Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Transcription

1 Supplementary Information to Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition Nadine Czudnochowski 1,2, *, Christian A. Bösken 1, * & Matthias Geyer 1 1 Max-Planck-Institut für molekulare Physiologie, Abt. Physikalische Biochemie, Otto-Hahn- Straße 11, Dortmund, Germany. 2 Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.G. ( matthias.geyer@mpi-dortmund.mpg.de). Supplementary Information contains: Supplementary Figures S1-S10

2 Supplementary Figure S1: Setup of a P-TEFb kinase assay using recombinant protein. (a) SDS PAGE display of Cdk9/CycT1 purified from baculovirus infected insect cells. Cdk9 contained an N-terminal hexahistidine tag for affinity purification. (b) ESI mass spectrometry of purified P-TEFb indicated mono-phosphorylation of Cdk9. The mass of human Cdk was calculated to 42,905.6 Da, whereas the determined mass showed an additional +80 Da corresponding to one phosphorylation group. (c) Size exclusion chromatography of P-TEFb indicated the homogeneity of the heterodimeric complex. Shown is the elution profile of an analytical Superdex 200 column. (d) Activity of P-TEFb for CTD substrate phosphorylation. A linear dependency of the kinase activity on the P-TEFb concentration was observed using excess CTD as substrate and ATP as the cosubstrate. The activity (in counts per minute) of radioactive labelled GST-CTD by transfer from [ 32 P]- -ATP to the substrate was measured in a filter binding assay. Data represent the mean ± s.d. from three independent experiments

3 Supplementary Figure S2: Time course of full length CTD phosphorylation by P-TEFb. A wild type GST-CTD containing all 52 hepta-repeats was fully phosphorylated by P-TEFb. To a reaction of 10 µm CTD (amino acids ) fused to GST and 3 mm ATP as co-substrate, 0.2 µm P-TEFb was added. In this assay, full saturation was achieved after one hour incubation time at 30 C. The P-TEFb mediated phosphorylations of the substrate lead to a large shift in the migration of GST-CTD

4 Supplementary Figure S3: Schematic depiction of a distributive or processive phosphorylation mechanism. (a) Cartoon of a distributive reaction mechanism. A random spreading of the phosphorylation events by the P-TEFb kinase occurs on the matrix of available modification sites on the substrate CTD molecules. (b) In contrast, in a processive reaction mechanism a succession of phosphorylation events is adhered to follow a defined direction and order. (c) Scheme of a phosphorylation pattern according to a distributive reaction mechanism. The histogram in the right hand panel indicates the number of substrate molecules sorted by the number of phosphorylations. (d) Schematic phosphorylation pattern according to a processive reaction mechanism. In contrast to the distributive mechanism, substrate molecules containing a high number of modifications are present significantly earlier, while at the same time substrate molecules that do not contain any modification ('null') persist significantly longer. The phosphorylation pattern generated by these two reaction mechanisms on the substrate matrices can therefore be well distinguished from the histograms of mass distribution

5 Supplementary Figure S4: P-TEFb is unable to phosphorylate CTD substrates continuously pre-phosphorylated at either Tyr1 or Thr4. (a) Design of CTD substrate peptides with phosphorylation marks continuously set at position Tyr1 (py1-ctd [3] ) or Thr4 (pt4-ctd [3] ). The consensus CTD peptide is displayed for clarity on top. Peptides were designed to be unambiguously assignable in the ESI-MS by alternating addition of one residue at the N- or C-terminus. (b) ESI-MS analyses of the py1-ctd [3] and pt4-ctd [3] peptides before and after 4h incubation with P-TEFb. Compared to the consensus CTD or the Ser7 prephosphorylated CTD, these peptides showed no significant increase in mass that would be indicative for a priming of such modified substrates as P-TEFb ligands. Only a small fraction of one additional phosphorylation is seen that might be unspecific

6 Supplementary Figure S5: Phosphorylation of CTD-peptides with full length P-TEFb. (a) Five synthesized CTD peptides consensus-ctd, ps2-ctd, ps5-ctd, ps7-ctd and K7-CTD containing each three hepta-repeats were incubated at a concentration of 100 µm with full length P- TEFb at a concentration of 0.1 µm and 1 mm ATP. The reaction was conducted for 15 min at 30 C and stopped by addition of EDTA. Whereas the pre-phosphorylated peptides ps2-ctd and ps5-ctd were not additionally phosphorylated by P-TEFb, cons. CTD, ps7-ctd and K7-CTD were highly phosphorylated. P-TEFb showed an increased activity for pre-phosphorylated ps7-ctd, but also K7- CTD was readily modified. (b) The phosphorylation kinetics of full length P-TEFb (Cdk , CycT ) was compared to P-TEFb with C-terminally truncated CycT1 (Cdk , CycT ) in a radioactive kinase assay using a MBP-CTD with 9 consensus hepta-repeats. To a concentration of 10 µm MBP-CTD [9] and 2 mm ATP, 0.2 µm P-TEFb was added. With a K cat /K M value of 1.57x10 3 M -1 s -1 for C-terminally truncated P-TEFb the kinase activity was about five-fold higher than those recorded for full length P-TEFb with a K cat /K M value of 287 M -1 s -1. Note, that in full length P-TEFb the Cdk9 subunit contained an N-terminally fused GST-His 6 affinity tag for purification. Data in panels a and b represent the mean ± s.d. from two independent experiments. (c) SDS PAGE analysis using full length CTD and full length P-TEFb. GST-CTD [52] substrate at a concentration of 10 µm was incubated with 3 mm ATP and 0.2 µm P-TEFb full length for 12 h at 30 C. To analyse the effect of HIV-1 Tat and TAR RNA on P-TEFb activity, about 2 µm Tat 1-86 or 2 µm Tat/TAR was added to P-TEFb prior to the reaction start. Despite the different activity, both enzymes achieve approximately the same number of phosphorylations on the CTD substrate as concluded from the similar migration of the phosphorylated product. HIV-1 Tat or Tat/TAR does not increase the number of phosphorylations on the full length CTD substrate

7 Supplementary Figure S6: Order reversal of Cdk7 and Cdk9 kinase modifications does not induce higher phosphorylation states in the CTD. (a) ESI-MS analysis of Cdk7 phosphorylations on a consensus GST-CTD [9] substrate with and without P-TEFb pre-phosphorylations. Incubation of the GST-CTD [9] substrate with the Cdk7/CycH/ MAT1 complex under saturating conditions overnight led to about six to eight phosphorylations of the CTD (upper line, left and right panel). Phosphorylation of the GST-CTD [9] substrate with P-TEFb (Cdk9/CycT1) resulted in 8 to 9 phosphorylations as described above (lower line, left panel). Subsequent incubation of such pre-phosphorylated substrate with Cdk7/CycH/MAT1 did not increase the number of phosphorylations to more than nine in total, corresponding to one phosphorylation per repeat. Thus, reversal in the order of reaction events first incubation with Cdk9 followed by incubation with Cdk7 on the CTD consensus repeats does not result in a Ser2/Ser5 double phosphorylation pattern. (b) Same experiment series as in a, now using a GST-K7-CTD [13] substrate, modified with a lysine at position 7 in all hepta-repeats. As before, no indication for a Ser2/Ser5 double phosphorylation in one CTD repeat could be observed. The S 5 PKY sequence might however comply with a bona fide substrate recognition motif for cyclin dependent kinases

8 Supplementary Figure S7: P-TEFb kinase activity, its interaction with substrates and inhibition by Hexim1. (a) Control experiments performed for the quantitative analysis of P-TEFb activity. The relative kinase activity at standard conditions (see Methods section) was set to 100% (lane 1). Lack of substrate CTD (lane 2) or P-TEFb enzyme (lane 3) abrogated the reaction mechanism. Likewise, GST protein instead of CTD substrate exhibited only slight phosphorylation counts. In contrast, addition of the solvent DMSO (lane 5) or of a protein of cytosolic localization with no function in transcriptional regulation (Ykt6, lane 6) did not impair the kinase activity of P-TEFb for its substrate CTD. Data represent the mean ± s.d. from three independent experiments. (b) Isothermal titration calorimetry experiment of 90 µm GST-CTD [9] injected from the syringe in 19 successive steps of 2 µl to 9 µm P-TEFb placed in the measurement cell. The isotherm showed no indication for a measureable binding affinity under these conditions, suggesting that the dissociation constant between P-TEFb and its consensus substrate CTD is at least higher than 20 µm. (c) SDS PAGE display of Hexim1 proteins exerted for the analysis of P-TEFb inhibition. Proteins were run on 18% polyacrylamide gels and stained with Coomassie blue

9 Supplementary Figure S8: Addition of HIV-1 Tat/TAR to P-TEFb does not increase the number of CTD substrate phosphorylations. ESI-MS analysis of GST-CTD [9] substrate molecules before start of the experiment (left panel), after incubation with P-TEFb for 5 h (middle panel), and after incubation with P-TEFb and HIV-1 Tat/TAR for 5 h (right panel). The maximal phosphorylation status of plus nine phosphate groups (+720 Da) corresponding to one phosphorylation per hepta-repeat was not exceeded by the presence of HIV-1 Tat/TAR

10 Supplementary Figure S9: Addition of HIV-1 Tat to P-TEFb does not change the substrate specificity for RNAPII CTD. Comparison of full length wild type GST-CTD phosphorylation by P-TEFb with and without HIV-1 Tat. (a+b) Same panels as in Figure 4c. Time course of full length GST-CTD phosphorylation by P-TEFb monitored over 20h and analysed by SDS PAGE coomassie staining. For clarity, the anti-ps2 Western blot is displayed again at the bottom with higher exposure times. (c+d) Same panels as in Figure 7d. Time course of full length GST-CTD phosphorylation by P-TEFb Tat monitored over 20h and analysed by SDS PAGE coomassie staining. Compared to P-TEFb alone, no significant increase in the kinetics of CTD is observed in the presence of Tat. For clarity, the anti-ps2 Western blot is displayed again at the bottom with higher exposure times

11 Supplementary Figure S10: Substrate specificity of P-TEFb for various CTD templates in the presence of the Super Elongation Complex (SEC) subunit AFF4. (a) Design of five CTD substrate peptides containing besides the consensus sequence various phosphorylation marks continuously set at either position Ser2, Ser5 or Ser7 or the prevailing variation Lys7. (b) AFF4 of the super elongation complex exhibits similar substrate specificities for P- TEFb mediated CTD phosphorylation as HIV-1 Tat. Substrate peptides at a concentration of 100 µm were incubated for 15 min at 30 C with 0.2 µm P-TEFb and 1 mm ATP. Human AFF was optionally added at concentrations of 2 µm. The reaction was stopped by adding 5 µl of 400 mm EDTA. As before P-TEFb exhibits a preference for a CTD substrate pre-phosphorylated at position Ser7, while the consensus CTD got readily phosphorylated. The presence of AFF4 always increased the catalytic activity of P-TEFb for the substrates, similarly as observed for Tat. However, radioactive counts were also increased when only AFF4 and P-TEFb was measured in the absence of CTD substrate (second column). Data represent the mean ± s.d. from measurements in duplicate