The entry of RNA polymerase II into a productive mode of elongation is controlled, in part, by the postinitiation activity of positive transcription elongation factor b (P-TEFb) (Marshall, N. F., and Price, D. H. (1995) J. Biol. Chem. 270, 12335-12338). We report here that removal of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II abolishes productive elongation. Correspondingly, we found that P-TEFb can phosphorylate the CTD of pure RNA polymerase II. Furthermore, P-TEFb can phosphorylate the CTD of RNA polymerase II when the polymerase is in an early elongation complex. Both the function and kinase activity of P-TEFb are blocked by the drugs 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and H-8. P-TEFb is distinct from transcription factor IIH (TFIIH) because the two factors have no subunits in common, P-TEFb is more sensitive to DRB than is TFIIH, and most importantly, TFIIH cannot substitute functionally for P-TEFb. We propose that phosphorylation of the CTD by P-TEFb controls the transition from abortive into productive elongation mode.

Rat brain phospholipase D1 (rPLD1) belongs to a superfamily defined by the highly conserved catalytic motif (H(X)K(X) 4 D, denoted HKD. RPLD1 contains two HKD domains, located in the N-and C-terminal regions. Deletion mutants of rPLD1 that contained only an N-or C-terminal HKD domain exhibited no catalytic activity when expressed in COS 7 cells. However, when N-terminal fragments containing one of the HKD domains were cotransfected with a C-terminal fragment containing the other HKD domain, PLD activity was restored. Furthermore, immunoprecipitation assays showed that the N-and C-terminal halves of rPLD1 were physically associated when expressed in COS 7 cells. In addition, deletion of 168 amino acids from the N terminus of rPLD1 significantly enhanced basal PLD activity while inhibiting the response to phorbol ester. Likewise, the coexpression of this truncated N-terminal half with the C-terminal half resulted in increased PLD activity. In summary, this study provides direct evidence that the enzymatic activity of rPLD1 requires the presence of the HKD domains in both the N-and C-terminal regions of the molecule. More importantly, the two halves of rPLD1 can associate, and this may be essential to bring the two HKD domains together to form an active catalytic center. These findings provide new insights into the catalytic mechanism of enzymes of the PLD superfamily. Phospholipase D (PLD)1 catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline (1). It also carrys out a phosphatidyl transfer reaction which is used as a specific measure of PLD activity (2). PLD activity has been detected in almost all organisms and is involved in a variety of signal transduction cascades (3, 4). PLD activity has been shown to be regulated by small G proteins, PKC, proteintyrosine kinases, and intracellular Ca 2ϩ. It was first cloned from plant (5) followed by yeast (6). To date, two types of mammalian PLD genes, termed PLD1 and PLD2, have been cloned (7-16). PLD1 has a low basal activity and responds to PKC and small G protein of the ADP-ribosylation factor and Rho families. On the other hand, PLD2 is constitutively active and shows little response to stimuli.Data base searches using PLD1 and other sequences reveal that PLD belongs to a superfamily (17-19) with four highly conserved regions (17). The most prominent conserved sequences reside in regions I and IV and contain the invariant motif, HXK(X) 4 D, denoted HKD. The HKD motif is found in other enzymes (17-19), including phosphatidyltransferases, poxvirus envelope proteins, a Yersinia murine toxin, and several endonucleases, including Nuc (20). Mutation of the HKD motifs residing in region I or IV renders human PLD1 and mouse PLD2 inactive (21). Studies of Nuc also suggest that the histidine in the HKD domain is directly involved in the catalytic reaction by forming a phosphoenzyme intermediate (22). However, the mechanism(s) by which the two HKD domains are organized spatially to form an active catalytic center is not clear. Vaccinia virus protein VP...

In the present study, we have shown by mutagenesis that conserved amino acids in the HKD domains are important for both the catalytic activity and the association between the two halves of rPLD1. Furthermore, we found that rPLD1 could be modified by Ser/Thr phosphorylation. The modification occurred at the N-terminal half of the enzyme, however, the association of the N-terminal domain with the C-terminal domain was required for the modification. The phosphorylation of the enzyme was not required for its catalytic activity or response to PKC␣ and small G proteins in vitro, although the phosphorylated form of rPLD1 was localized exclusively in the crude membrane fraction. In addition, we found that the individually expressed Nand C-terminal fragments did not interact when mixed in vitro and were unable to reconstruct PLD activity under these conditions. It is concluded that the association of the N-and C-terminal halves of rPLD1 requires their co-expression in vivo and depends on conserved residues in the HKD domains. The association is also required for Ser/Thr phosphorylation of the enzyme.