The peptidyl-prolyl isomerase Pin1 interacts with hSpt5 phosphorylated by Cdk9

We identify and characterize several phosphorylated forms of the hSpt5 subu nit of the DRB sensitivity-inducing factor (DSIF). A 175-kDa phosphorylated form of hSpt5 is bound to nuclei of interphase HeLa cells. This form is ra pidly dephosphorylated when cultured cells are exposed to various drugs bel onging to distinct chemical families. All these compounds are known to inhi bit the protein kinase Cdk9, which phosphorylates in vitro hSpt5 and Rpb1, the largest subunit of RNA polymerase II. The efficiency to promote the dep hosphorylation of both proteins matches their capacity to inhibit purified Cdk9 kinase, suggesting that Cdk9 is the major kinase phosphorylating hSpt5 and Rpb1 in vivo. We show that Cdk9 phosphorylates both the CTR1 and the C TR2 domains of recombinant hSpt5. These domains contain numerous serine-pro line and threonine-proline residues similar to those found in the carboxyl- terminal domain (CTD) of Rpb1. The structural homology between hSpt5 CTRs a nd the Rpb1 CTD is further highlighted by the presence on both proteins of a phosphoepitope recognized by the monoclonal antibody CC-3. Of particular interest, the peptidyl-prolyl isomerase Pin1 interacts with Cdk9-phosphoryl ated hSpt5. Cdk9 dependent phosphorylation of Rpb1 and hSpt5 followed by Pi n1 interaction might thus contribute to the regulation of transcription, pr e-mRNA maturation, and the dynamics of these proteins in interphase and mit osis. (C) 2001 Academic Press.