Direct targeting and regulation of RNA polymerase II by cell signaling kinases.

Distinct phosphorylation marks are placed on the carboxyl-terminal domain (CTD) of RNA polymerase II (Pol II) during different stages of gene transcription. These phospho-CTD marks function as a molecular recognition code for the recruitment of stage-specific effector proteins. Querying ~80% of the human kinome, we identified 117 kinases that phosphorylate the CTD with a high degree of positional selectivity. The unifying characteristic linking these diverse kinases is that they selectively regulate Pol II at signal-responsive genes. An example of such "direct-at-gene" Pol II regulation is displayed by epidermal growth factor receptor (EGFR), a cell surface receptor tyrosine kinase. More broadly, our atlas of CTD kinases implicates Pol II as a direct regulatory end point for signal-transducing kinases that govern cellular physiology and contribute to the etiology of numerous diseases. Editor's summary: RNA polymerase II (Pol II) function is regulated by phosphorylation at specific residues within its intrinsically disordered C-terminal domain (CTD). Traditionally, only a few closely related kinases were known to target two positions within the heptapeptide repeats of the CTD. Dabas et al. now report that more than 100 additional cell signaling kinases spanning diverse families can selectively phosphorylate previously uncharacterized "orphan" residues of the CTD (see the Perspective by Young). This expanded atlas of the CTD kinome provides a foundation for investigating how signaling kinases directly regulate polymerase function across different physiological and pathological contexts. —Di Jiang INTRODUCTION: Distinct stages of gene transcription are marked by distinctive phosphorylation patterns on the structurally malleable C-terminal domain (CTD) of the RNA polymerase II (Pol II). These stage-specific phospho-patterns function as a molecular recognition code, which is read by different effector proteins to enable productive transcription, cotranscriptional RNA processing, and access to chromatinized genomic DNA. A handful of cyclin-dependent kinases (CDKs) constitute the "canonical" CTD kinases that phosphorylate either the second or the fifth serine residue of the YS2PTS5PS hepta-peptide repeats of the CTD at all Pol II–transcribed genes. Phosphorylation of Ser5 by CDK7 during transcription initiation engages capping enzymes, whereas phosphorylation of Ser2 by CDK9 and CDK12 facilitates productive elongation and cotranscriptional processing of the nascent RNA. Dysfunction of these CTD kinases is linked to the etiology of a diverse array of human diseases. RATIONALE: Although the functional roles of phospho-Ser2 and phospho-Ser5 and the kinases that place those marks have been extensively studied, the functions of the three so-called "orphan" residues (Tyr1, Thr4, and Ser7) and the identity of kinases that modify those sites remain poorly defined. Unbiased mass spectrometric mapping of the CTD revealed that the orphan sites are phosphorylated, and non-CDK kinases, such as HRR25, PLK3, and ABL1, modify those residues. Notably, unlike Ser2 or Ser5, whose phosphorylation has broad effects, mutations of the orphan residues or inhibition of kinases that act on them selectively disrupt the expression of limited sets of genes. The pathways mapped to those genes suggest that of the ~250 phospho-acceptor residues that are densely packed within ~360 residues of the human CTD, nearly 150 orphan sites may be used by other kinases to selectively regulate distinct sets of functionally coherent genes. To bridge the knowledge gap and identify previously unknown CTD-active kinases, we used three orthogonal kinome testing platforms in conjunction with machine learning algorithms to predict kinase-substrate pairings. RESULTS: Of the 427 human kinases surveyed (~80% of the kinome), 117 were identified by high-throughput screening and validated by immunoblotting assays that revealed specific residues phosphorylated by each kinase. Rather than indiscriminate phosphorylation, most of the CTD-active kinases showed notable positional specificity. Orphan residues emerged as preferred substrates of signal transduction kinases, many of which are known to bind and phosphorylate transcription factors (TFs), chromatin, and other nuclear proteins. Unexpectedly, receptor tyrosine kinases (RTKs) emerged as particularly robust CTD kinases. Although their role in signaling at the plasma membrane is well documented, many RTKs, such as epidermal growth factor receptor (EGFR), traffic to the nucleus under physiological and pathological conditions. Our imaging, genomic, biochemical, and functional studies of EGFR reveal a previously unrecognized role in hyperphosphorylating Pol II and selectively regulating the expression of the earliest signal-responsive genes. CONCLUSION: Our study provides a comprehensive atlas of the CTD-active kinome. Most of these diverse CTD kinases play integral roles in signal transduction. We propose that signal-activated CTD kinases, guided by client TFs, phosphorylate and regulate Pol II at signal-responsive target genes. Phosphorylation of orphan residues in the CTD enables selective regulation of Pol II function without perturbing transcription across the genome. The extent to which signaling kinases leverage their ability to phosphorylate the CTD to confer additional layers of signal-responsive gene regulation remains to be explored. Signal-transducing kinases act on RNA Pol II at target genes.: Signaling molecules bind receptors and trigger molecular cascades to regulate signal-responsive genes. These cascades use kinases (K) that bind sequence-selective TFs. TF-associated kinases then phosphorylate the CTD of RNA Pol II to regulate its function. These kinases belong to diverse classes, which determine their preference for specific positions within the CTD hepta-peptide repeats. TK, tyrosine kinase; TKL, tyrosine kinase–like; STE, homologs of yeast sterile/STE kinases; CK1, casein kinase; AGC, PKA/PKG/PKC; CAMK, calcium- and calmodulin-dependent protein kinases; CMGC, CDK/MAP/GSK/CLK; P, Pro; S, Ser; T, Thr; Y, Tyr. [ABSTRACT FROM AUTHOR]