C-terminal Repeat Domain Kinase I Phosphorylates Ser2 and Ser5 of RNA Polymerase II C-terminal Domain Repeats

Janice C Jones,Hemali P Phatnani,Timothy A Haystead,Justin A Macdonald,S Munir Alam,Arno L Greenleaf

doi:10.1074/jbc.m402218200

Abstract

The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5. A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern. We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3'-end formation. Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5. We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2. When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD. We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates.

Highlights

The C-terminal domain (CTD)1 of the largest subunit (Rpb1p) of budding yeast RNA polymerase II (RNAPII) is composed of about 26 tandem repeats of Tyr1-Ser2-Pro3-Thr4Ser5-Pro6-Ser7 (YSPTSPS)
We sought to determine the positional specificity of phosphorylation by yeast C-terminal repeat domain (CTD) kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3؅-end formation
Specificity of Monoclonal Antibodies 8WG16, H5, and H14 — Commercially available monoclonal antibodies that are regarded as phosphorylation specific have commonly been employed to determine in vivo CTD phosphorylation patterns and the positional specificity of phosphorylation by CTD kinases

Summary

EXPERIMENTAL PROCEDURES

Materials—Materials and sources were as follows: H5, H14, and 8WG16 antibodies (Covance), DEAE-Sepharose Fast Flow (Amersham Biosciences), MacroPrep CM Support (Bio-Rad), ImmunoPure Immobilized streptavidin (Pierce), protease inhibitor mixture The 300 – 450 mM KCl fractions contained a peak of kinase activity and CTDK-I proteins These fractions were further purified on an immunoaffinity resin made with a rabbit antibody raised against and affinitypurified against a peptide (“S2 peptide”; LSRYNDTSFQTSSRYQGSRY) unique to the N terminus of the Ctk protein (see Ref. 27). Purification of Phosphorylated CTD Peptide Products—Kinase reactions (25 ␮l) were diluted to 100 ␮l in 0.1% trifluoroacetic acid and loaded onto a C18 HPLC column. Phosphoamino Acid Analysis—CTD peptide substrates were phosphorylated in an in vitro kinase assay (as described above). Determination of Antibody-CTD Affinities Using Surface Plasmon Resonance Measurements (BIACORE)—For immobilization of CTD peptides to the streptavidin sensor chip surface, biotinylated CTD peptides (Table I; nonphospho, 2-phospho, 5-phospho, and 2 ϩ 5-phospho peptide) were diluted to ϳ5 ng/ml in phosphate-buffered saline with 0.05% Tween 20. Raw ascites fluid containing monoclonal antibody H5, H14, or 8WG16

TABLE I Description of CTD substrates

RESULTS

TABLE II

DISCUSSION