ENHANCED PHOSPHORYLATION OF THE C-TERMINAL DOMAIN OF RNA-POLYMERASE-II UPON SERUM STIMULATION OF QUIESCENT CELLS - POSSIBLE INVOLVEMENT OF MAP KINASES

The largest subunit of RNA polymerase (RNAP) II contains at it C-termi nus an unusual domain comprising tandem repeats of the consensus seque nce Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain (CTD) can unde rgo phosphorylation at multiple sites giving rise to a form of the enz yme designated RNAP IIO. The unphosphorylated form is designated RNAP IIA. The largest subunits of RNAPs IIO and IIA are designated IIo and IIa, respectively. In quiescent NIH 3T3 fibroblasts, subunits IIo and IIa are present in comparable amounts. Upon serum stimulation, the amo unt of subunit IIo increases markedly and remains elevated for several hours. The increase of subunit IIo also occurs in transcription-inhib ited cells and, therefore, is not a consequence of serum-activated tra nscription. This observation suggests that serum stimulation activates a CTD kinase and/or inhibits a CTD phosphatase. This hypothesis is su pported by the finding that serum stimulates phosphorylation of a beta -galactosidase-CTD fusion protein expressed in these cells. Furthermor e, an enhanced CTD kinase activity was discovered in lysates from seru m-stimulated fibroblasts and was found to copurify with MAP kinases on a Mono Q column and to bind to anti-MAP kinase antibodies. The idea t hat MAP kinases phosphorylate the CTD in vivo is supported by the obse rvation that subunit IIa, but not subunit IIb which lacks the CTD, is phosphorylated at multiple sites by purified MAP kinase. Consequently, the MAP kinases are a new class of CTD kinases which appear to be inv olved in the phosphorylation of RNAP II following serum stimulation. T his phosphorylation may contribute to the transcriptional activation o f serum-stimulated genes.