10.1101/gr.849004. [PMC free article] [PubMed] [CrossRef] [Google Scholar] 43. splicing factors, highlighting the role of CDK9 in transcription-coupled splicing events. Based on comparison to data, our findings suggest that cellular context fundamentally impacts the activity of CDK9 and specific selection of its substrates. and [3]. Given the large number of kinases and their limited specificity, protein phosphorylation apparently undergoes several layers of regulation. Recruitment of kinases and control of their activity substantially contribute to the regulation of protein phosphorylation [4]. The question of the number of kinases that can participate in phosphorylation of a target site is usually difficult to solution. Kinases can be removed by genetic knockout or by RNA interference-mediated downregulation. Alternatively, the activity of kinases can be inhibited by chemical inhibitors of varying specificity [5]. Notably, such inhibitors are of high therapeutical interest, as many kinases are involved in human malignancy [6]. However, all these approaches usually do not represent a direct proof for the phosphorylation MAPK6 of a substrate by a specific kinase studies recognized multiple novel substrates of CDK9 and previously unknown phospho-acceptor sites [11, 12]. However, such methods cannot provide information about the activity of CDK9 in a cellular context. We have recently produced a human B cell collection expressing an analog-sensitive CDK9 (CDK9as). These cells are homozygous for F103A mutations at CDK9 gene loci, which renders them sensitive to inhibition by a specific adenine analog. By using this cell collection, we previously analyzed the effects Epibrassinolide of CDK9 inhibition in cells and exhibited that CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases [13]. Here, we combined this analog-sensitive cell collection with phosphoproteomics to study the cellular substrates of CDK9 in a quantitative way. RESULTS Analog-sensitive CDK9 cells allow for quantitative phosphoproteomics CDK9as cells were recently used to study the effects of CDK9 inhibition on nascent transcription in cells [13]. Here, we utilized this cell collection to study substrates of CDK9 in a cellular context and quantitate the contribution of CDK9 to individual phosphosites (Supplementary Physique 1A). First, we analyzed RNA Pol II phospho-CTD levels at different time points of 1-NA-PP1 treatment by western blot (Supplementary Physique 1B). Reduction of phosphorylation levels was poor after 15 min but very strong after 2 h of inhibition. Thus, we next decided to treat CDK9as with 1-NA-PP1 for one hour followed by quantitative phospho-proteomics using SILAC (Physique 1A). Three paired replicates were analyzed and 1102 common phosphosites were detected. Phosphosites showed strong correlation among all replicates and Pearson correlation coefficients ranged from r = 0.71 to r = 0.89 (Figure 1B and Supplementary Figure 2). We recognized 120 phosphosites as significantly decreased (substrates Specificity of kinase inhibitors as well as the study of kinase substrates is typically performed methods allow identification of potential CDK9 substrates, they cannot provide information about the activity of CDK9 in cells. Thus, we compared our cellular set of CDK9 substrates to the results of the Fisher lab that decided CDK9 substrates using a combined analog-sensitive and chemical approach [11]. Of 120 cellular substrates, four (HS90B, IWS1, PRRC2A, SRRM2) could be co-identified in the dataset, but only for HS90B we found a matching phosphosite on S255 (Physique 3A). The minimal overlap of cellular and data suggests, that analysis alone limits the understanding of kinases and their inhibitors Epibrassinolide that can be received in such experiments. Open in a separate window Physique 3 (A) Venn diagram depicting the overlap between cellular (this study, CDK9as SILAC) and (11) CDK9 substrates. (B) Model: The study of protein kinases and their substrates fundamentally differs when performed outside of cellular context. Conversation Quantitative phosphoproteomics puts CDK9 in the center of co-transcriptional events The canonical role of CDK9 as the kinase subunit of P-TEFb in the release of promoter-proximal pausing of RNA Pol II is usually well established and has been demonstrated Epibrassinolide in various studies [8C10]. Surprisingly, our list of CDK9 substrates did not contain several of those substrates, that are mostly linked with the canonical role of CDK9, including Pol II CTD, NELF, and DSIF. This might be explained by the complex nature of our sample, in which peptides of these proteins may be masked by others that are more abundant. Importantly, we did not include any fractionation to enrich for specific proteins in our sample preparation to maintain an unbiased approach, and to specifically identify those phosphopeptides that are quantitatively most important. Several high confidence substrates identified in the present study are associated with events in early transcription as well, including Mediator subunit.