deletion strategy to disrupt the N-terminal mitochondrial-targeting motif – to specifically address the mitochondrial role of AKAP10 in the event that multiple isoforms were expressed. Certainly, we were unable to discriminate between various isoforms in our zebrafish knockdown experiments. We have also considered the possibility that human AKAP10 mutations may have non-mitochondrial effects that would account for the observed cardiovascular phenotype (please see point 1 by reviewer #2). To address these reviewer concerns, we have, now, included a discussion in the main text of our revised manuscript as well as the corresponding Figure Legend to explicitly state that our CRISPR deletion strategy eliminates the N-terminal motif.2) In subsection “Mitochondrial PKA is localized to the outer mitochondrial membrane via AKAP10” it states that AKAP10 has long been recognized as a downstream target for the GATA1 erythroid lineage transcription factor. This is not immediately apparent in the Fujiwara et al. 2009 Mol Cell paper (text or supplementary figures). Please clarify.AKAP10 mRNA was found to be upregulated by Fujiwara, T, et al., Mol Cell, 2009 in Supplementary Table 3. There were two probe sets for AKAP10 – one found a significant induction while the other did not. Based on this, and the lack of an obvious GATA-1 binding site in the immediately upstream AKAP10 promoter, very little follow-up work was performed. We would also like to note that expression profiling of maturing erythroid cells independently performed by Zhang, Socolovsky, Gross and Lodish, 2003, also found a similar induction of AKAP10 transcript as murine red blood cells differentiated . We have now included this second reference to further support our results. In addition, we have also re-phrased that sentence to emphasize that these previous reports were high-throughput studies that require detailed analysis of raw data sets.We apologize for the confusion. 3) On the basis of other studies it is quite possible that deletion of AKAP10 results in the up-regulation of other mitochondrial anchoring proteins. This is an easily performed set of experiments. Also, the authors should incorporate analysis of WAVE1 they cite the paper that identified it at the mitochondria but never follow up on this.akap10 in zebrafish. To date, we have failed to get germline transmission of a null allele despite evidence of a deletion event in the parental generation. As the reviewer noted, we hypothesize that there are compensatory responses (such as upregulation of other AKAPs), in murine models not found in zebrafish. While our efforts, so far, have been met with disappointment, we believe that it may actually pave the way for the use of zebrafish to study AKAP mechanisms since adaptive responses may be different than currently used cell culture and murine model systems. Our analysis is only preliminary and beyond the scope of the current work.We thank the reviewer for bringing this to our attention since it has important implications on our future work. Over the past two years, we have tried exhaustively to use CRISPR genome editing to delete 4) The experiments in We apologize for our oversight. We have now performed additional sets of experiments to address this issue . It has 5) The data linking PKA and Stat5 seems rather weak. The authors need to deftly describe how biochemical effects at the mitochondria with PKA relate to changes in nuclear CREB phosphorylation. Especially since Cam Kinases, Mist1 & Mist 2 and Akt are just as potent modulators of Ser 133 on CREB.This is a similar concern raised by reviewer 2 point 8. In our work, we use Ser133 CREB phosphorylation as a simply marker for PKA activity. We understand that other kinases have been reported to also phosphorylate the same site and our work with the PKA inhibitor, 14-22 , provideAs the reviewer has noted, previous work suggests that CREB is phosphorylated in the nucleus after passive diffusion of active PKAc into the nucleus . This suggests that unlike the mitochondrial matrix that appears to have a distinct pool of PKA , the interplay between cytosolic PKA activation and nuclear CREB responses is much more indiscriminate. Our new data shows that the phospho- STAT5/PKAc complex can only be detected in the cytosolic, non-mitochondrial compartment , indicatThe manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) All reviewers were concerned about the quality of We have now elaborated on an explanation for the low level of phosphorylation in vitroin our Discussion section. We increased incubation time from 10 minutes to 30 minutes for [γ-32P]-ATP labeling experiments to help reach maximal phosphorylation and have now included a note regarding this procedural modification in both the main text as well as the “Materials and methods” section.We have also repeated the experiment for Overall, we have independently repeated these results several times and using complimentary approaches. We strongly believe that we have provided compelling evidence that FECH is a PKA target.2) Given that the sample is only 10% phosphorylated, interpretation of the kinetic data in All enzyme kinetic constants are a function of temperature. Our kinetic assays were performed at room temperature while single time-point experiments were carried out at 37oC. This is a major caveat that limits the interpretation of in vitroresults. It is not technically feasible to perform a radioactive labeling assay at the desired 30-second intervals at 37oC. We have included a brief note regarding this caveat in our revised work (Results section).Although the extent of Fech phosphorylation at the non-physiological room temperature was ~10%, this apparent modest reaction is at the terminus of a multi-step signalling cascade starting with Epo stimulation. Therefore, one would expect an “amplification effect” which would greatly magnify the apparent small changes in direct phosphorylation. Examples of such amplification effect would be the coagulation cascade or the complement system.3) If possible, the manuscript would be strengthened by including a native gel for the in vitro phosphorylated protein, looking for a gel shift to confirm the quantification reported. Also, would it be possible to separate the phospho protein by ion exchange chromatography?E. coli . If we are successful, we will be able to not only perform more detailed enzymatic assays but also obtain more structural data regarding how phosphorylation may influence enzymatic activity. These efforts would require more in-depth analysis that are, however, beyond the scope of the current manuscript and beyond the reasonable, allocated time frame for a revision.We thank the reviewers for their interest in our work. Actually, we are trying to develop a system where native phosphoproteins are produced by engineered


Escherichia coli as a fusion with maltose binding protein (MBP), and can be purified to homogeneity after proteolytic removal of MBP by affinity and size-exclusion chromatography. The purified protein can be fully activated in vitro by phosphorylation with protein kinases ERK2 and PDK1. Compared to full-length RSK2 purified from insect host cells, the bacterially expressed and phosphorylated murine RSK2 shows the same levels of catalytic activity after phosphorylation, and sensitivity to inhibition by RSK-specific inhibitor SL0101. Interestingly, we detect low levels of phosphorylation in the nascent RSK2 on Ser386, owing to autocatalysis by the C-terminal domain, independent of ERK. This observation has implications for in vivo signaling, as it suggests that full activation of RSK2 by PDK1 alone is possible, circumventing at least in some cases the requirement for ERK.Ribosomal S6 kinases (RSK) play important roles in cell signaling through the mitogen-activated protein kinase (MAPK) pathway. Each of the four RSK isoforms (RSK1-4) is a single polypeptide chain containing two kinase domains connected by a linker sequence with regulatory phosphorylation sites. Here, we demonstrate that full-length RSK2—which is implicated in several types of cancer, and which is linked to the genetic Coffin-Lowry syndrome—can be overexpressed with high yields in  There are two catalytic domains: the N-terminal kinase domain (NTKD), which belongs to the AGC family and which is the biologically active module that phosphorylates downstream protein targets; and the C-terminal kinase domain (CTKD), with homology to the calmodulin-dependent family [The four isoforms of the ribosomal S6 p90 protein kinase RSK1-4), along with the two closely related isoforms of the mitogen- and stress-activated protein kinase MSK1-2), constitute a unique family of Ser/Thr kinases, which are made up of single polypeptide chains harboring two Ser/Thr kinase catalytic domains in tandem -4, along. All the, constitt family , 9. The Recently, there has been a surge in interest in the molecular physiology and inhibitor design for the RSK kinases, and particularly for RSK2. This is because the level of RSK2 expression and phosphorylation is significantly higher in a subset of MAPK driven cancer cell lines as compared to non-cancer controls, and RSK2 is therefore considered to be a viable cancer drug target –13, specRSK2 is also involved in a hematopoietic transformation: when compared to the wild type, knockout mice lacking RSK2 showed much higher survival rate upon induction of myeloma by transplantation of oncogenic bone marrow . Similarvia phosphorylation of the Na+/H+ exchanger isoform 1 both in the heart and in brain [+/H+ exchange activity in vascular smooth muscle found in hypertension, based on the ability of angiotensin II to stimulate RSK in an ERK and Ca2+-dependent fashion and activate this exchanger [Cancer is not the only pathological state in which RSK kinases play a part. Mutations in the gene coding for RSK2 have been associated with the Coffin-Lowry Syndrome . Anotherin brain –29. RSKsxchanger . Thus, sxchanger –35 and ixchanger .E. coli. However, these studies were not able to reveal details of any conformational changes that in the full-length protein would be associated with the complex phosphorylation cascade and activation process. The full-length RSK kinases are typically overexpressed in eukaryotic systems, often in insect cells, with low yield and at considerable cost. Moreover, these recombinant proteins are already phosphorylated in situ and active. Although expression of full-length, His-tagged RSK2 in E. coli has been reported, little or no information has been provided about purification procedures, yield, purity or specific activity [E. coli, utilizing MBP as a fusion protein to boost expression yield and a His6-tag to facilitate purification. Using this recombinant protein, mass spectrometry and western blots, we were able to confirm the canonical activation mechanism, although we discovered that the CTKD of RSK2 is able to phosphorylate Ser386 in the absence of ERK1/2 , thus making it possible for PDK1 to activate NTKD independently of ERK1/2. Ser386 is the only site partly phosphorylated in the nascent enzyme, and it can be readily and efficiently dephosphorylated using Mn2+ dependent Lambda Protein Phosphatase (Lambda PP).Biophysical studies of the RSK kinases and structure-based drug discovery have been limited by the difficulties in the preparation of homogeneous and pure samples of recombinant protein. Structural studies focused on the isolated NTKD and CTKD domains, which can be produced in activity , 37. HerRabbit polyclonal antibodies raised against pSer227, pThr365/pSer369, and pThr577 of human RSK2 were purchased from Santa Cruz Biotechnology Inc. Rabbit monoclonal antibodies produced against pSer386 of human RSK-2 were purchased from Cell Signaling Technology. RSK-2 (E-1) mouse monoclonal IgG were purchased also from Santa Cruz Biotechnology Inc.Fluorophore-labeled secondary antibodies Alexa Fluor® 680 goat anti-rabbit IgG (H+L) and IRDye®800CW Goat anti-Mouse were purchased respectively from Life Technologies™ and LI-COR. Lambda Protein Phosphatase was procured from New England BioLabs® Inc. RSK2 kinase from Sf9 cells and ADP-Glo kinase assay were purchased from Promega. Active PDK1 was purchased from EMD Millipore and active ERK2 was expressed and purified as described elsewhere .AY083469.1) was amplified from mouse brain cDNA prepared in our laboratory  and cloned into pMBPHisParallel2 and pHisParallel2 vectors [600~4.0. Thereafter the temperature was lowered to 16°C, expression was induced by the addition of isopropyl β-d-1-thiogalactopyranoside to 0.3 mM after which the cultures were grown for a further 16 hrs; cells were harvested by centrifugation and the cell pellets frozen at −80°C. For purification, cell pellets were thawed, re-suspended in 100 ml of lysis buffer , and lysed with EmulsiFlex-C3 homogenizer. Polyethylene imine was added to the lysate to a final concentration of 0.2% and the lysate was centrifuged at 80,000xg for 35 min. Clarified lysate was loaded onto 5 ml Ni-NTA column , washed with lysis buffer without inhibitors and benzonase and eluted with a linear gradient of imidazole. Fractions containing RSK2 were pooled together and applied on a 4 ml gravity-flow amylose column, which was subsequently washed with Buffer A . Proteins were eluted into the same buffer containing 10 mM maltose and immediately applied onto a 3 ml gravity-flow Ni-NTA column. The column was washed with 100 ml of Buffer A to remove maltose and bound proteins were eluted with Buffer A containing 250 mM imidazole. To cut off the MBP-(His)6 tag TEV protease has been added to a final concentration of 0.05 mg/ml and samples were dialyzed overnight against buffer A. To separate the MBP-(His)6 tag protein samples were passed through the 4 ml gravity-flow amylose column and flow-through fractions were pooled together, concentrated, and purified on Sephadex 200 size exclusion column equilibrated with Buffer A.Full-length mouse RSK2  in 20 mM Tris buffer containing 500 mM NaCl, 10 mM of MgCl2, and 200 μM of ATP at room temperature for 2 hrs. Aliquots of activation reaction were taken at different time points and analyzed for site-specific phosphorylation by immunoblot analysis. .Samples (~50 ng) of RSK2 were resolved on polyacrylamide gels, transferred to Immobilion R-FL polyvinylidine difluoride membrane (Millipore), which were then blocked with the Odyssey Blocking Buffer (TBS) LI-COR. The membranes were subjected to phospho-RSK2  and total RSK-2 antibodies diluted in blocking odyssey buffer (1:8000). Next the membranes were washed 3 times in TBS with 0.05% Tween 20. Primary antibodies were visualized using fluorophore-labeled secondary antibodies conjugated to either Alexa Fluor® 680 or IRDye®800CW, and scanned with an Odyssey infrared imaging system (LI-COR Biosciences).2). The enzymatic activity of RSK2 was determined by measuring the amount of ATP consumed in the reaction using ADP-Glo system (Life Technologies) and a luminescence plate reader . In some experiments SL0101 at concentrations ranging between 0.1 and 100 μM was added to the reaction.Activated RSK2 was assessed for enzymatic activity at room temperature using 0.2 mg/ml (160 μM) of S6 peptide and either 10 or 100 μM of ATP in a kinase buffer  in 50 mM ammonium bicarbonate on ice for 30 min. Any excess enzyme solution was removed and 20 μL 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in a 100 μL aliquot of 50% acetonitrile/5% formic acid. This extract was evaporated to 15 μL for MS analysis.The LC-MS system consisted of a ThermoFisher Velos Orbitrap ETD mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm x 75 μm id Phenomenex Jupiter 10 μm C18 reversed-phase capillary column. 5 μL of the extract was injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 μL/min over 1.3 hours. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the rapid switching capability of the instrument acquiring a full scan mass spectrum to determine peptide molecular weights followed by product ion spectra (20) to determine amino acid sequence in sequential scans. This mode of analysis produces approximately 40000 MS/MS spectra of ions ranging in abundance over several orders of magnitude. The data were analyzed by database searching using the Sequest search algorithm.m) of protein samples was determined using a fluorescence-based assay by monitoring the fluorescence intensity of SYPRO®Orange dye in the presence of the protein as a function of temperature. RSK2 at 0.2 mg/ml  was mixed with 1x SYPRO®Orange Protein Gel Stain (Life Technologies) in 50mM Tris pH.8.0, 500mM NaCl, 5mM 2-mercaptoethanol. The thermostability experiments were carried out in StepOnePlus™ Real-Time PCR System (Applied Biosystems) with a temperature gradient from 20 to 90°C in the reaction volume of 10 μl. The first derivative of the resultant curve was calculated in order to determine the melting temperature (Tm) for both nascent and activated RSK2. In separate experiments 40x stock solutions of the RSK inhibitors, BI-D1870 and SL0101 were prepared in ethylene glycol and added to RSK2 prior to thermal shift assay. Corresponding control experiments were carried out in the presence of 2.5% of ethylene glycol.The melting temperature (TDynamic light scattering (DLS) experiments were carried out at 10°C in a Dyna-Pro Temperature-controlled Microsampler (Wyatt Technology Corporation). RSK2 at ~0.7 mg/ml was analyzed in 50 mM Tris pH.8.0, 500 mM NaCl, 5mM 2-mercaptoethanol in the reaction volume of 25 μl. Samples were centrifuged for 10 minutes at 4°C and ~20000xg, immediately before measurements. Each sample was measured 20 times with 5-s acquisitions and the data were averaged. Polydispersity and hydrodynamic radius were calculated from fitting the autocorrelation data using the regularization method.The following RSK2 variants were generated, expressed and purified: S227A, T365A, S386A, T557A and K451A, T577A. All mutations were introduced using the QuickChange Site-Directed Mutagenesis Kit (Agilent Technologies) and confirmed by DNA sequencing. The respective protein variants were purified and activated as described above for the full-length wild type protein.E. coli was exposed to 400 U of lambda phosphatase (New England BioLabs® Inc.) in 1x NEBuffer for PMP  supplemented with 1mM MnCl2. Incubation was performed at RT for 30 minutes and checked using Western Blotting analysis.RSK-2 protein (5μM) purified from 2. Protein phosphorylation was preserved by transferring the frozen tissue into 10% trichloroacetic acid in acetone at -80°C for freeze substitution, as described previously [g for 10 min, the samples were subjected to PAGE electrophoresis and Western blotting.All animal experiments were performed according to protocols reviewed and approved by the University of Virginia Institutional Animal Care and Use Committee and were in compliance with United States Public Health Service and Department of Agriculture guidelines for laboratory animal welfare. Five month old males C57Bl/J6 mice were euthanized and the abdominal aortae from the iliac branches to the region of the kidney were removed, placed in ice-cold HEPES-buffered Krebs solution, cleaned of their adventitia and equilibrated at 37°C in the HEPES-buffered Krebs solution for 1 hr. Aortas were incubated with MEK inhibitor U0126 (10μM) or PDK1 inhibitor GSK2334471 (10μM) or DMSO control for another hour followed by stimulation with TXA2 receptor mimetic U46619 (1μM) for 5 min and snap frozen in liquid Neviously . The triE. coli as a fusion protein with a His6-tag [E.coli with the N-terminal His6 tag and to purify this protein by affinity chromatography on Ni-NTA column using a linear gradient of imidazole for elution . The fusion protein eluted from the amylose column was re-applied onto Ni-NTA column, extensively washed to remove maltose bound to MBP, eluted with imidazole, digested with TEV protease, and passed through the amylose column again to remove MBP. The final purification step by size exclusion chromatography on Sephadex 200 yielded ~3 mg of purified RSK2 per liter of E.coli culture as determined by optical absorbance of the protein at 280 nm. When purified protein was analyzed by analytical size exclusion chromatography, it showed an asymmetric profile with a single peak roughly corresponding to a molecular weight of a monomeric RSK2 . Resolving RSK2 sample on a size exclusion column disrupts a large fraction of RSK2 dimers due to dilution, and shearing forces; nevertheless an asymmetric peak . We found that at 0.7 mg/ml the protein is predominantly a homodimer, with the hydrodynamic radius 5.0 nm of and a corresponding molecular weight of ~ 145 kDa . This was consistent with the protein being properly folded in solution. Furthermore, in the presence of BI-D1870 and SL0101, both of which are specific inhibitors of RSK2 [.To ascertain proper folding of the protein, we used the thermal stability assay (TSA) to detect temperature-induced, denaturing transition . We obse of RSK2 , 45, 46,in vitro phosphorylation at the canonical regulatory sites with activated recombinant ERK2 and commercial PDK1 kinases . The protein undergoes expected phosphorylation: Thr365, Thr577 and Ser386 are phosphorylated rapidly. Half-maximum phosphorylation levels were achieved within the first 5 minutes of the reaction. In contrast, Ser227 within the activation loop is phosphorylated at a lower rate. Interestingly, we observed low level phosphorylation on Ser386 even at 0 min . To ascertain if Ser386 is phosphorylated in the nascent protein, we carried out a mass spectrometry analysis of both nascent and phosphorylated samples. Several peptides containing Ser386 showed phosphorylation . While mass spectrometry is not a qualitative method, the result confirms that the western blot result is indeed due to partial phosphorylation, which we estimate at ~10%. In theory, phosphorylation could occur in E.coli due to endogenous kinases, but the fact that only Ser386 was phosphorylated hinted at the possibility that RSK2 may undergo autocatalytic phosphorylation. To verify this hypothesis, and to monitor possible interdependence between the phosphorylation events, we expressed and purified several variants of RSK2, i.e. T577A, K451A/T577A, T365A and S227A. Western blots using anti-pS386 antibodies  confirmed that the wild type protein is partly phosphorylated in its nascent state, and fully phosphorylated in the presence of ERK2 and PDK1. . As expected, the negative control (S386A) and the catalytically impaired K451A/T577A variants showed no phosphorylation . The T577A variant showed impaired phosphorylation on Ser386 in the presence of ERK2/PDK1, showing that the non-phosphorylated activation loop in CTKD impeded, but did not eliminate catalytic activity. The S227A variant was slightly less susceptible to phosphorylation on Ser386 compared to the wild type, and the T365A mutation had no effect.Next, we tested the susceptibility of recombinant RSK2 to in vivo there is a pool of Ser386-phosphorylated RSK2 that is activated by PDK1 alone; this pool may increase upon upregulation of RSK2 expression in pathological cases, e.g. in cancer cells.The autophosphorylation of RSK2 on Ser386 is not completely unexpected. It has been reported, that upon the removal of the C-terminal α-helix, i.e. from residue 697, the CTKD of RSK2 becomes constitutively active in the absence of ERK . Our worin vivo by various proteomics studies (http://www.phosphosite.org), but their functional significance is not known.Finally, mass spectrometry data for the phosphorylated sample revealed new target sites for ERK1/2 –Thr362 and Ser375. The phosphorylation of these sites has been previously reported .Because our objective was to obtain fully dephosphorylated, inactive protein, we assessed the possibility of enzymatic removal or residual phosphorylation of pSer386 using protein phosphatase λ (ppλ), a potent enzyme with broad activity towards phosphoserine, phosphothreonine and phosphotyrosine. Indeed, ppλ was able to dephosphorylate Ser386 fully, as judged by western blots . From these data we determined specific activity of RSK2 to be 258 nmol/min/mg, only slightly lower than 370 nmol/min/mg determined for fully activated human RSK2 purified from insect cells [50 value for SL0101 is estimated to be in the low μM range [50 ~ 8 μM . This is in a good agreement with earlier studies that used RSK2 overexpressed in insect cells.To complete the study, we assessed the enzymatic activity of the phosphorylated RSK2 using ADP-Glo™ kinase activity kit. This end-point assay allows a highly sensitive quantification of ATP consumed in the phosphorylation reaction. Using 0.2 mg/ml (0.16 mM) of S6 peptide as a substrate and 0.1 mM ATP we could easily detect conversion of ATP to ADP. Under conditions of low ng amounts of RSK2, the percentage of converted ATP was proportional to the amount of RSK2 in the reaction ct cells . The higct cells , 46, 48.ct cells , 49. The. Under resting conditions there is detectable phosphorylation of sites S227, T365, S386 and T577, that is significantly increased by U46619 stimulation in the absence but not presence of MEK inhibitors, in agreement with the scheme in Fig 1. In keeping with the above in vitro results on recombinant RSK, there was detectable phosphorylation at S386 even with inhibition of MEK/ERK/MAPK-induced phosphorylation under control as well as in the presence of U46619 supporting the concept of RSK susceptible to ERK-independent phosphorylation on Ser277. To ascertain that that the bands in our Western blots are indeed RSK phospho-Ser227 and phospho-Thr577 rather than non-specific labeling, we have also run an additional experiment in which we pre-absorbed the phospho-Ser227 and the phospho-Thr577 antibodies with phospho-Ser227 and phospho-Thr577 blocking peptides, which abolishes interaction  and can be specifically and selectively phosphorylated in vitro by ERK and PDK1. A fully dephosphorylated and inactive RSK2 can be easily prepared by dephosphorylation in vitro using Lambda Protein Phosphatase. In contrast, RSK2 from eukaryotic hosts, e.g. insect cells, is expected to be phosphorylated on multiple sites, due to basal activity of kinases of ERK/MAPK pathway, resulting in a potentially heterogeneous sample. When fully activated, bacterially-expressed RSK2 is suitable for functional studies including high throughput screening of chemical libraries for drug discovery. The autocatalytic properties of RSK2 suggest that the protein undergoes slow modification on Ser386, making it susceptible to ERK-independent phosphorylation on Ser277. This has potential implications for signal transduction in vivo.In conclusion, we report a new, efficient method for production of wild-type, full length inactive RSK2 kinase in S1 FileFigure A. Purification of His-tagged RSK2. (A) Optical absorption profile of His-tagged RSK2 eluted from Ni-NTA column and the analysis of collected fractions on Coomassie-stained SDS-PAGE gel. (B) Untagged RSK2 binds to Ni-NTA column. Proteins were separated by SDS-PAGE and stained by Coomassie brilliant blue. Pooled fractions after second purification of RSK2 on Ni-NTA were visualized before (lane 1) and after (lane 2) digestion with TEV protease to remove the His-tag. Untagged RSK2 was passed through the Ni-NTA column: lanes 3, 4 and 5 show proteins in unbound, wash and bound fractions respectively. The fact that untagged RSK2 binds to Ni-NTA was also verified by using the protein obtained after the removal of MBP-(His)6-tag (data not shown). Figure B. Analytical size-exclusion of purified RSK2. A 0.4 ml sample of RSK2 (0.6 mg/ml) was resolved on Superdex 200 column. Five 1 ml fractions with positive optical absorbance at 280 nm were collected and analyzed on Coomassie-stained polyacrylamide gel. Approximate positions of molecular weight standards are shown as arrows in the upper part of the figure. An asymmetric nature of the peak is likely to be due to a weak dimerization of RSK2. Figure C. Thermal stability of nascent and activated RSK2. Purifed non-phosphorylated (A) and in vitro phosphorylated (B) RSK2 were assessed for thermostability using thermofluor assay described in Materials and Methods. Figure D. Western Blot detection of phosphoprotein levels in wild-type and mutant RSK2 by ERK or PDK1. Samples of recombinant RSK2 were assayed for phosphorylation of Ser386 in the nascent form and after incubation with ERK2 or PDK1. The bottom panel shows the total amount of the protein identified by a generic antibody to RSK2 (see text for further details). The splitting of the bands is occasionally observed due to heterogeneity of the sample; each row shows a single membrane. Figure E. Demonstration of specificity of the antibody used for detection of phosphorylated RSK2 at the Ser227 site. A) Western blot of serial dilutions of mouse abdominal aorta lysate incubated with anti-pSer227 or anti-pThr577 RSK2 antibodies (upper panels) or with anti-pSer227 or anti-pThr577 RSK2 antibodies pre-absorbed with blocking peptides of the same sequences used to generate the antibodies (lower panels). Tubulin bands indicate protein loading for each lane. pSer227 RSK2 antibody, sc-12445R, blocking peptide, sc-12445P, pThr 577 RSK2 antibody, sc-16407R, blocking peptide, sc-16407P, Santa Cruz. B) Western blots of mouse aortic smooth muscle cells from Rsk2-/- and wild type mice stimulated with serum and pre-incubated with RSK2 inhibitors LJH685 (1μM) plus LJI308 (1μM) or the PDK1 inhibitor GSK2334471 (1 μM). Cells were starved overnight and treated with inhibitors for 1hr followed by stimulation with 5% fetal serum for 5 min. Following electrophoresis, membranes were reacted with anti-pSer227 RSK2 antibody or anti-tubulin antibody as a protein loading control. Note that serum stimulation increases phosphorylation at Ser227, indicative of RSK2 activation and that this is markedly suppressed in the presence of LJH685/LJI308 and GSK2334471 (upper panel) and that in the absence of RSK2 no band is detected (lower panel), indicative of the specificity of the anti-pSer227 RSK2 antibody used in this study.(DOCX)Click here for additional data file.