Oncogene (1998) 17, 3093 ± 3102 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Upregulation of cyclin T1/CDK9 complexes during T cell activation Judit Garriga1,2, Junmin Peng4, Matilde ParrenÄ o1,2, David H Price4, Earl E Henderson1,3 and Xavier GranÄ a*,1,2 1Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, Pennsylvania 19140, USA; 2Department of Biochemistry, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, Pennsylvania 19140, USA; 3Microbiology and Immunology, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140, USA and 4Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA Cyclin T1 has been identi®ed recently as a regulatory date in response to intracellular or extracellular signals subunit of CDK9 and as a component of the transcrip- in mammalian cells, although it has been shown that tion elongation factor P-TEFb. Cyclin T1/CDK9 com- the levels of cyclin C mRNA are stimulated by serum plexes phosphorylate the carboxy terminal domain and cytokines (Lew et al., 1991; Liu et al., 1998). All (CTD) of RNA polymerase II (RNAP II) in vitro. Here cyclin/CDK pairs involved in transcription are able to we report that the levels of cyclin T1 are dramatically phosphorylate the C-terminal domain (CTD) of RNA upregulated by two independent signaling pathways polymerase II (RNAP II) in vitro. Multiple kinases triggered respectively by PMA and PHA in primary seem to phosphorylate the CTD of RNAP II in vivo in human peripheral blood lymphocytes (PBLs). Activation a manner that appears to be essential for progression of these two pathways in tandem is sucient for PBLs to through dierent transcriptional stages (Dahmus, enter and progress through the cell cycle. However, the 1996). In this regard, transcriptional initiation and expression of cyclin T1 is not growth and/or cell cycle elongation can be distinguished by the level of CTD regulated in other cell types, indicating that regulation of phosphorylation. cyclin T1 expression is dependent on tissue-speci®c A positive transcription elongation factor (P-TEFb) signaling pathways. Upregulation of cyclin T1 in has been described that enables RNA polymerase II to stimulated PBLs results in induction of the CTD kinase enter productive elongation by phosphorylating the activity of the cyclin T1/CDK9 complex, which in turn CTD and counteracting negative factors which limit correlates directly with phosphorylation of RNAP II in polymerase processivity (Marshall et al., 1996; Mar- vivo, linking for the ®rst time activation of the cyclin T1/ shall and Price, 1995). Cloning of the subunits of P- CDK9 pair with phosphorylation of RNAP II in vivo.In TEFb has revealed that it is a cyclin/CDK pair (Peng addition, we report here that endogenous CDK9 and et al., 1998a; Wei et al., 1998; Zhu et al., 1997). The cyclin T1 complexes associate with HIV-1 generated Tat small subunit of Drosophila P-TEFb was cloned using in relevant cells and under physiological conditions sequence information obtained from the puri®ed (HIV-1 infected T cells). This, together with our results protein (Zhu et al., 1997) and was found to be the showing that HIV-1 replication in stimulated PBLs homolog of the human CDC2-related kinase PI- correlates with the levels of cyclin T1 protein and TALRE (GranÄ a et al., 1994a). We have reported associated CTD kinase activity, suggests that the previously that PITALRE is a primarily nuclear Ser/ cyclin T1/CDK9 pair is one of the HIV-1 required host Thr-Pro-directed kinase (Garriga et al., 1996b) with cellular cofactors generated during T cell activation. activity that is not cell cycle regulated in HeLa and ML-1 (GranÄ a et al., 1994a). Initial studies found most Keywords: PITALRE; CDK; RNA polymerase II; of the active PITALRE in high molecular weight HIV; Tat; peripheral blood lymphocytes complexes containing a number of unidenti®ed proteins. We had suggested that one of these proteins, p95, might be a positive regulatory subunit because the kinase activity of ectopically overexpressed Introduction PITALRE seemed to be limited by the amount of p95 (Garriga et al., 1996a). Subsequently, the large subunits Cyclins are the regulatory subunits of cyclin dependent of both Drosophila (Peng et al., 1998b) and human kinases (CDKs). Several of these complexes have been (Peng et al., 1998a; Wei et al., 1998) P-TEFb were identi®ed hereto with roles in cell cycle regulation and cloned and found to be cyclin proteins. Because the transcription (GranÄ a and Reddy, 1995; Jones, 1997; kinase subunit (PITALRE) required the cyclin subunit Morgan, 1997). The activity of cyclin/CDK pairs with for activity, PITALRE was renamed CDK9 and the roles in cell cycle control is generally regulated by large subunits were called T-type cyclins. growth factors or is dependent on the cell cycle stage. Recent evidence clearly implicates P-TEFb in Tat However, no changes in the activity of cyclin/CDK transactivation of the HIV-1 promoter. Tat pull-down pairs involved in transcription have been reported to experiments provided early evidence of a Tat-associated kinase (TAK) (Herrmann and Rice, 1993, 1995). Tat acts by binding to the nascent transcript containing the TAR sequence and increasing the processivity of RNAP II (Jones, 1997; Jones and Peterlin, 1994). Since *Correspondence: X GranÄ a Received 23 September 1998; revised 29 October 1998; accepted 2 the latter function is similar to that of P-TEFb November 1998 (Marshall et al., 1996; Marshall and Price, 1995) it Upregulation of cyclin T1/CDK9 during T cell activation J Garriga et al 3094 was not surprising that CDK9 was found to associate it is associated with components of the basal with the activation domain of Tat in vitro and in HeLa transcriptional machinery as was found for CDK7 cells overexpressing Tat (Yang et al., 1997; Zhu et al., and CDK8 (Morgan, 1997). To this end, we utilized 1997). Additionally, P-TEFb was found to be essential antibodies to various subunits of basal transcriptional for Tat transactivation both in vitro (Zhu et al., 1997) factors and to RNAP II. Coincidentally, a polyclonal and in vivo (Mancebo et al., 1997). antibody (s235) raised to a peptide corresponding to Multiple cyclin partners for human CDK9 have the carboxyl-terminal end of the large subunit of been identi®ed allowing for dierent forms of P-TEFb TFIIF, RAP74, was found to cross-react strongly and (Peng et al., 1998a). Two genes (T1 and T2) encode speci®cally with the PITALRE-associated protein p95 cyclin T1, T2a and T2b with the latter two diering (see Materials and methods). As demonstrated in from each other only at the carboxyl-terminus due to Figure 1a, s235 and anti-CDK9 antibodies immuno- dierential splicing (Peng et al., 1998a). P-TEFb, precipitate a complex containing p95 and CDK9 in the comprised of CDK9 and any one of the cyclin T absence of competing antigenic peptides (left and lower subunits, is able to stimulate transcription in vitro and panels). A dierent antibody raised to the amino- in vivo from a CMV promoter when co-transfected terminus of RAP74 (anti-RAP74-NT) recognized with CDK9 in HeLa cells (Peng et al., 1998a). While RAP74 in a total protein extract and also in vitro this work was in progress a direct interaction between translated RAP74 (Figure 1a, right panel). However, Tat and cyclin T1 was demonstrated in vitro (Wei et RAP74, was not detected in either CDK9 or s235 al., 1998). This interaction enhances the anity and immunoprecipitates. To ascertain whether the protein speci®city of Tat binding to the HIV RNA element complexes brought down by anti-CDK9 and s235 TAR. It is not clear yet if cyclin T2a or T2b can exhibited similar kinase activities, we performed kinase associate with Tat. assays in the presence or absence of exogenous Activation of T-cells allows strong transcriptional substrates as described in Materials and methods. activation of the HIV promoter and induces productive The kinases immunoprecipitated by both antibodies replication of the virus (Kaufman et al., 1987; Nabel exhibited similar speci®city in that both could and Baltimore, 1987; Siekevitz et al., 1987; Tong- phosphorylate the CTD of RNAP II (Figure 1b, left Starksen et al., 1987). It has also been shown that a panel) and pRB (not shown) and without any CTD activity that associates with GST-Tat is exogenous substrate phosphorylation of p95 and upregulated upon activation of T-cells (Nekhai et al., autophosphorylation of CDK9 was observed with 1997; Yang et al., 1997). Since CDK9 has been found both immunoprecipitates (Figure 1b, right panel). to associate with Tat in vitro and in transient Since the origin of the s235 antibody is not essential transfection experiments in cell lines that support Tat to the results reported here, further characterization of transactivation, it is likely that induction of the GST- this antibody will be reported elsewhere (see Materials Tat-associated CTD activity upon activation of and methods). primary peripheral blood lymphocytes (PBLs) results Three cyclin partners for human CDK9 have from activation of a cyclin/CDK9 complex. However, recently been identi®ed and designated cyclins T1, the mechanism of activation of such a complex in T2a and T2b (Peng et al., 1998a). Since p95 has a stimulated PBLs remains unknown. A number of molecular weight similar to these cyclins, we consid- dierent possibilities can be envisioned including ered whether p95 could correspond to one of these upregulation of the protein levels of a rate limiting proteins. We performed immunoprecipitations with subunit (either by the cyclin, the CDK or both) and anti-CDK9, anti-cyclin T1 and anti-cyclin T2 antibo- activation of preexisting subunits and/or complexes by dies followed by Western blot with s235 (Figure 1c).