NOS1, on those brain alterations.Inhibition deficits have been suggested to be a core cognitive impairment in attention‐deficit/hyperactivity disorder (ADHD). Exploring imaging patterns and the potential genetic components associated with inhibition deficits would definitely promote our understanding of the neuropathological mechanism of ADHD. This study aims to investigate the multimodal imaging fusion features related to inhibition deficits in adults with ADHD (aADHD) and to make an exploratory analysis of the role of inhibition‐related gene, NOS1 ex1f‐VNTR genotypes, and inhibition.Specifically, multisite canonical correlation analysis with reference plus joint independent component analysis (MCCAR + jICA) was conducted to identify the joint co‐varying gray matter volume (GMV) and the functional connectivity (FC) features related to inhibition in 69 aADHD and 44 healthy controls. Then, mediation analysis was employed to detect the relationship among inhibition‐related imaging features, NOS1 ex1f‐VNTR on inhibition via FC component . In addition, the results indicated a significant indirect effect of GMV on the inhibition via FC component .Inhibition‐directed multimodal imaging fusion patterns of aADHD were reduced GMV and FC in inhibition network and increased GMV and FC in default mode network. The results showed a significant indirect effect of NOS1 ex1f‐VNTR genotypes and GMV might influence the inhibition through the mediation effect of the aforementioned FC (NOS1/GMV→FC→Inhibition).The findings suggested that reduced GMV and FC in inhibition network and increased GMV and FC in default mode network were jointly responsible for inhibition deficits in aADHD. Both the  NOS1 ex1f‐VNTR genotypes and GMV might influence the inhibition through the mediation effect of the aforementioned FC, which established a potential ‘gene‐brain structure/ function‐cognition’ pathway.The study suggested reduced gray matter volume (GMV) and functional connectivity (FC) in inhibition network and the increased GMV and FC in default mode network were jointly responsible for inhibition deficits in ADHD. Both the  Four aADHD and two controls were excluded because they failed to finish the MRI scan. Finally, 69 aADHD and 44 age‐ and sex‐matched healthy controls (HC) were included for analyses in this study. All aADHD were recruited from the outpatient clinics of Peking University Sixth Hospital and HC were recruited by advertisement. All participants underwent the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders‐Fourth Edition (DSM‐IV) Axis I Disorders (SCID‐I)All participants met the following criteria: (1) right‐handed; (2) no history of head trauma with a loss of consciousness; (3) no history of neurological disorders or other severe disease; (4) no current diagnosis of major depressive disorder, schizophrenia, clinically significant panic disorder, bipolar disorder, pervasive developmental disorders, or mental retardation; (5) no excessive head movements (>3.0 mm of translation or 3 degrees of rotation in any direction); and (6) a full‐scale intelligence quotient (FIQ) above 80 . Furthermore, participants with any history of psychiatric disorders were also excluded as healthy controls.never to 4 = always) were summed to yield inattentive, hyperactive/impulsive and total scores.ADHD core symptoms were evaluated by the ADHD Rating Scale‐IV (ADHD RS‐IV).Inhibit” factor score from Behavior Rating Inventory Executive Function—Adult Version (BRIEF‐A).never, 2 = sometimes, 3 = often) to evaluate their performance in daily life.Inhibition function was assessed by the “2.2MRI data were acquired on a GE Signa 3 T Horizon HDx system  at the Centre for Neuroimaging Sciences, Peking University Sixth Hospital. Participants were required to lie in the supine position and remain still and relaxed with their eyes closed but not falling asleep during the resting‐state fMRI scanning. The parameters on the GE scanner were repetition time (TR) = 2000 ms, echo time (TE) = 30 ms, flip angle = 90°, matrix = 64 × 64, field of view (FOV) = 220 mm × 220 mm, 43 axial slices, slice thickness = 3.2 mm, slice gap = 0 mm in resting‐state fMRI. The following parameters were in T1 on GE scanner: TR = 6.7 ms, TE = Min Full, flip angle = 8°, 180 slices, slice thickness = 1.0 mm, slice gap = 0 mm, FOV = 256 mm × 256 mm, and matrix = 256 × 256.2.3http://rfmri.org/DPARSF) according to standard preprocessing procedures. The GMV was analyzed by voxel based morphometry (VBM) and sMRI data were re‐oriented, segmented into white matter, gray matter, and cerebrospinal fluid using a unified segmentation approach,3, smoothing using a 4 mm Gaussian kernel, temporal band‐pass filtering (0.01 to 0.1 Hz), nuisance signal regression , and head motion scrubbing.The T1‐weighted structural MRI (sMRI) and resting‐state functional MRI (fMRI) data were analyzed by the Data Processing Assistant for Resting‐State fMRINOS1 ex1f‐VNTR were performed according to the methods described in Rief et al.+/25R+ indicated the carriers with two 25R, while 25R+/25R− as carriers with one 25R, and 25R−/25R− as carriers without 25R.The peripheral blood genotyping of the 2.4https://www.nitrc.org/projects/fit) and the methodology paper.t‐tests were further performed on mixing coefficients of each component for each modality. We aimed to investigate the joint independent component (IC), which is significantly correlated with inhibition, co‐varying among modalities and group discriminative. The brief flowchart of the supervised data fusion strategy could be found in Figure Multisite canonical correlation analysis with reference plus joint independent component analysis (MCCAR + jICA) is a supervised, goal‐directed model that enables a priori information  as a reference to guide multimodal data fusion. This model can maximize the inter‐modality covariation and precisely identify the co‐varying multimodal component closely related to reference information, which may not be detected by a blind (unsupervised) multimodal fusion approach. In this study, the GMV feature and FC feature were jointly analyzed by “MCCAR + jICA” in which “inhibition” was set as the reference to guide the joint decomposition of this two MRI features. For the code and more information about this method, please refer to the Fusion ICA Toolbox  was performed to explore the potential genetic effects of 2.6NOS1 ex1f‐VNTR genotypes, mediation analysis was further conducted to better delineate the gene‐sMRI/fMRI‐inhibition pathways.Once a joint GMV‐FC fusion pattern was identified to be group discriminative as well as associated with inhibition performance, and inhibition exhibited significant difference in different http://wwwafhayescom/public/process) in SPSS v.23 was used to conduct these analyses. Bootstrapping was used to estimate the confidence interval (CI) of sampling distributions and the indirect effects of mediators. In this study, bootstrapping sampled from the data 5000 times and provided 95% CI of indirect effects. If the 95% CI does not contain zero, it suggests that the mediating effect is significant at p < 0.05 level. In all mediation analysis, age and gender were controlled as covariates.PROCESS macro  were transformed into Z scores, visualized at |Z| > 3 as in Figure P = 2.3 × 10−4†; FC: p = 0.0019†; Figure r = 0.44, p = 2.8 × 10−5†; FC: r = 0.50, p = 1.3 × 10−6†; Figure The joint component was found to be both significantly group discriminating , hyperactive/impulsive , and total scores  after controlling for age, gender, and diagnosis. Increased IC_ref loadings in GMV were significantly correlated with higher inattentive  and marginally significantly correlated with total scores  after controlling for age, gender, and diagnosis.In addition to “inhibition” function, we also conducted correlation analyses for ADHD core symptoms and other multiple inhibition‐related cognitive domains such as emotional control, shifting, and self‐monitoring which constituted the BRI of BRIEF_A to further identify the reliability of the findings to some extent. As shown in Figure Both GMV loadings and FC loadings showed significantly positive correlation with self‐monitoring and BRI in ADHD patients after controlling for age, gender, and diagnosis. Moreover, GMV loadings were significantly correlated with emotional control  and ADHD diagnosis was found for inhibition function . When analyzing in ADHD and controls separately, significant association was only found in aADHD . As shown in Figure +/25R+ carriers was significant lower than that in 25R−/25R−  or 25R+/25R− carriers . No significant and/or marginally significant genetic effects of other NOS1 ex1f‐VNTR genotypes  on inhibition function were found.Marginally significant interaction of F = 2.97, p = 0.058). When analyzing separately, marginal significant association only existed for aADHD . Post hoc analysis showed that FC in 25R+/25R+ carriers was significant lower than that in NOS1 ex1f‐VNTR with 25R−/25R−  and 25R+/25R−   and marginally significantly correlated with FC  after controlling for age, gender, and diagnosis. Specifically, the correlations between inhibition and GMV loadings were significant in aADHD but not in HC  after controlling for age and gender. The correlations between FC and GMV loadings were marginally significant in aADHD but not in HC  after controlling for age and gender.Besides, GMV was significantly correlated with inhibition (3.4NOS1 ex1f‐VNTR (25R) genotypes, FC, GMV and inhibition in aADHD.Based on the above findings of “gene‐cognition,” “gene‐brain,” and “brain‐cognition” relationship, the following mediation analyses were conducted among the NOS1 ex1f‐VNTR (25R) genotypes to FC was significant , and the path from FC to inhibition was significant , but the path from the NOS1 ex1f‐VNTR (25R) genotypes to inhibition did not reach statistical significance. Figure NOS1 ex1f‐VNTR on the inhibition via FC component . However, the direct effect from NOS1 ex1f‐VNTR to inhibition after the addition of FC to the model was not significant , which suggested that NOS1 ex1f‐VNTR affected inhibition completely through the mediation effect of the aforementioned FC (NOS1→FC→Inhibition), not directly.In the mediation model of NOS1 ex1f‐VNTR (25R) genotypes, FC, and inhibition, the path from the p = 0.029], the path from FC to inhibition was significant , and the path from the GMV to inhibition was significant . Figure →FC→Inhibition).In the mediation model of GMV, FC, and inhibition, the path from the GMV to FC was significant [B = 0.36 (SE = 0.16), 4NOS1 ex1f‐VNTR genotypes in aADHD. First, we attempt to reveal how abnormally structural and functional neuroimaging features jointly contribute to inhibition impairment in aADHD. To the best of our knowledge, this is the first study to explore the inhibition‐guided multimodal brain imaging fusion in aADHD. Second, we explored how genetic variants and brain structural/functional alterations worked relatedly to cause inhibition deficit by constructing a potential “gene‐brain‐cognition” pathway. Our findings indicated that both the inhibition network and the DMN were involved in inhibition deficits for aADHD. In addition, the FC might mediate the relationship of the functional genetic variants, NOS1 ex1f‐VNTR and inhibition, as well as the relationship of the GMV and inhibition in aADHD.This study provided several new insights into the relationships between inhibition performance, related brain structural/functional alterations, and Recently, more and more studies were designed to explore brain morphometric and functional abnormalities and alterations in aADHD. aADHD‐related morphometric alterations and abnormal activation emerged in large‐scale networks, predominantly located within the frontoparietal, visual, dorsal attention, and default mode networks and so on.In the study, a supervised, goal‐directed fusion model was used to explore the underlying structure‐function fusion pathogenesis of inhibition in aADHD. “Inhibit” score was used as a specific prior reference to guide the fusion in this study. Results showed that the gradual addition of GMV and FC variables could improve the interpretation level of inhibition variability (more details see the supplementary results and Table →FC→Inhibition” model. As expected, the GMV of the abovementioned regions could play a role not only directly but also through the mediation of FC in inhibition impairment. As far as we know, this is the first attempt to guide multimodal imaging data fusion with cognitive scores as a reference to seek potential multimodal neural markers of inhibition deficits in ADHD.Not only reduced GMV and FC within the inhibition network, but also increased GMV and FC within the DMN were involved in inhibition deficits in aADHD. This implicated the abnormalities observed in ADHD patients affected not only isolated brain structure regions but also the functional connectivity between these brain regions, thus providing a structure‐function fusion neural mechanism for inhibition deficits in aADHD. A central assumption of neuroscience is that brain structure could predict and/or is linked to brain function.NOS1 ex1f‐VNTR with impaired inhibition function in aADHD, which was consistent with previous report in some extent.NOS1 ex1f‐VNTR during inhibition process.NOS1 gene.NOS1 allele carriers,NOS1 ex1f‐VNTR affected inhibition through the mediation of FC of the inhibition network and DMN.In our present study, we have found a marginal genetic association of There are some limitations in current study. Firstly, the sample size in genetic subgroup is relatively small, which might lead to marginal findings in genetic association analyses. In addition, we did not perform analyses for males and females separately, but just set as covariate. A larger sample size may lead to more robust results and help us to illustrate the potential gender‐specific relationship in the future. Secondly, fusion modal included only two modalities of GMV and FC in this study. Inhibition may also be affected by white matter integrity, local spontaneous brain activity, or cerebral blood flow. The fusion of these modalities would provide more informative knowledge of inhibition and could be studied in the future.5NOS1 ex1f‐VNTR variant and the GMV might affect inhibition through the mediation of the abnormal FC of these brain regions, identified a potential gene‐brain‐cognition pathway for inhibition in aADHD. This multimodal fusion method may be helpful in searching for meaningful biological markers and cross‐information links in multiple neuroimaging modalities. Integration of multimodal brain imaging and individual‐level genetic data delineates a new way for elucidating the pathophysiology and provides potential biomarkers of inhibition impairments.Our results showed that reduced GMV and FC in the inhibition network and increased GMV and FC in the DMN were jointly responsible for inhibition deficits in aADHD. More importantly, both the functional All authors reported no potential conflicts of interest.Supplementary MaterialClick here for additional data file.


The positive transcription elongation factor b (P-TEFb) is a critical coactivator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells. In eukaryotic cells, coding gene expression starts with transcription of DNA to RNA by RNA polymerase II (RNAPII) in the nucleus. This process consists of initiation, promoter clearance, capping, elongation, and termination upon which the transcribed single-strand RNA is cleaved and poly-adenylated before transport to the cytoplasm . ClearanThe pause of RNAPII is caused by two factors, the negative elongation factor (NELF)  and DRB In the organism, the kinase activity of P-TEFb is kept under a tight control to maintain the appropriate state of growth and proliferation of cells. To ensure this balance, a large complex, known as the 7SK small nuclear ribonucleoprotein (7SK snRNP), sequesters a large amount of P-TEFb (from 50 to 90% in different cells) in an inactive state . 7SK snRIn activated and proliferating cells, high levels of P-TEFb are found. In contrast, they are vanishingly low in resting cells, especially monocytes and memory T cells. While levels of CDK9 persist, those of CycT1 are greatly reduced. At the same time, transcripts for CycT1 and CDK9 remain high in all these cells . Based oPreviously, residues in the N-terminal region of CycT1 , including cyclin boxes, positions 30–248)  were fouMutant CycT1 proteins were poorly expressed in 293T cells . WhereasWe further examined whether all these threonine residues are important for binding to CDK9 . Thus, mSince threonine residues are potential phosphorylation sites, we examined whether phosphorylation of Thr143 and/or Thr149 in CycT1 contributes to P-TEFb assembly. 293T cells ectopically expressing CycT1  or CycT1An online database for phosphorylation site prediction  scores Thr143 and Thr149 above the threshold value (default 0.5), indicating that these threonines are potential phosphorylation sites . To examTo further demonstrate that these two threonines in CycT1 are phosphorylated in vivo, we performed phospho-peptide mapping analyses by the validated in-gel phospho-staining with a specific phosphoprotein dye (Phospho-Tag). For the purpose of tryptic peptide mapping, we chose a further truncated CycT1 protein to position 192 (CycT1(192)). In contrast to two predicted tryptic fragments of equivalent size for CycT1(280) protein, only one large tryptic peptide with a predicted mass 4.3 kD  is found in the CycT1(192) protein, all others being of much smaller size. These predictions were generated by the Expasy website. Next, WT CycT1(192) and mutant CycT1(192)TT143149AA proteins were expressed in 293T cells with or without okadaic acid, and purified by IP. WB assay of the IPed proteins gave similar band shift as WT CycT1(280) and its mutant proteins compare . Indeed,Finally, purified WT CycT1(192) and mutant CycT1(192)TT143149AA proteins were subjected to trypsin digestion, followed by separation by 4–20% SDS-PAGE. Tryptic peptides containing phosphorylated residues were detected by silver and in-gel Phospho-Tag staining. As presented in Escherichia coli (elec) and polar solvation (ΔEsolv−polar) energies, which is consistent with the stabilizing interactions described in previous figures. Further, we performed molecular mechanics/generalized born surface area calculations (MM-GBSA)  . Since Phia coli . To undehia coli . As presMM-GBSA) , which pMM-GBSA) . We thenWT CDK9 or mutant CDK9K68A proteins were coexpressed with WT CycT1(280) or mutant CycT1(280)Q73A proteins in the presence of bortezomib for 12 hr before co-IP. As presented in In resting and memory T cells, levels of CycT1 are vanishingly low. In previous sections, we discovered that Thr143 and Thr149 are phosphorylated in the stable P-TEFb complex. Using kinase prediction programs (NetPhos 3.1), these residues lie in separate PKC consensus sites. While Thr143 received the highest score, Thr149 could also be a target for PKC or other kinases. These findings implied that PKC family members are kinases that phosphorylate Thr143 and/or Thr149.To examine whether PKC promotes the phosphorylation and stability of CycT1, several PKC inhibitors were introduced to different cells . Of thesTo examine whether PKC inhibition antagonizes the phosphorylation of CycT1(280) in the presence of the high concentration of okadaic acid, cells expressing the exogenous CycT1(280) protein in the presence of bortezomib were treated with the same set of kinase inhibitors before adding okadaic acid for another 1.5 hr. Levels of threonine phosphorylation of CycT1(280) were compared by WB after IPs with anti-HA antibodies. As presented in To validate further the specificity of such negative regulation by PKC inhibitors, Jurkat and activated primary CD4+ T cells were also treated with different PKC inhibitors at increasing amounts for 12 hr. As presented in Staurosporine and bisindolylmaleimide IX were also used in activated primary CD4+ T cells to confirm further our findings . In thesAnalysis of target specificities of our PKC inhibitors indicated that PKCα, PKCβ, PKCε represent candidate PKC isoforms responsible for the phosphorylation of CycT1. To validate that these PKC isoforms can target CycT1 for phosphorylation and promote P-TEFb assembly, their Flag-epitope-marked versions were expressed in 293T cells. Different dominant (kinase) negative mutant PKC isoforms were also coexpressed with WT CycT1(280) or mutant CycT1(280)TT143149AA proteins in the presence of bortezomib for 12 hr. Co-IPs were performed with anti-Flag antibodies. As presented in To confirm further that PKCα and PKCβ contribute to the assembly and stability of P-TEFb, WT PKCα or mutant PKCαK386R proteins were coexpressed with CycT1(280) and CDK9 in the presence of bortezomib (12 hr) in 293T cells. Interactions between these proteins were analyzed by co-IPs with anti-HA antibodies. As presented in Finally, we confirmed the phosphorylation of Thr143 and Thr149 of CycT1 by PKC in vitro. WT CycT1(280) and mutant CycT1(280)TT143149AA proteins were expressed in 293T cells and isolated by pull-down with HA-Ab-conjugated beads. They were incubated in the presence or absence of purified active PKC proteins (PKCα and PKCβ) in vitro. Phosphorylated proteins were separated by SDS-PAGE and detected by in-gel Phospho-Tag staining as in Previous papers demonstrated that isoforms of PKC are inactive or absent in resting cells . MoreoveWe observed previously that levels of CycT1 increase significantly in resting CD4+ T cells with the addition of bortezomib . NeverthIt was demonstrated that treatment of T cells with calcium ionophores such as ionomycin also depletes PKC and induces anergy . TherefoIn this study, we found that mutations of two critical residues in CycT1  impair its binding to CDK9. Phosphorylation of these residues promotes CycT1:CDK9 interactions. Structural modeling indicated that phosphates on Thr143 and Thr149 in CycT1 increase intramolecular and intermolecular binding to specific residues in CycT1 and CDK9, respectively, which contributes to P-TEFb assembly. This prediction was confirmed experimentally. Thr143 and Thr149 are located in PKC consensus sequences. Indeed, PKC inhibitors inhibited CycT1 phosphorylation. Of PKC isoforms, PKCα and PKCβ were found not only to bind to CycT1 but to promote CycT1:CDK9 interactions and stabilize CycT1 in vivo and in vitro. Finally, depleting PKC or its inactivity led to CycT1 dissociation from CDK9 and its degradation in transformed cell lines as well as primary activated and anergic T cells. We conclude that the assembly and stability of P-TEFb are potentiated by the phosphorylation of CycT1, which is regulated by PKCα, PKCβ, and PP1.Ours is the first study that addresses the reversible phosphorylation of CycT1 that helps to regulate P-TEFb. Based on previous mutageneses of CycT1, three threonine residues  could affect CycT1 stability and P-TEFb assembly. They are conserved in over 142 mammalian species and in CycT2 . Moreover, previous reports indicated that these residues play critical roles in P-TEFb function . Of thesAlthough unphosphorylated CycT1 dissociates from P-TEFb and is degraded, CDK9 remains and is stabilized by HSP70 and HSP90 . This obGlobal mRNA sequencing studies examine changes in levels of specific transcripts between and in different states, that is, activation, proliferation, and differentiation of cells . HoweverFinally, since our study revealed families of kinases and phosphatases that affect P-TEFb, it is possible that the use of more targeted phosphatase inhibitors could block this transition to quiescence and terminal differentiation of cells. Their use might even prevent the establishment of proviral latency. Although the phosphomimetic substitution of one of these two threonine residues was not successful (data not presented), it is possible that further modifications of CycT1 and CDK9 could create a constitutively active P-TEFb complex. If successful, modified P-TEFb complexes could be studied for effects on immune responses as well as latency induction and reversal in many different scenarios. The other approach would be to identify the E3 ligase that is responsible for the degradation of the unphosphorylated CycT1 protein. To this end, bortezomib and other proteasomal inhibitors have been examined already for the reversal of HIV latency in resting CD4+ T cells . Taking HA-CycT1 (h:CycT1), CDK9-Flag (CDK9:f), and plasmids containing mutated CycT1 or CDK9 sequences were constructed by cloning PCR fragments containing the coding sequences of CycT1 and CDK9 into pcDNA3.1 vector with indicated epitope tags. PKC plasmids  were obtained from Addgene, and their coding sequences were subcloned into the pcDNA3.1 vector containing the Flag epitope tag.All antibodies, chemicals, and proteins are listed in the Key resources table.2. Resting CD4+ T cells were purified from bulk PBMCs by using Dynabeads Untouched Human CD4 T Cells Kit (Thermo Fisher Scientific). Selected CD4+ T cells were activated by Dynabeads Human T-Activator CD3/CD28 kit (Thermo Fisher Scientific) and were maintained in RPMI 1640 with 10% FBS, containing 30 U/ml IL-2 (Sigma-Aldrich).Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Corning) with 10% fetal bovine serum (FBS) (Sigma-Aldrich), Jurkat cells and peripheral blood mononuclear cells (PBMCs) were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Corning) with 10% FBS at 37°C and 5% COTransfection of plasmid DNA was conducted in 293T cells using Lipofectamine 3000 (Life Technologies) and X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer’s instructions.293T cells were treated with 2 μM bortezomib for 12 hr, Jurkat cells were treated with 100 nM bortezomib for 12 hr, and activated CD4+ T cells were treated with 50 nM bortezomib for 12 hr before the cell lysis. 293T cells were treated with 5 nM or 1 μM okadaic acid for 1.5 hr, or with 150 nM calyculin A before the cell lysis. 293T cells, Jurkat cells, and activated CD4+ T cells were treated with MEK 1/2 inhibitor (MEK 1/2i) or different PKC inhibitors for 12 h before the cell lysis.g). The supernatant was precleared and incubated with indicated primary antibodies or control IgG overnight. Mixtures were then incubated with protein G-Sepharose beads for additional 2 hr, followed by five times’ wash with RIPA buffer (500 mM NaCl). Co-IP samples and input (1% of whole-cell lysates) were subjected to WB as described previously  supplemented with the protease and phosphatase inhibitors, then for one-time sonication , followed by a 10 min centrifugation . The band shift of the phosphorylated CycT1 proteins was confirmed by the actual size change in comparison to untreated conditions (no upper/phosphorylated bands detected).WBs were visualized by enhanced chemiluminescence (ECL) (PerkinElmer) produced by HRP-conjugated secondary antibodies, and chemiluminescent signals were directly captured by LI-COR image analyzer. Band intensities of WBs were quantified using Image Studio software (LI-COR). Relative protein expression in whole-cell lysates was calculated by normalizing the indicated proteins with loading control β-actin. Relative protein-protein interactions in IP and co-IP were calculated by normalizing the IPed proteins with indicated antibodies targeted proteins. Quantification data were presented as fold change over values obtained with control samples.g, 10 min). The supernatant was precleared and incubated with HA-Ab-conjugated beads for 4 hr, followed by washing with RIPA buffer (500 mM NaCl) for three times and twice with RIPA buffer without detergents. Proteins associated with anti-HA-beads were eluted by incubation with the HA peptide (1 mg/ml). The eluted proteins were subjected to trypsin digestion using Rapid Trypsin kit (Promega) according to the manufacturer’s instructions and separated in SDS-PAGE gel for silver staining by using Pierce Silver Stain Kit (Thermo) or phospho-staining by using Phospho-Tag Phosphoprotein Gel Stain Kit (ABP Biosciences) according to the manufacturer’s instructions.293T cells were lysed with RIPA buffer on ice. Lysates were sonicated once  and cleared by centrifugation . Reaction mixtures were then subjected to SDS-PAGE gel, followed by in-gel Phospho-Tag staining.WT CycT1(280), mutant CycT1(280)TT143149AA, and EGFP proteins were expressed in 293T cells, isolated by HA-Ab-conjugated beads, and eluted from the beads by the HA-peptide (1 mg/ml). Eluted proteins were incubated with or without purified constitutively active PKC proteins (4 μg PKCα and PKCβ1) at 30°C for 1 hr in kinase buffer  CD25-Va2+CD4+ T cells were sorted from combined lymphoid organs (spleens and lymph nodes) of OTII or W131AOTII mice (8–12 weeks of age). The cells (106 cells) were washed with PBS, the supernatant was aspirated, and the pelleted cells were lysed as described above, then subjecting to WB assay.W131AOTII mice were described previously . Control143,149), CycT1 phosphorylated at Thr143 (PThr143), and CycT1 phosphorylated at Thr149 (PThr149). Models of the four P-TEFb complexes were built based on the crystallographic structure of the human P-TEFb complex (PDB code 3MI9) using the AmberTools leap tool. All systems were neutralized with Na+ and Cl- ions and solvate is a cubic box with periodic boundary conditions. Simulations were performed using the AMBER19 force-field  and root mean-squared fluctuation (RMSF) to monitor system equilibration and assess the local flexibility of the systems, respectively.For each production trajectory, the AmberTools The binding energies of the four P-TEFb complexes were calculated by the MM-GBSA method , impleme This study addresses an important question regarding the regulation of positive transcription elongation factor b (P-TEFb), which is an important regulator of gene expression targeted by the HIV Tat protein. The authors propose a novel mechanism with the potential to explain the differences in P-TEFb regulation between proliferating and quiescent cells, which might in turn have important implications for antiviral therapy. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Eric J Wagner as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.Thank you for submitting your article "Assembly and levels of P-TEFb depend on reversible phosphorylation of cyclinT1" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Test the effect of phosphomimetic mutants because if these mutants function as predicted, it could add significant strength to the study.2) provide stronger evidence that Thr143 and Thr149 are indeed phosphorylated in vitro and in vivo.Reviewer #1:In proliferating cells, the levels of p-TEFb comprised of CDK9 and Cyclin T1 are high to coincide with ongoing transcriptional activation. However, in resting cells the levels of Cyclin T1 are vanishingly low but the mechanism underlying this is poorly understood. The authors set out to understand how Cyclin T1 stability is regulated and uncovered a key role for two specific threonine, which are subject to phosphorylation by PKC and de-phosphorylation by PP1. The major strengths of the paper include determining that phosphorylation of the two threonines in Cyclin T1 not only contribute to its stability but are required for interaction with CDK9. Moreover, the authors show that PKC activity is a key regulated component in the transition from resting to activation that triggers p-TEFb assembly. The implication of this work is that it provides an explanation for long-sought question as to how transcriptional activation potential is regulated in specific cell types – notably memory T cells.Overall, the data presented is of high quality and the presentation of the results is logical and well-described. In my view, the conclusions drawn are justified by the data presented. My suggestions below are meant to only strengthen the manuscript further.Figure 2 – referring to 'panels' in the text is confusing as it was unclear what panel 1/2/3 were specifically referring toFigure 2- the authors state that the pThr signal overlayed onto the upper band for CyCT1 but this is not easily ascertained based upon how the figures are constructedFigure 2- the statement that OA enhanced co-IP by ~5 fold for the full length and truncation mutant is not convincing as stated. First of all, it doesn't look like OA enhanced CyCT coIP much at all in panel 2A and in 2B, that enhancement appears minor. Can the authors state explicitly how this was quantified and if it was done so using replicate IPs so that reproducibility can be determined?Figure 2C – The T143/T149A mutant is convincing that the pThr signal is diminished. But it is confusing why the input CycT1 signal in the T143/T149AA is identical to the wt-280 protein . This would suggest that mutation of these two threonines does not substantively alter overall phosphorylation of the 280 cycT1 protein. This result almost seems ignored, can the authors clarify this interpretation?Figure 3A: the authors could consider a slightly different or additional perspective in their image. It is difficult to see the K68 interaction with T149 as the side chains are right on top of each other.Figures3B/C/D: similar comment to above in terms of how the fold reduction in binding is calculated. The data in panel C/D look very convincing but the data in panel B is not as robust – yet the 'fold-reduction' is calculated as very close for B/C?Figure 4: overall, the data shown is quite compelling but there is one overarching concern I have. The data in 4C demonstrates that treatment with staurosporine eliminates any pThr signal present on CycT1. While this is convincing, it is also apparent that all aberrantly migrating CycT1 that appears after OA treatment is collapsed into a single band suggesting that all phosphorylation of CycT1 is sensitive to staurosporine. This doesn't seem likely given the authors showed in panel 3C (bottom) that TT143,149AA, which diminishes pThr signal has no apparent impact to the remaining phosphorylations of CycT1 as the aberrant migrating form is unchanged. This speaks to the concern of staurosporine specificity but could be mitigated if the authors also conduct a PKC RNAi experiment followed by transfection of their HA-CycT1.Reviewer #2:This study addresses an important question regarding the regulation of positive transcription elongation factor b-P-TEFb, the complex of cyclin-dependent kinase 9 (CDK9) and cyclin T1. In proliferating cells, this complex is the major rate-limiting regulator of transcription elongation by RNA polymerase II (RNAPII) and target of HIV Tat protein-which co-opts P-TEFb to drive expression of viral genes-and is negatively regulated by an inhibitory 7SK ribonucleoprotein (RNP) complex. This axis does not appear to operate in quiescent cells, including resting or exhausted T cells that are refractory to Tat, apparently because of decreased levels of cyclin T1 protein despite high levels of the mRNA. The authors propose a novel regulatory mechanism with the potential to explain the differences in P-TEFb regulation between proliferating and quiescent cells: phosphorylations on two Thr residues in cyclin T1 that stabilize its binding to Cdk9, which are placed by protein kinase C (PKC) isoforms and removed by protein phosphatase 1 (PP1), and which become dephosphorylated in quiescent cells, leading to disassembly of the P-TEFb complex and subsequent proteasomal degradation of the free cyclin. This is an attractive hypothesis, both because it would solve a longstanding puzzle in the field and yield insights into potential antiviral drug targets, and because the proposed mechanism of CDK regulation it posits is novel and surprising (possibly unprecedented). The data are for the most part consistent with the proposed model but fall short of proving it; many of the functions of the individual players or modifications, and relationships between them, are inferred rather than demonstrated. There are questions of specificity-about the kinase and phosphatase inhibitors used, and about the phosphorylated residues being targeted-that would need to be answered more definitively before this mechanism can be considered validated. Below I list my major, specific concerns and comments:1. The implication of PKC and PP1, the two key enzymatic players in the P-TEFb regulatory network proposed here, rests heavily on experiments with inhibitory small molecules that are at best only partially selective for their intended targets. For example, staurosporine, the "PKC inhibitor" used for most of their experiments, is a relatively promiscuous protein kinase inhibitor. Likewise, okadaic acid is a potent inhibitor of PP2A with reduced but significant potency towards PP1 and other phosphoprotein phosphatase (PPP) family members; the authors' argument for its PP1-specificity in this setting is that it influences cyclin T1 stability at high but not low concentrations. I do not find this reasoning compelling; at the higher concentrations needed to inhibit PP1 in vivo, PP2A would also be more completely inactivated.2. The use of dominant negative, kinase-dead variants of PKC somewhat allays the concerns regarding specificity of the kinase inhibitors, and suggests that PKC activity might indeed be influencing cyclin T1 phosphorylation state, but a demonstration that PKC can directly phosphorylate cyclin T1, specifically on Thr143 and Thr149, is missing, and would be needed to justify some of the stronger conclusions drawn here .3. There is in fact no demonstration that Thr143 and Thr149 are actually phosphorylated in vivo or in vitro, only that their mutation to Ala diminishes reactivity of cyclin T1 with anti-pThr antibodies. This could be an indirect effect on phosphorylation of a different residue due to conformational changes caused by the mutations. The authors state, on p. 20 of Discussion, that these sites "were missed" in previous studies but to justify that assertion would need to show unambiguously that they are indeed phosphorylated . Importantly, recombinant, highly active and apparently stable P-TEFb can be purified-and crystallized-in the absence of these phosphorylations . There is precedent for phosphorylations (on the CDK) being required to stabilize CDK-cyclin complexes in vivo but dispensable in vitro or in overexpression conditions, but to conclude that is the case here would require proof that these residues are indeed phosphorylated in vivo.4. There is a circularity to the logic behind the choice of PKC isoforms to target specifically (p. 13): Thr143 and Thr149 looked like PKC sites, leading the authors to test a panel of PKC inhibitors that were specific for the isoforms they selected for further studies. A more cogent argument would be based on both positive and negative data, i.e., inhibitors of different isoforms (and different kinases entirely) not having the same effects.5. Selectivity in phosphatase inhibition is notoriously hard to achieve. Nonetheless, there are small molecules with some selectivity for PP1 over PP2A (e.g. tautomycetin), greater selectivity for PP2A (e.g. cytostatin) or ~equal potency towards both ; the authors should try these inhibitors and/or targeted depletion of PP1 catalytic (or regulatory) subunits to test PP1 involvement more rigorously.6. The authors should test potentially phosphomimetic substitutions of these residues -a manipulation they allude to in the Discussion but did not attempt. Although Glu is not guaranteed to mimic pThr, the upside if it does would be considerable: if a constitutive negative charge is sufficient to stabilize P-TEFb, it should increase cyclin T1 in quiescent cells (perhaps conferring sensitivity to Tat) and, if the proposed model is correct, make cyclin T1 in proliferating cells resistant to degradation induced by PKC inhibition.7. The study would be greatly strengthened by measurements of CDK9 activity after the various manipulations tested, i.e., cyclin T1 mutations, PKC ablation, PP1 inhibition.8. The effectiveness of PKC inhibitors or dominant-negative alleles should be gauged with measurements of phosphorylation on known PKC substrates, if phosphospecific antibodies are available for these targets.9. The second sentence of the abstract is ambiguously worded: it should be made clearer that what is absent in quiescent cells is P-TEFb, not 7SK RNP (if that is what the authors mean).10. Pp. 2-3, non-expert readers might not appreciate distinction between promoter clearance and promoter-proximal pause release (and thus between functions of CDK7 and -9), which are conflated to a significant degree in the authors' description.11. P. 3, first paragraph, sentence starting "CDK9 is…" "DRB" should be "DRB sensitivity inducing factor (DSIF)" (referred to in the next sentence).12. P. 6 and elsewhere, the authors should avoid the nomenclature "T3A" and T2A" to refer to different combinatorial mutations  because it will be confused with single point mutations, i.e., of "Thr3" or "Thr2" (which are nonexistent). I suggest the more conventional shorthand "3TA" and "2TA" to refer to these alleles.13. I am just a bit skeptical about the quantification of immunoblot signals: some of the numbers reported for fold-differences do not match the visual evidence . They appear to be using ECL (rather than fluorescence) for detection and quantification, so these numbers should probably be taken with a grain of salt. I would recommend the authors provide more detail about the quantification, e.g. the raw data for the blot scanning, preferably with error bars.14. There is a sentence fragment on p. 13, starting with "Since the catalytic domains…"15. The precedent of cyclin E degradation cited on p. 20 is interesting but not quite apt: in that case degradation is promoted by phosphorylation of cyclin E (on a sequence motif known as a phosphodegron), and I am not aware of any requirement for cyclin E phosphorylation in binding to CDK2, so its relevance to the proposed mechanism of cyclin T1 degradation is not clear.Reviewer #3:The manuscript by Huang and colleagues describes two phosphorylations sites in human Cyclin T1, which regulate the interaction to CDK9 and contribute to the binding and stability of Cyclin T1 in quiescent and terminally differentiated cells. The author describe that the pool of free Cyclin T1 is rapidly degraded when it is not phosphorylated on these two respective sites in the cyclin boxes of cyclin T1. The PP1 phosphates inhibitor okadaic acid leads to P-TEFb stabilization, as less CycT1 is degraded, whereas kinase inhibitors targeting PKC result in P-TEFb disassembly and degradation of CycT1. Using a set of different kinase inhibitors, the authors find that PKC-α and PKC-β are responsible for CycT1 phosphorylation.The findings described in this study bring the transcription regulating cyclins, as CycT1, closer to the cell cycle regulating cyclins, which are long known for the tight regulation between expression upregulation and ubiquitination downregulation. These findings are potentially important, as they could shift our view on the transcriptional cyclins and the need for transcriptional CDK regulators , if gene expression and degradation is indeed another layer of regulation of these cyclins.E. coli exhibiting no phosphorylation and binds properly well to CDK9. It would be very beneficial and in support of this study to know the dissociation constants for the interaction partners in a CycT1 phosphorylated and non-phosphorylated state.There is one major conundrum to this study: Multiple biochemical and structural studies on CDK9 and Cyclin T1 have been performed, often by co-expression and co-purification of these two subunits. In none of them (to my knowledge) a phosphorylated T143 or T149 residue was identified. The sentence "Unphosphorylated CycT1 dissociates from P-TEFb and is degraded." (page 20 and similar in the abstract) seems therefore farfetched. If pT143 and pT149 stabilizes the complex in such a profound degree, why are researchers able to form the non-phosphorylated complex in the first place? CycT1 can be expressed from I am surprised that a phosphorylated threonine mimetic, as glutamic acids (E) or aspartic acid (D), was not analyzed in these experiments. To this reviewers' opinion the authors should analyze how a double mutation of CycT1 (T143E/T149E) acts on CDK9 binding, P-TEFb complex formation and the stability of Cyclin T1. Using only the T143A/T149A mutant but not a phosphorylation mimetic is a lack in the design of the study.Throughout the entire manuscript, the authors are unprecise and ambiguous with predictions  and 'real' experimental findings. E.g.: Page 18, Discussion, 3 sentence: "Structural analyses revealed that phosphates on Thr143 and Thr149 in CycT1 increase intramolecular and intermolecular binding to specific residues in CycT1 and CDK9, respectively, which potentiates P-TEFb assembly and stabilizes CycT1." In Figure 3A, a model is displayed but not an experimentally determined structure with phosphorylated residues, as 3MI9 does not contain any phosphorylation in Cyclin T1. The figure legend to 3A should clearly say this. The description in the main text is okay, but the text of the figure legend is clearly misleading and almost fraud. This is a model only!Another example is on page 8: "An online database for phosphorylation site prediction  scores Thr143 and Thr149 above the threshold value (default 0.5), indicating that these threonines are potential phosphorylation sites . To verify that Thr143 and Thr149 are the main phosphorylation sites in CycT1, …" The second sentence starting with "To verify …." suggests, that the first observation are real data but not simply predictions. Moreover, the prediction has been made on a 43-residue sequence but not the full CycT1 structure  as I understand. This does not account for any tertiary structure etc. and is therefore not to the highest standard. I would not consider this analysis good evidence for phosphorylation sites.There are clearly dissociation constants missing for the statement that the threonine phosphorylation in cyclin T1 is increasing its binding capacity to CDK9 and the stability of the cyclin. From the model it seems that pT143 could also only act indirectly as it forms internal contacts within the cyclin but is not in the direct interface to the kinase.eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Matthias Geyer (Reviewer #3).Thank you for submitting your article "Assembly and levels of P-TEFb depend on reversible phosphorylation of cyclin T1" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.There was agreement among the Reviewers that the revised submission had been improved and the new data presented mostly addressed previously noted critiques. However, there was still a shared concern that stronger data is needed to demonstrate that these two threonine residues are phosphorylated in cells. In light of these counter-balancing thoughts, the Editors have decided on an additional round of revision to address this specific concern. We highlight essential revisions below and provide specific feedback from the Reviewers.Essential revisions:1) Conduct mass spectrometry on preferably full-length Cyclin T1 to demonstrate phosphorylation at T143 and T149 or provide convincing evidence for their phosphorylation using phospho-specific antibodies if such a reagent is in hand.Reviewer #2:This is a revision of a manuscript I reviewed previously. The authors have addressed one of my major concerns by providing evidence that PKC can phosphorylate a cyclin T1 fragment in T143/149-dependent fashion in vitro . I am less persuaded by the evidence implicating PP1 in dephosphorylating cyclin T1, but this is largely owing to the technical difficulty of proving phosphatase specificity in cell-based experiments. Taken together, there is substantial reason to believe, based on the data presented, that presence or absence of PKC-dependent signaling is a determining factor in P-TEFb activity levels in different settings (e.g. resting or exhausted versus activated T cells).My biggest concern remains, however, that the mechanism the authors propose for this regulation-phosphorylation of two specific residues on cyclin T1 that stabilize an interaction with CDK9-is only incrementally closer to being validated in the revised version. The new data addressing this point were obtained in a complicated setup, in which a severely truncated cyclin T1 fragment (missing residues needed for interaction with CDK9) was expressed in 293T cells and then pulled down and detected in gels by a commercial phosphoprotein-staining reagent, either intact  or after tryptic digestion (2E). Mutation of T143 and T149 to A abolished the signal, which is promising but still preliminary evidence that these sites might be phosphorylated in intact cyclin T1 under physiologic conditions. This does not address the concern, also expressed by Reviewer 3, that this requirement-and the phosphorylations themselves-have escaped previous detection. The authors dismiss this concern by pointing out that structures of P-TEFb were solved for complexes expressed in insect cells, but this is not persuasive; in those structures, phosphorylation on T186 of the CDK9 activation segment was readily observed . Fully active, stable P-TEFb complexes can also be reconstituted from purified CDK9 expressed in insect cells and cyclin T1 purified from bacteria. Finally, a quick check of the PhosphoSite online data base did not turn up either of these sites. So I must reiterate my initial position that, because the authors are proposing a novel and highly detailed mechanism for regulating an essential transcription regulator, the existence, necessity and sufficiency of T143 and T149 phosphorylation simply must be proven and not merely inferred. Typically, a study such as this would include dispositive supporting data obtained via mass spectrometry, phospho-specific antibodies, or both, to gain acceptance at a top-tier journal.Reviewer #3:The authors present a thoroughly revised version of their manuscript that addresses the main concerns from the previous review process. Particularly the second point regarding stronger evidence that T143 and T149 are directly phosphorylated is now supported by additional data. However, I miss any new data within the revised manuscript regarding the first main point on the use of phosphomimetic mutants in Cyclin T1. The authors state in their reply letter that the CycT1 T149D or T149E mutants maintained CDK9 binding, T143 to E or D instead did not. I think that this first mutant site T149(E/D) that sustains binding clearly strengthen their findings. Why don't they show these binding data, or did I miss it? The contribution of the T149 position to the interaction would be anyhow more indirect as shown from the modelling and it is well accepted, that these phosphomimetic mutants must not work. But as one of them seems to work, I am confused that these data are not contained in the manuscript. The new molecular dynamics simulations and MM-GBSA binding energy calculations are nice complementary data but cellular binding/precipitation data would be very convincing. The other points are well addressed. Essential revisions:1) Test the effect of phosphomimetic mutants because if these mutants function as predicted, it could add significant strength to the study.We created phosphomimetic mutants for Thr143 and Thr149. While Thr149 to aspartic or glutamic acid restored this binding, the same substitutions for Thr143 did not. Indeed, we agree with Reviewer 2 that not all phosphomimetic substitutions work, which also happened in our hands.Arabidopsis thaliana. J Biol Chem. 2013 Mar 15;288(11):7519-7527.). It’s also worth considering what phosphorylated amino acids are doing in a given protein. For example, hosphor-specific antibodies do not recognize substituted aspartates or glutamates – indicating quite different structural epitopes represented by these substitutions . The most recent publication also demonstrated that phosphomimetic substitutions were not able to restore the activation loop of RSK ).After investigating many published phosphorylation-related papers, there emerges no consistent picture for acidic amino acid substitutions to mimic phosphorylated serine, threonine or tyrosine residues. As we know, the charge densities, distributions and pKas of the carboxyl group of aspartic acid or glutamic acid are quite different from a phosphate group. There are some good examples such as MEK and PKB/Akt where such substitutions work quite well (to ~10-30% of fully phosphorylated wild type proteins). In other cases, it doesn't work in related proteins such as MKK4 (Thr234)  kinase kinases, which control stomata development in elec) and polar solvation (ΔEsolv−polar) energies. This finding is consistent with the stabilizing interactions described in Figures 1 and 2. Thus, the phosphorylation of Thr143 and Thr149 in CycT1 is thermodynamically advantageous for interactions between CDK9 and CycT1 (Table S1).Consistent with our further Molecular Mechanics/Generalized Born Surface Area calculations (MM-GBSA) for the P-TEFb complex,, the predominant contribution to the increased binding energy of the complex comes from electrostatic (ΔE2) Provide stronger evidence that Thr143 and Thr149 are indeed phosphorylated in vitro and in vivo.In addition to existing IP-WBs and kinase inhibitors experiments that provideextensive confirmation of this phosphorylation, we performed additional in-gel phosphoprotein staining studies for the direct phosphorylation of Thr143 and Thr149. First, the WT CycT1(280) or CycT1(192) rather than their AA mutant counterparts contained significantly higher levels of phospho-threonines in cells . Second, in-gel phosphoprotein staining of a trypsin peptide from the WT CycT1(192) and its AA mutant counterpart demonstrated specific phosphorylation of Thr143 and Thr149 in cells . Third, we present the direct PKC phosphorylation of Thr143 and Thr149 in vitro . Taken together, existing and added data provide overwhelming evidence that Thr143 and Thr149 are phosphorylated in vivo and in vitro. They add substantially to all conclusions of our work.eLife. Below are our point-by-point responses to each comment.We believe that the revised manuscript addresses all points raised by the reviewers and therefore is suitable for publication in Reviewer #1:In proliferating cells, the levels of p-TEFb comprised of CDK9 and Cyclin T1 are high to coincide with ongoing transcriptional activation. However, in resting cells the levels of Cyclin T1 are vanishingly low but the mechanism underlying this is poorly understood. The authors set out to understand how Cyclin T1 stability is regulated and uncovered a key role for two specific threonine, which are subject to phosphorylation by PKC and de-phosphorylation by PP1. The major strengths of the paper include determining that phosphorylation of the two threonines in Cyclin T1 not only contribute to its stability but are required for interaction with CDK9. Moreover, the authors show that PKC activity is a key regulated component in the transition from resting to activation that triggers p-TEFb assembly. The implication of this work is that it provides an explanation for long-sought question as to how transcriptional activation potential is regulated in specific cell types – notably memory T cells.Overall, the data presented is of high quality and the presentation of the results is logical and well-described. In my view, the conclusions drawn are justified by the data presented. My suggestions below are meant to only strengthen the manuscript further.Figure 2 – referring to 'panels' in the text is confusing as it was unclear what panel 1/2/3 were specifically referring to.We apologize for the confusion. In the revised manuscript, we added an explanation about panel numbers .Figure 2- the authors state that the pThr signal overlayed onto the upper band for CyCT1 but this is not easily ascertained based upon how the figures are constructedWe added more detailed explanation about how WBs were processed in the manuscript. To detect CycT1 pThr signals by IP-WB, we first stained with anti-pThr antibodies, then the membranes were stripped and reblotted with anti-HA antibodies to detect HA-CycT1 (unphosphorylated and phosphorylated forms). Also, the band shift of the phosphorylated CycT1 protein was confirmed by the actual size change in comparison to the untreated condition (no upper/phosphorylated bands detected). Combining all these data, the upper bands of CycT1 detected by anti-pThr antibodies represent phosphorylated CycT1 proteins .Figure 2- the statement that OA enhanced co-IP by ~5 fold for the full length and truncation mutant is not convincing as stated. First of all, it doesn't look like OA enhanced CyCT coIP much at all in panel 2A and in 2B, that enhancement appears minor. Can the authors state explicitly how this was quantified and if it was done so using replicate IPs so that reproducibility can be determined?All WB images were captured by the LI-COR imaging system. Luminescent signals corresponding to each protein band were processed with their analysis program. They were within the linear range of measurements according to Li-COR instructions. Relative protein-protein interactions in co-IP were calculated by normalizing the IPed proteins with indicated antibodies targeted proteins. Quantification data were presented as fold changes over values obtained with control samples  . For example, in Figure 2A, relative CycT1:CDK9 interactions were calculated by normalizing intensities of IPed CycT1 bands with IPed CDK9 bands. Data are from three independent experiments with excellent reproducibility. In addition, we repeated experiments by using low and high concentrations of okadaic acid as well as calyculin A, which is another protein phosphatase inhibitor. These new co-IPs are provided in the revised Figures 2A and 2B.Figure 2C – The T143/T149A mutant is convincing that the pThr signal is diminished. But it is confusing why the input CycT1 signal in the T143/T149AA is identical to the wt-280 protein . This would suggest that mutation of these two threonines does not substantively alter overall phosphorylation of the 280 cycT1 protein. This result almost seems ignored, can the authors clarify this interpretation?Our interpretation of the data is that there are additional phosphorylation sites in CycT1 that are not involved in P-TEFb assembly and/or CycT1 degradation, and these phosphorylation sites are also stabilized by okadaic acid and/or calyculin A. They could contribute to the much clearer band shift of CycT1. Indeed, we compared levels of other phosphorylated residues between WT CycT1(280) and mutant CycT1(280)TT143149AA, and they were only slightly different between these two proteins, supporting this interpretation. New data in the revised manuscript address this issue more directly .Figure 3A: the authors could consider a slightly different or additional perspective in their image. It is difficult to see the K68 interaction with T149 as the side chains are right on top of each other.We updated the structural analysis and figure in the revised manuscript . They present a better view of the linkage between Thr149 in CycT1 and Lys68 in CDK9.Figures3B/C/D: similar comment to above in terms of how the fold reduction in binding is calculated. The data in panel C/D look very convincing but the data in panel B is not as robust – yet the 'fold-reduction' is calculated as very close for B/C?Band intensities were quantified as described above. Additionally, we also repeated experiments using the mutant CycT1 Q73A protein and the data are consistent with our conclusion. The WB in Figure 3B was replaced with new data in the revised manuscript.Figure 4: overall, the data shown is quite compelling but there is one overarching concern I have. The data in 4C demonstrates that treatment with staurosporine eliminates any pThr signal present on CycT1. While this is convincing, it is also apparent that all aberrantly migrating CycT1 that appears after OA treatment is collapsed into a single band suggesting that all phosphorylation of CycT1 is sensitive to staurosporine. This doesn't seem likely given the authors showed in panel 3C (bottom) that TT143,149AA, which diminishes pThr signal has no apparent impact to the remaining phosphorylations of CycT1 as the aberrant migrating form is unchanged. This speaks to the concern of staurosporine specificity but could be mitigated if the authors also conduct a PKC RNAi experiment followed by transfection of their HA-CycT1.+ T cells . In addition, experiments with dominant negative PKCs and the new in vitro PKC kinase assay  also support the involvement of PKC in this process.Thanks for pointing out the concerns about the specificity of the PKC inhibitor staurosporine. First, staurosporine is a very strong and general PKC inhibitor, which can neutralize robustly increased phosphorylation effects by protein phosphatase inhibitors. Indeed, staurosporine removed all threonine phosphorylation and the resulting CycT1 proteins collapsed into one single band. As mentioned above, our interpretation is that there are multiple phosphorylation sites that are sensitive to staurosporine, and among them are Thr143 and Thr149 that are involved in P-TEFb assembly and CycT1 degradation. To obtain results with more specific PKC inhibitors, we employed two other PKC inhibitors  and one non-PKC inhibitor MEK 1/2i which also inhibited interactions between CycT1 and CDK9, and diminished phosphorylation of Thr143 and Thr149 , confirming that these residues are phosphorylated by PKC, which is important for P-TEFb assembly. Meanwhile, with other three PKC inhibitors (not inhibiting PKCα and PKCβ), only these selected PKC inhibitors were able to decrease the endogenous levels of CycT1 proteins in activated primary CD4Reviewer #2:This study addresses an important question regarding the regulation of positive transcription elongation factor b-P-TEFb, the complex of cyclin-dependent kinase 9 (CDK9) and cyclin T1. In proliferating cells, this complex is the major rate-limiting regulator of transcription elongation by RNA polymerase II (RNAPII) and target of HIV Tat protein-which co-opts P-TEFb to drive expression of viral genes-and is negatively regulated by an inhibitory 7SK ribonucleoprotein (RNP) complex. This axis does not appear to operate in quiescent cells, including resting or exhausted T cells that are refractory to Tat, apparently because of decreased levels of cyclin T1 protein despite high levels of the mRNA. The authors propose a novel regulatory mechanism with the potential to explain the differences in P-TEFb regulation between proliferating and quiescent cells: phosphorylations on two Thr residues in cyclin T1 that stabilize its binding to Cdk9, which are placed by protein kinase C (PKC) isoforms and removed by protein phosphatase 1 (PP1), and which become dephosphorylated in quiescent cells, leading to disassembly of the P-TEFb complex and subsequent proteasomal degradation of the free cyclin. This is an attractive hypothesis, both because it would solve a longstanding puzzle in the field and yield insights into potential antiviral drug targets, and because the proposed mechanism of CDK regulation it posits is novel and surprising (possibly unprecedented). The data are for the most part consistent with the proposed model but fall short of proving it; many of the functions of the individual players or modifications, and relationships between them, are inferred rather than demonstrated. There are questions of specificity-about the kinase and phosphatase inhibitors used, and about the phosphorylated residues being targeted-that would need to be answered more definitively before this mechanism can be considered validated. Below I list my major, specific concerns and comments:1. The implication of PKC and PP1, the two key enzymatic players in the P-TEFb regulatory network proposed here, rests heavily on experiments with inhibitory small molecules that are at best only partially selective for their intended targets. For example, staurosporine, the "PKC inhibitor" used for most of their experiments, is a relatively promiscuous protein kinase inhibitor. Likewise, okadaic acid is a potent inhibitor of PP2A with reduced but significant potency towards PP1 and other phosphoprotein phosphatase (PPP) family members; the authors' argument for its PP1-specificity in this setting is that it influences cyclin T1 stability at high but not low concentrations. I do not find this reasoning compelling; at the higher concentrations needed to inhibit PP1 in vivo, PP2A would also be more completely inactivated.As mentioned above (to Reviewer 1), the revised manuscript, we added data with more specific PKC inhibitors including bisindomaleinide IX and HBDDE and a non-PKC kinase inhibitor (MEK1/2 inhibitor) as well as with another protein phosphatase inhibitor, calyculin A. They all support our conclusions. Moreover, we now include data with low concentrations of okadaic acid that have no effect on CycT1 phosphorylation and P-TEFb assembly. This concentration inhibits PP2A, not PP1. While we do agree with the reviewer's comment "at the higher concentrations needed to inhibit PP1 in vivo, PP2A would also be more completely inactivated", the fact that there is no such effect with low concentrations of okadaic acid clearly indicates that PP2A has a minor, if any, involvement in this process. Elucidation of PP isotypes involved in biological processes is very difficult because of the large numbers of isoforms and subunits and the lack of highly specific phosphatase inhibitors. However, using high vs low concentration of okadaic acid to separate between PP1 and PP2A is generally accepted by the field; indicated papers are listed below:1). Pei JJ, et al., Okadaic-acid-induced inhibition of protein phosphatase 2A produces activation of mitogen-activated protein kinases ERK1/2, MEK1/2, and p70 S6, similar to that in Alzheimer’s disease. Am J Pathol. 2003.2). Mailhes JB, et al,. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induces premature separation of sister chromatids during meiosis I and aneuploidy in mouse oocytes in vitro. Chromosome Res. 2003.3). Swingle M, Ni L, Honkanen RE. Small-molecule inhibitors of ser/thr protein phosphatases: specificity, use and common forms of abuse. Methods Mol Biol. 2007.4). A. B. Dounay and C. J. Forsyth, “ Okadaic Acid: The Archetypal Serine / Threonine Protein Phosphatase Inhibitor”, Current Medicinal Chemistry 2002.5). Takai A, et al., Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship. Biochem J. 1992.6). Lourdes Garcia, et al., PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress. FEBS Letters. Volume 523, Issues 1–3, 2002.7). Swingle M, Ni L, Honkanen RE. Small-molecule inhibitors of ser/thr protein phosphatases: specificity, use and common forms of abuse. Methods Mol Biol. 2007.2. The use of dominant negative, kinase-dead variants of PKC somewhat allays the concerns regarding specificity of the kinase inhibitors, and suggests that PKC activity might indeed be influencing cyclin T1 phosphorylation state, but a demonstration that PKC can directly phosphorylate cyclin T1, specifically on Thr143 and Thr149, is missing, and would be needed to justify some of the stronger conclusions drawn here .We performed direct phosphorylation studies in vivo and in vitro . They are included in the revised manuscript. See also comments to the editor above.3. There is in fact no demonstration that Thr143 and Thr149 are actually phosphorylated in vivo or in vitro, only that their mutation to Ala diminishes reactivity of cyclin T1 with anti-pThr antibodies. This could be an indirect effect on phosphorylation of a different residue due to conformational changes caused by the mutations. The authors state, on p. 20 of Discussion, that these sites "were missed" in previous studies but to justify that assertion would need to show unambiguously that they are indeed phosphorylated . Importantly, recombinant, highly active and apparently stable P-TEFb can be purified-and crystallized-in the absence of these phosphorylations . There is precedent for phosphorylations (on the CDK) being required to stabilize CDK-cyclin complexes in vivo but dispensable in vitro or in overexpression conditions, but to conclude that is the case here would require proof that these residues are indeed phosphorylated in vivo.We agree and we performed additional studies to confirm the phosphorylation of Thr143 and Thr149 in vivo and in vitro. See answers to comment 2 and to the editor, above. Moreover, examining the literature and talking to direct participants in these crystallographic studies, no-one has been able to express P-TEFb from bacteria. Johnston and Tahirov (as well as Alber and Hurley) groups used P-TEFb purified from baculovirus, where these residues in CycT1 are most likely already phosphorylated.4. There is a circularity to the logic behind the choice of PKC isoforms to target specifically (p. 13): Thr143 and Thr149 looked like PKC sites, leading the authors to test a panel of PKC inhibitors that were specific for the isoforms they selected for further studies. A more cogent argument would be based on both positive and negative data, i.e., inhibitors of different isoforms (and different kinases entirely) not having the same effects.+ T cells for further confirmation of our conclusions. As presented in the new figure , the MEK 1/2 inhibitor had no effect on endogenous CycT1 protein levels. Three new PKC inhibitors (not inhibiting PKCα and PKCβ)  also did not decrease CycT1 protein levels. In contrast, other PKC inhibitors (PKCα and PKCβ related) demonstrated consistent effects as presented in Figure 4—figure supplement 1D. Taking all these data together, we can draw the conclusion that phosphorylation of Thr143 and Thr149 in CycT1 is PKC dependent.Better descriptions are provided in the revised manuscript. Moreover, we did include more PKC inhibitors for comparison, and one non-PKC inhibitor (MEK 1/2 inhibitor) as the negative control. As the new Figures 4B and 4C demonstrate, the MEK1/2 inhibitor had no effect on interactions between CycT1 and CDK9 and Threonine phosphorylation levels of CycT1. In contrast, other more specific PKC inhibitors bisindolylmaleimide IX and HBDDE decreased these interactions and phosphorylation levels. Moreover, the MEK1/2 and other PKC inhibitors were also tested in primary CD45. Selectivity in phosphatase inhibition is notoriously hard to achieve. Nonetheless, there are small molecules with some selectivity for PP1 over PP2A (e.g. tautomycetin), greater selectivity for PP2A (e.g. cytostatin) or ~equal potency towards both ; the authors should try these inhibitors and/or targeted depletion of PP1 catalytic (or regulatory) subunits to test PP1 involvement more rigorously.In the revised manuscript, we added effects of calyculin A, as suggested. Furthermore, low concentrations of okadaic acid did rule out the involvement of PP2A, compared to the high concentration of okadaic acid and calyculin A .6. The authors should test potentially phosphomimetic substitutions of these residues -a manipulation they allude to in the Discussion but did not attempt. Although Glu is not guaranteed to mimic pThr, the upside if it does would be considerable: if a constitutive negative charge is sufficient to stabilize P-TEFb, it should increase cyclin T1 in quiescent cells (perhaps conferring sensitivity to Tat) and, if the proposed model is correct, make cyclin T1 in proliferating cells resistant to degradation induced by PKC inhibition.We indeed tested the phosphomimetic substitutions of Thr143 and Thr149 in CycT1. While T149E restored the binding, neither T143E nor T143D rescued interactions between CycT1 and CDK9. As the reviewer acknowledges, phosphomimetic mutants, particularly those of CDKs, do not always mimic the actual phosphorylation. Molecular dynamics simulations and MM-GBSA binding energy calculations indicates that phosphorylation of Thr143 and Thr149 do have the largest effects in binding stability. Therefore, adding one negative charge on Thr143 might not be sufficient to restore the intra-molecular interaction with Q73. Based on this assumption, we constructed various reciprocal mutant CycT1 proteins and are testing them for their ability to restore P-TEFb assembly without phosphorylation. However, these new substitutions are beyond the scope of the current study and could create a non-degradable P-TEFb complex, which will involve significant further experimentation. We also addressed this point in the response to the editor, above.7. The study would be greatly strengthened by measurements of CDK9 activity after the various manipulations tested, i.e., cyclin T1 mutations, PKC ablation, PP1 inhibition.The presented study identifies the mechanism of P-TEFb assembly (CycT1:CDK9 interaction). Since CycT1 unbound CDK9 protein is not enzymatically active, measuring CDK9 activity under these conditions will not be informative.8. The effectiveness of PKC inhibitors or dominant-negative alleles should be gauged with measurements of phosphorylation on known PKC substrates, if phosphospecific antibodies are available for these targets.Since PKC inhibitors and dominant negative PKC proteins have been studied extensively, we believe that it is not necessary to repeat them here.9. The second sentence of the abstract is ambiguously worded: it should be made clearer that what is absent in quiescent cells is P-TEFb, not 7SK RNP (if that is what the authors mean).Thanks for the corrections. We specified that CycT1 protein levels are greatly diminished in quiescent cells, which leads to the loss of P-TEFb, which does not translate to other components, such as 7SK RNA or HEXIM1.10. Pp. 2-3, non-expert readers might not appreciate distinction between promoter clearance and promoter-proximal pause release (and thus between functions of CDK7 and -9), which are conflated to a significant degree in the authors' description.We appreciate the comment. In the revised manuscript, we use the term "promoter-proximal pause-release" throughout, which describes the step of pausing after promoter clearance. Promoter clearance is when RNAPII moves past the promoter after CDK7 had phosphorylated Ser 5, then RNAPII pauses by NELF/DSIF, then promoter-proximal pause-release occurs when CDK9 phosphorylates NELF/DIS and Ser 2.11. P. 3, first paragraph, sentence starting "CDK9 is…" "DRB" should be "DRB sensitivity inducing factor (DSIF)" (referred to in the next sentence).We corrected the sentence accordingly.12. P. 6 and elsewhere, the authors should avoid the nomenclature "T3A" and T2A" to refer to different combinatorial mutations  because it will be confused with single point mutations, i.e., of "Thr3" or "Thr2" (which are nonexistent). I suggest the more conventional shorthand "3TA" and "2TA" to refer to these alleles.Thanks for the suggestions. We only used the T3A for the triple threonines mutations, and other single or double threonine mutations are named as TT143149AA, T143A and T149A to avoid confusions. We explained this briefly in the first part of results, which is also in the description of Figure 1A.13. I am just a bit skeptical about the quantification of immunoblot signals: some of the numbers reported for fold-differences do not match the visual evidence . They appear to be using ECL (rather than fluorescence) for detection and quantification, so these numbers should probably be taken with a grain of salt. I would recommend the authors provide more detail about the quantification, e.g. the raw data for the blot scanning, preferably with error bars.As we explained to Reviewer #1 , all WB images were captured by the LI-COR imaging system, and the luminescence was quantified and calculated accordingly. There was no scanning/photoshopping involved in this process. A detailed description of quantifying band intensities of whole lysates, co-IP and IP WB is added in the 'Materials and methods’ of the revised manuscript.14. There is a sentence fragment on p. 13, starting with "Since the catalytic domains…"Thank you for pointing out this omission. This sentence fragment has been corrected in the revised manuscript.15. The precedent of cyclin E degradation cited on p. 20 is interesting but not quite apt: in that case degradation is promoted by phosphorylation of cyclin E (on a sequence motif known as a phosphodegron), and I am not aware of any requirement for cyclin E phosphorylation in binding to CDK2, so its relevance to the proposed mechanism of cyclin T1 degradation is not clear.In the quoted manuscript, the phosphorylated Cyclin E protein is targeted by SCF E3 ligase complex for degradation. In this section, we wanted to compare the instability of unbound Cyclin E and unstable CycT1 disassociated from CDK9 by its dephosphorylation. This degradation is also important for various cellular functions, as Cyclin E controls the key steps in the cell cycle.Reviewer #3:The manuscript by Huang and colleagues describes two phosphorylations sites in human Cyclin T1, which regulate the interaction to CDK9 and contribute to the binding and stability of Cyclin T1 in quiescent and terminally differentiated cells. The author describe that the pool of free Cyclin T1 is rapidly degraded when it is not phosphorylated on these two respective sites in the cyclin boxes of cyclin T1. The PP1 phosphates inhibitor okadaic acid leads to P-TEFb stabilization, as less CycT1 is degraded, whereas kinase inhibitors targeting PKC result in P-TEFb disassembly and degradation of CycT1. Using a set of different kinase inhibitors, the authors find that PKC-α and PKC-β are responsible for CycT1 phosphorylation.The findings described in this study bring the transcription regulating cyclins, as CycT1, closer to the cell cycle regulating cyclins, which are long known for the tight regulation between expression upregulation and ubiquitination downregulation. These findings are potentially important, as they could shift our view on the transcriptional cyclins and the need for transcriptional CDK regulators , if gene expression and degradation is indeed another layer of regulation of these cyclins.E. coli exhibiting no phosphorylation and binds properly well to CDK9. It would be very beneficial and in support of this study to know the dissociation constants for the interaction partners in a CycT1 phosphorylated and non-phosphorylated state.There is one major conundrum to this study: Multiple biochemical and structural studies on CDK9 and Cyclin T1 have been performed, often by co-expression and co-purification of these two subunits. In none of them (to my knowledge) a phosphorylated T143 or T149 residue was identified. The sentence "Unphosphorylated CycT1 dissociates from P-TEFb and is degraded." (page 20 and similar in the abstract) seems therefore farfetched. If pT143 and pT149 stabilizes the complex in such a profound degree, why are researchers able to form the non-phosphorylated complex in the first place? CycT1 can be expressed from E. coli where post-translational modifications (PTM) are not preserved. Although structural studies by Geyer group used CycT1 expressed in E. coli, these studies did not include CDK9. In fact, there are no studies with P-TEFb expressed from E. coli. This finding has been confirmed by our direct communications with Prof. Price, Alber and Hurley.As described in the answer to Reviewers 1 and 2, all P-TEFb structures  were obtained after the purification of the complex from the eukaryotic (insect S9 cells) cells, where CDK9 and CycT1 are likely phosphorylated. Also, enzymatically active P-TEFb complex can be purified from eukaryotic cells, but not from elec) and polar solvation (ΔEsolv−polar) energies, which is consistent with the previous co-IPs between CycT1/mutant CycT1 and CDK9/mutant CDK9 proteins . Furthermore, new Figure 2D demonstrates that with okadaic acid treatment, total phosphorylation levels in purified WT CycT1(280) are much higher than in its purified mutant CycT1(280)TT143149AA protein after phospho-staining. Taken together, Thr143 and Thr149 in CycT1 are actually phosphorylated in vivo, and such phosphorylation is necessary for P-TEFb assembly in cells. See also additional comments to the editor and Reviewers 1 and 2.In addition, the new table S1 demonstrates that after further Molecular dynamics simulations and MM-GBSA binding energy calculations, the phosphorylation of Thr143 and Thr149 in CycT1 results in a thermodynamically more stable CycT1:CDK9 association. The predominant contribution to the increased binding energy of the complex comes from electrostatic  or aspartic acid (D), was not analyzed in these experiments. To this reviewers' opinion the authors should analyze how a double mutation of CycT1 (T143E/T149E) acts on CDK9 binding, P-TEFb complex formation and the stability of Cyclin T1. Using only the T143A/T149A mutant but not a phosphorylation mimetic is a lack in the design of the study.Please see our answer to the editor and Reviewers 1 and 2.Throughout the entire manuscript, the authors are unprecise and ambiguous with predictions  and 'real' experimental findings. E.g.: Page 18, Discussion, 3 sentence: "Structural analyses revealed that phosphates on Thr143 and Thr149 in CycT1 increase intramolecular and intermolecular binding to specific residues in CycT1 and CDK9, respectively, which potentiates P-TEFb assembly and stabilizes CycT1." In Figure 3A, a model is displayed but not an experimentally determined structure with phosphorylated residues, as 3MI9 does not contain any phosphorylation in Cyclin T1. The figure legend to 3A should clearly say this. The description in the main text is okay, but the text of the figure legend is clearly misleading and almost fraud. This is a model only!Another example is on page 8: "An online database for phosphorylation site prediction  scores Thr143 and Thr149 above the threshold value (default 0.5), indicating that these threonines are potential phosphorylation sites . To verify that Thr143 and Thr149 are the main phosphorylation sites in CycT1, …" The second sentence starting with "To verify …." suggests, that the first observation are real data but not simply predictions. Moreover, the prediction has been made on a 43-residue sequence but not the full CycT1 structure  as I understand. This does not account for any tertiary structure etc. and is therefore not to the highest standard. I would not consider this analysis good evidence for phosphorylation sites.elec) and polar solvation (ΔEsolv−polar) energies. Results of following IP-WBs support the model . In addition, the NetPhos search was first a prediction, not providing actual evidence, but subsequent experiments using mutations in these sites verified this prediction. In the revised manuscript, we made this point clearer.We apologize the confusion. In the revised manuscript, we clarified the prediction and actual experimental findings. First of all, the description of Figure 3A is changed greatly and we emphasized that this is merely a model to provide a better understanding of which residues in the CycT1:CDK9 complex are targeted by Thr143 and Thr149. We also added more predictions based on molecular dynamics simulations and MM-GBSA binding energy calculations , which further indicate that the phosphorylation of Thr143 and Thr149 in CycT1 results in a thermodynamically more stable CDK9:CycT1 association. The predominant contribution to the increased binding energy of the complex comes from electrostatic . I am less persuaded by the evidence implicating PP1 in dephosphorylating cyclin T1, but this is largely owing to the technical difficulty of proving phosphatase specificity in cell-based experiments. Taken together, there is substantial reason to believe, based on the data presented, that presence or absence of PKC-dependent signaling is a determining factor in P-TEFb activity levels in different settings (e.g. resting or exhausted versus activated T cells).We added additional phosphatase inhibitors in the revised manuscript. Examining the literature, we find that experimental procedures followed in our work represent the accepted gold standard. The plethora of PP1 and PP2 subunits renders most other approaches difficult if not impossible.My biggest concern remains, however, that the mechanism the authors propose for this regulation-phosphorylation of two specific residues on cyclin T1 that stabilize an interaction with CDK9-is only incrementally closer to being validated in the revised version. The new data addressing this point were obtained in a complicated setup, in which a severely truncated cyclin T1 fragment (missing residues needed for interaction with CDK9) was expressed in 293T cells and then pulled down and detected in gels by a commercial phosphoprotein-staining reagent, either intact  or after tryptic digestion (2E). Mutation of T143 and T149 to A abolished the signal, which is promising but still preliminary evidence that these sites might be phosphorylated in intact cyclin T1 under physiologic conditions. This does not address the concern, also expressed by Reviewer 3, that this requirement-and the phosphorylations themselves-have escaped previous detection. The authors dismiss this concern by pointing out that structures of P-TEFb were solved for complexes expressed in insect cells, but this is not persuasive; in those structures, phosphorylation on T186 of the CDK9 activation segment was readily observed . Fully active, stable P-TEFb complexes can also be reconstituted from purified CDK9 expressed in insect cells and cyclin T1 purified from bacteria. Finally, a quick check of the PhosphoSite online data base did not turn up either of these sites.We agree that the phosphorylation of T186 was observed in the crystal structure of P-TEFb, but important others in CDK9 and CycT1 were not. In addition, CycT1 from position 1-280 is fully functional in cells when presented to RNA polymerase via RNA (many papers from our and other groups). The full length CycT1 1-726 is only required for DNA presentation. There, the histidine-rich stretch in CycT1 binds to the CTD and directs the modification of RNA polymerase from the distance. In our 2002 manuscript, we discovered that promoters for the most part initiate transcription and enhancers elongate transcription, precisely because of this extended C-terminal half of CycT1. Thus, CycT1 1-280 is a fully functional P-TEFb when presented to RNA polymerase as it begins to synthesize RNA. That is precisely how Tat works! However, CycT1 1-280 has two almost identical large tryptic digests, which necessitated a further truncation to create a unique, identifiable fragment that is phosphorylated by the gold standard for the direct mapping of phorphorylated residues in proteins, that of comparing wild type and mutated peptides for phosphorylated residues. As explained in greater detail below, it would have been impossible to demonstrate the phosphorylation of these adjacent threonines in any other way than with a truncated, validated CycT1 protein. We went to great lengths to explain this logic in the revised manuscript.To the second point, no one has been able to reconstitute P-TEFb from bacterially expressed components. We contacted Alber/Hurley and Tahirov groups, especially those responsible for generating P-TEFb in insect cells and they confirmed this finding. Importantly, they also investigated only P-TEFb lacking the C-terminal half of CycT1, which is unstructured and resisted all attempts at crystallization. Other validated (published) phosphorylated residues in CDK9 also did not appear in these structures.There is a suggestion throughout this review that we did not follow precedent. The only reason this study was undertaken is because of two publications, which revealed that T143, T149 and two other residues in CycT1 were essential for P-TEFb. In our manuscript, we could eliminate contributions of these 2 other residues, which left T143 and T149. Several independent prediction protocols, all freely available on line  revealed that they are bona fide phosphorylation sites, especially for PKC.R is a proteosomal inhibitor that revolutionized the treatment of multiple myeloma). In these studies, they played key roles in reversing HIV latency, especially when combined with PKC agonists. Thus, our findings are not new to us but follow abundant precedent in the literature!In separate work by us and others, we found that CycT1 was degraded in resting and terminally differentiated cells, which was prevented by MG132 and bortezomib  and 2. are spaced close together, which would be difficult if not impossible to access and distinguish by rather bulky antibodies. As to mass spectrometry, we consulted two experts as well. Upon reading the paper, they thought our data were convincing, internally consistent and exhaustive. They pointed out that mass spectrometry is notoriously bad with two adjacent (closely positioned) phosphorylated residues. Agreeing that sampling of free and complexed CycT1 (unphosphorylated and phosphorylated) would present another challenge of abundance and representation. Another problem was with sample preparation and removal of phosphate groups during isolation and purification of P-TEFb. Intrinsic to mass spectrometry, they mentioned acidity, competition for ionization, suppression of signals, unidentifiable peaks and incomplete peptide maps. There are many other considerations, too many to enumerate here . Some relate directly to the published data on CycT1, where only 2 phosphorylated sites were observed in the full-length protein . Using traditional mapping of phosphorylated residues by radiography, Jones and Zhou groups found many more sites in CDK9 and CycT1. Our Figure 2 reveals that there are also additional sites in the truncated CycT1. In conclusion, both experts urged strongly that any mass spectrometric findings would have to be validated by other methods, which they thought we did already. Indeed, they were puzzled that these other accepted and validated measures were not accepted by this reviewer.