Chemical Tools To Decipher Regulation of Phosphatases by Proline Isomerization on Eukaryotic RNA Polymerase II

Abstract

Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts.

Figure 1

Locked proline isosteres. (A) Native proline containing motif in the trans-proline configuration (left) and trans-locked proline isostere (pSer-Ψ[(E)C=CH]-Pro) (right). (B) Native proline containing motif in the cis-proline configuration (left) and cis-locked proline isostere (pSer-Ψ[(Z)C=CH]-Pro) (right). Sequences of synthesized peptides incorporating isosteres are provided in Supplementary Table 1.

Figure 2

Drosophila melanogaster Ssu72 + Symplekin analysis using locked proline peptides. (A) Kinetic analysis of Ssu72 + Symplekin against native, cis-locked, and trans-locked Pro6 containing phospho-Ser5 peptides. Ssu72 + Symplekin shows considerable activity against the cis-locked (goldenrod) compound with significantly lower activity against the native peptide (tomato). Activity against the trans-locked peptide was not detected above background (blue). Data are from three experimental replicates, error bars indicate standard deviation (n = 3). (B) 2F_o – F_c map about cis-locked peptide (goldenrod) contoured to 1σ. Density accounts for residues analogous to Ser2 through Ser7 of a consensus CTD heptad repeat. (C) Surface depiction of Ssu72 + Symplekin. The image has been rotated with respect to panels B and D about a vertical axis along the Ser5 position by ~90° counterclockwise and tilted toward the viewer by an additional ~90°. The locked-proline isostere fits into a restrictive hydrophobic pocket. (D) Alignment of complex crystal structures of Ssu72 + Symplekin containing cis-locked (goldenrod, PDB ID 4ygx) and native (tomato, PDB ID 4imj) peptides. Residues numbered to indicate position in consensus CTD heptad repeat.

Figure 3

Human Scp1 analysis using locked proline peptides. (A) Kinetic analysis of Scp1 against native, cis-locked, and trans-locked Pro6 containing phospho-Ser5 peptides. Scp1 shows comparable and high activity against native (tomato) and trans-locked (blue) peptides. Activity against cis-locked peptide (goldenrod) is observed but nearly 10-fold smaller than that observed for native and trans-locked substrates. Data are from three experimental replicates; error bars indicate standard deviation (n = 3). (B) 2F_o – F_c map about cis-locked peptide (goldenrod, left) and trans-locked (blue, right) contoured to 1σ. Density accounts for residues analogous to Ser2 through Pro6 of a consensus CTD heptad repeat. (C) Alignment of complex crystal structures of Scp1 containing cis-locked (goldenrod, PDB ID 4yh1) and trans-locked (blue, PDB ID 4ygy) peptides. The structures align well except at the Pro6 location, where they are flipped 180° relative to one another. Residues numbered to indicate position in consensus CTD heptad repeat. (D) Surface depiction of Scp1 and cis-locked peptide (goldenrod). (E) Surface depiction of Scp1 and trans-locked peptide (blue).

Figure 4

Proline isomer specificity of Fcp1. (A) Kinetic analysis of Fcp1 against native, cis-locked, and trans-locked Pro3 containing phospho-Ser2 peptides. Fcp1 shows comparable and higher activity against native (tomato) and trans-locked (blue) peptides. Activity against cis-locked peptide (goldenrod) is observed but lower than that observed for native and trans-locked substrates. Native and trans-locked data are from three experimental replicates; error bars indicate standard deviation (n = 3). cis-Locked data is from one experimental replicate (n = 1). (B) Fcp1/Pin1 coupled assay. All trials show comparable activity with or without Pin1 supplementation. Error bars indicate standard deviation (n = 3).

Figure 5

In vitro reconstruction Pin1 mediated Ssu72 enhancement. (A) Western blot against TFIIH phosphorylated GST-CTD dephosphorylated by Ssu72 with or without Pin1. Phospho-Ser5 (pSer5) was monitored for reactions containing Ssu72 alone (top) or Ssu72 + Pin1 (bottom) over the indicated time course. Mouse IgG heavy chain (Ctrl) was introduced during reaction quenching to provide a loading control. (B) Quantification of Western blot. Phospho-Ser5 bands were first normalized to loading control and then relative to the respective zero time point for each condition. Blot and quantification represent one experimental replicate of three independent experimental replicates, all displaying increased dephosphorylation upon Pin1 supplementation.

Figure 6

Impact of Pin1 knockdown on CTD phosphorylation states in HeLa cell lines. (A) Western blot analysis of phosphorylated Ser2 (top) and phosphorylated Ser5 (bottom) in HeLa cell lines transformed with either empty vector (left) or shPin1 containing vector (right). Blots were performed using three biological replicates of both the empty vector control and shPin1 containing vector cells. Control and knockdown protein sample pairs were prepared in parallel. The three paired sample sets were analyzed on three separate blots. (B) Quantification of Western blot. Phosphorylated Ser5 levels increase 30–60% upon Pin1 knockdown. Quantification of Western blot was performed by first normalizing control and shPin1 samples to the endogenous loading control (β-actin). The shPin1 samples were then normalized to the paired vector control samples. Significance was assessed using Welch’s t test (n = 3).

Figure 7

Model of differentiated regulation mediated by proline isomerization of CTD in RNA polymerase II dephosphorylation. Trans-preferred or specific phosphatases, Scp1 and Fcp1, have substrate consistently available due to the thermodynamic preference for trans-proline in the CTD. Therefore, these enzymes bypass regulation by prolyl isomerases like Pin1. However, cis-specific phosphatases like Ssu72 rely on a minor substrate pool containing the cis-proline isomer and quickly deplete their available substrate. Prolyl isomerases, like Pin1, can restore the equilibrium between cis and trans isomers and replenish substrate pools. This regulatory switch provides for proper RNA polymerase II CTD phosphorylation levels and normal transcription termination. Upon Pin1 disruption or knockdown, global transcription defects, like read-through, may occur.