Peripheral Blood Lymphocytes Mechanisms During Activation Of

Cyclin T1 Expression Is Regulated by Multiple Signaling Pathways and Mechanisms during Activation of Human Peripheral Blood Lymphocytes This information is current as of September 27, 2021. Renée M. Marshall, Dominic Salerno, Judit Garriga and Xavier Graña J Immunol 2005; 175:6402-6411; ; doi: 10.4049/jimmunol.175.10.6402 http://www.jimmunol.org/content/175/10/6402 Downloaded from

References This article cites 61 articles, 38 of which you can access for free at:

http://www.jimmunol.org/content/175/10/6402.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cyclin T1 Expression Is Regulated by Multiple Signaling Pathways and Mechanisms during Activation of Human Peripheral Blood Lymphocytes1

Rene´e M. Marshall, Dominic Salerno, Judit Garriga, and Xavier Gran˜a2

Stimulation of primary human T lymphocytes results in up-regulation of cyclin T1 expression, which correlates with phosphorylation of the C-terminal domain of RNA polymerase II (RNAP II). Up-regulation of cyclin T1 and concomitant stabilization of cyclin-dependent kinase 9 (CDK9) may facilitate productive replication of HIV in activated T cells. We report that treatment of PBLs with two mitogens, PHA and PMA, results in accumulation of cyclin T1 via distinct mechanisms. PHA induces accumulation of cyclin T1 mRNA and protein, which results from cyclin T1 mRNA stabilization, without significant change in cyclin T1 promoter activity. Cyclin T1 mRNA stabilization requires the activation of both calcineurin and JNK because inhibition of either precludes cyclin T1 accumulation. In contrast, PMA induces cyclin T1 protein up-regulation by stabilizing cyclin T1 protein, Downloaded from -apparently independently of the proteasome and without accumulation of cyclin T1 mRNA. This process is dependent on Ca2؉ independent protein kinase C activity but does not require ERK1/2 activation. We also found that PHA and anti-CD3 Abs induce the expression of both the cyclin/CDK complexes involved in RNAP II C-terminal domain phosphorylation and the G1-S cyclins controlling cell cycle progression. In contrast, PMA alone is a poor inducer of the expression of G1-S cyclins but often as potent as PHA in inducing RNAP II cyclin/CDK complexes. These findings suggest coordination in the expression and activation of RNAP II kinases by pathways that independently stimulate gene expression but are insufficient to induce S phase entry in primary T http://www.jimmunol.org/ cells. The Journal of Immunology, 2005, 175: 6402–6411.

yclin T1 is one of four regulatory cyclins (cyclin T1, T2a, that binds a RNA structure in the nascent HIV transcript called T2b, or K) that bind to and activate cyclin-dependent trans-activation response and recruits the cyclin T1/CDK9 com- C kinase 9 (CDK9)3 (1–4). Each T-type cyclin/CDK9 complex but not other T-type cyclin/CDK9 complexes (17, 18). Tat- plex constitutes a distinct positive transcription elongation factor b mediated recruitment of the cyclin T1/CDK9 complex stimulates (P-TEFb) (1, 3). These complexes have been implicated in stim- processive transcription by RNAP II by phosphorylating its CTD

ulating elongation upon initiation, of otherwise paused transcripts, and negative transcription elongation factors (reviewed in Ref. 19). by guest on September 27, 2021 by phosphorylating the C-terminal domain (CTD) of RNA polymer- CD4-positive T lymphocytes are one of the primary targets for ase II (RNAP II) and negative transcription elongation factors (5–12). HIV and are fundamental to viral pathogenesis. Interestingly, HIV It has also been proposed recently that the Saccharomyces cerevisiae replicates more efficiently in activated, as opposed to resting, T and Drosophila melanogaster CDK9 orthologs recruit polyadenyla- cells, as the viral genome integrates poorly in quiescent T cells. tion factors linking transcription with RNA processing (13, 14). Sur- Once integrated, replication depends on viral and host cellular fac- prisingly, in these studies CDK9/Ctk1 was not required for transcrip- tors, which may become limiting, as these cells become resting tional elongation (13, 14). memory cells (20). GST-Tat pull-down experiments suggested that The cyclin T1/CDK9 complex is also known as Tat-associated during activation of PBLs, TAK activity was up-regulated (21). kinase (15, 16). Tat is a HIV protein essential for viral replication Subsequently, we and others (22–24) showed that cyclin T1 expression was up-regulated following stimulation of PBLs by mitogens, such as PMA and PHA, as well as by costimulation with Fels Institute for Cancer Research and Molecular Biology and Department of Bio- anti-CD3/anti-CD28 Abs or cytokines. Cyclin T1 protein up-reg- chemistry, Temple University School of Medicine, Philadelphia, PA 19140 ulation correlated with hyperphosphorylation of RNAP II and HIV Received for publication April 14, 2005. Accepted for publication August 18, 2005. replication in PMA/PHA-treated PBLs (22). Studies using flavopiri- The costs of publication of this article were defrayed in part by the payment of page dol, a potent inhibitor of CDK9 activity, have suggested that P-TEFb charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. activity is required for transcription of several genes (25). HIV rep- 1 This work was supported by Department of Defense, Breast Cancer Research Pro- lication is also very sensitive to flavopiridol and a dominant-negative gram Grant DAMD 17-02-1-0576 (to R.M.M.), National Institutes of Health (NIH) form of CDK9 (25–27). Thus, up-regulation of cyclin T1 expression R01 AI45450 (to X.G.), National Institute of Allergy and Infectious Diseases Career during T cell activation may serve to keep pace with the increase in Development Award Grant K02 AI01823 (to X.G.), and W. W. Smith Grant A9802/ 9901 (to X.G.). The facilities used for this work were supported in part by Shared transcription and/or increased recruitment of the cyclin T1/CDK9 Resources for Cancer Research Grant R24 CA88261-01 and General Clinical Re- complex by inducible transcription factors to specific promoters. The search NIH Grant M01 RR00349. increase in cyclin T1 expression may also facilitate HIV replication 2 Address correspondence and reprint requests to Dr. Xavier Granã, Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, during reactivation of T cells harboring integrated latent HIV AHP Building, Room 308, 3307 North Broad Street, Philadelphia, PA 19140. E-mail genomes. address: [email protected] Primary T cells are fully activated by engagement of the TCR 3 Abbreviations used in this paper: CDK9, cyclin-dependent kinase 9; P-TEFb, pos- and CD28 costimulation (reviewed in Ref. 28). This process is itive transcription elongation factor b; CTD, C-terminal domain; RNAP II, RNA mimicked by costimulation with mitogens, PHA and PMA, which polymerase II; PKC, protein kinase C; CHX, cycloheximide; VSV-G, vesicular sto- ϩ matitis virus-G; LTR, long terminal repeat; snRNA, small nuclear RNA. raise intracellular Ca2 levels and activate protein kinase C (PKC),

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 6403 respectively (28). Given the potential importance of cyclin T1 obtained from Cell Signaling Technology. Rb-826 (PC644-50UG) was up-regulation in the expression of cellular genes and HIV replica- purchased from Oncogene. tion during T cell activation/reactivation, we have dissected the Northern blot analysis signaling pathways required and the mechanisms responsible for up-regulation of cyclin T1 via two distinct T cell mitogens, which Total RNA was isolated using the QIAshredder and RNeasy Mini kit from also stimulate HIV replication (22). We demonstrate that PHA and Qiagen. Prime RNase inhibitor (Eppendorf) was added at a final concentration of 1.0 U/␮l. Five micrograms of total RNA was resolved by form- PMA independently up-regulate cyclin T1 protein levels through aldehyde-denaturing 1% agarose electrophoresis and transferred overnight different mechanisms. We have also identified key signaling steps to nitrocellulose (Hybond-N; Amersham Biosciences) by capillarity in required for cyclin T1 up-regulation by each mitogen. In this re- 10ϫ SSC as described previously (36). Membranes were incubated overnight with random-primer labeled probes at 42°C in hybridization solution gard, PHA-mediated up-regulation of cyclin T1 mRNA and pro- ϫ ϫ (50% formamide, 5 SSC, 1 Denhardt, 100 mg/ml SDS, 20 mM NaPO4, tein requires the activation of both calcineurin and JNK, as inhi- and 10 ␮g/ml denatured herring sperm DNA). Membranes were washed bition of either event prevents cyclin T1 accumulation. In contrast, at 42°C twice with 2ϫ SSC/0.1% SDS and then with 0.1 SSC/0.1% SDS until PMA-mediated stabilization of cyclin T1 protein requires calcium- the wash was free of radioactivity. Bands were visualized by autoradiography. independent PKCs but does not require ERK activity. Our results JNK assay also show coordination of the expression of RNAP II cyclin/CDKs with signals that induce gene expression. Cells were lysed in JNK lysis buffer (25 mM HEPES (pH 7.6), 0.1% Triton X-100, 300 mM NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA with 20 mM ␤-glycerophosphate, 2.0 ␮M DTT, 0.2 mM sodium vanadate, 2 ␮g/ml Materials and Methods leupeptin, and 4 ␮g/ml aprotinin freshly added). Two hundred micrograms Cell culture of lysate was incubated with ϳ5 ␮g of GST-c-Jun (1-79)-bound-Sepharose Downloaded from beads (see below) rocking for2hat4°C. c-Jun-bound complexes were PBLs were isolated as previously described with some modifications (22). pelleted and washed five times with cold PBS and then incubated in 40 ␮l Briefly, 500 ml of whole blood was collected from donors and layered onto of JNK reaction buffer (20 mM HEPES (pH 7.6), 10 mM MgCl ,20mM Ficoll-Paque Plus (Amersham Biosciences) and centrifuged at 650 ϫ g for 2 ␤-glycerophosphate, 0.1 mM sodium vanadate, and 20 ␮M[␥-32P]-ATP 45 min. The mononuclear layer was isolated, transferred to a fresh tube, (5000 cpm/pmol) (PerkinElmer)) for 20 min at 30°C. The reaction was and washed twice with room temperature PBS and resuspended in RPMI stopped by addition of 2ϫ Laemmli’s sample buffer. Samples were resolved 1640 supplemented with 10% heat-inactivated FBS at a concentration of

by 12% SDS-PAGE, and gels were stained, dried, and visualized by auto- http://www.jimmunol.org/ 1.5 ϫ 106 cells/ml. PBLs were obtained after monocyte depletion using radiography. GST-c-Jun (1-79) was expressed and purified as described plastic adherence and adjusted to a concentration of 1.0 ϫ 106 cells/ml, previously (37). plated in 10-cm plates, and incubated overnight at 37°C and 5% CO2. The next day PBLs were activated with PHA (Sigma-Aldrich) at a final con- Transduction of PBLs with luciferase reporter viral vectors centration of 1 ␮g/ml and/or PMA (Sigma-Aldrich) at a final concentration of 1 ng/ml, unless otherwise indicated. To activate PBLs via CD3/CD28 co- PCEIII-CMV-LacZ vector was derived from pHRЈ-CMV-LacZ (38) as de- stimulation, 1 ml of PBS containing 10 ␮g/ml human anti-CD3 (217669; scribed previously (39). PCEIII.cyclinT1.Luc was generated by removing EMD Biosciences) was incubated in 6-cm plates at 37°C for 30 min to allow the cyclin T1 promoter-luciferase cassette from PGL3.cyclinT1.Luc by di- the Ab to attach to the plastic. PBS was removed and cells plated at 1.0 ϫ 106 gesting with XbaI/KpnI. The cyclin T1 promoter spans 2220 bp upstream cells/ml, and 10 ␮g/ml anti-CD28 (D23648; EMD Biosciences) was immedi- of the ATG initiation codon and was fully sequenced (40). PCEIII-CMV- ately added to the appropriate plates. Cells were collected 48 h later. LacZ was digested ClaI/XhoI to remove the CMV-LacZ cassette. Both by guest on September 27, 2021 digests were blunt ended and ligated. 293T cells were cotransfected with Pharmacological inhibitors PCEIII.cyclinT1.Luc (transfer), gag/pol (packaging), and vesicular stoma- titis virus-G (VSV-G) (envelope) constructs to generate cyclin T1-lucif- The following pharmacological inhibitors were used to inhibit particular erase lentiviruses or NL4-3.Luc.R-E- (41, 42) and VSV-G envelope con- signaling pathways: SP600125 (10 ␮M), a JNK inhibitor (BIOMOL) (29); structs to generate long terminal repeat (LTR)-luciferase viruses by FK506 (20 nM), a calcineurin inhibitor (Calbiochem) (30); PD98059 (30 ϩ calcium-phosphate precipitation. Medium was changed 16–20 h following ␮M), a MEK inhibitor (Calbiochem) (30); and rottlerin (5 ␮M),aCa2 - transfections, and medium containing viruses was collected at 24 and 48 h independent PKC inhibitor (Calbiochem) (31). In addition, cycloheximide later. The virus containing medium was centrifuged at 652 ϫ g for 5 min (CHX) (50 ␮g/ml) (Sigma-Aldrich) was used to inhibit protein translation and filter sterilized using a 0.22-␮m filter to remove cellular debris. The (32), actinomycin D (1 ␮g/ml) was used to inhibit cellular transcription for viral medium was ultracentrifuged at 25,000 rpm for 90 min at 4°C. Pellets the RNA stability assays (33) and lactacystin (10 ␮M) (Sigma-Aldrich) to were resuspended in 1 ml of PBS/0.1% BSA and then subjected to a second inhibit proteasome activity (34). round of ultracentrifugation to further concentrate the sample. Pellets were ␮ ϫ 6 Protein assays then resuspended in 200 l of PBS/0.1% BSA. A total of 2.0 10 PBLs was resuspended in 500 ␮l of RPMI 1640 and 10% heat-inactivated FBS Whole cell lysates were prepared under nondenaturing and denaturing con- in 24-well plates. Sequabrene (Sigma-Aldrich) was added to a final conditions. Cells were lysed in nondenaturing lysis buffer (50 mM Tris-HCl centration of 8 ␮g/ml, virus was added, cells were centrifuged at 244 ϫ g (pH 7.4), 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton X-100, for 90 min, and then incubated at 37°C. After 16 h of infection, 1.5 ml of 0.1 mM sodium vanadate, 1 mM PMSF, 10 ␮g/ml leupeptin, 4 ␮g/ml medium was added to bring the concentration to 1.0 ϫ 106 cells/ml, cells aprotinin, and 40 ␮g/ml pepstatin) as reported previously (22). Protein were activated with PHA (1 ␮g/ml) or vehicle and collected 48 h later. concentration was determined by the method of Bradford in the cell lysates. Cells were lysed under nondenaturing conditions and luciferase activity Denaturing lysis conditions were described earlier (35) and modified as measured using the Promega luciferase assay kit as per manufacturer follows. Cells were lysed by adding denaturing lysis buffer (125 mM Tris directions. (pH 6.8), 2.0% SDS, 0.1 M DTT, and 10% glycerol) plus protease inhibitors (Complete Mini Protease Inhibitor Cocktail Tablet; Roche), vortexing Results briefly, immediately boiling for 5 min at 100°C, and then briefly vortexing again. DNA was sheared by passing lysate through a 21G11/2 needle using Cyclin T1 protein expression is independently up-regulated in a 1-cc syringe three times. Debris was pelleted by centrifugation at PBLs by signals that activate T cells ϫ 13,000 g for 10 min at room temperature. Lysates were transferred to It has been previously shown that cyclin T1 protein expression is fresh tubes, and Bradford assays were performed to determine protein concentration. Standards were normalized for SDS concentration. For Western regulated in PBLs stimulated with PMA and/or PHA as well as via blot analysis, 20 ␮g of protein was resolved by SDS-PAGE and transferred costimulation of CD4 positive cells with anti-CD3/anti-CD28 Abs to polyvinylidene difluoride membrane in 10 mM CAPS (pH 11.0) con- or cytokines (22–24). However, these findings were later chal- taining 10% methanol. The following Abs were used: anti-cyclin T1 (SC- lenged by Martin-Serrano et al. (35), who suggested that the ap- 10750), CDK9 (SC-484), cyclin H (SC-609), cyclin D2 (SC-593), cyclin D3 (SC-182), cyclin E (SC-247), cyclin A (SC-596), p53 (SC-6243), and parent increase in cyclin T1 protein levels was likely due to pro- total RB SC-50 were purchased from Santa Cruz Biotechnology. Total teolytic degradation during the preparation of the cell lysates. We ERK1/2 (9102), phospho-ERK1/2 (9101), and Rb-780 (9307) Abs were have carefully re-examined this issue and found that cyclin T1 is 6404 T CELL MITOGEN-SPECIFIC REGULATION OF CYCLIN T1 Downloaded from http://www.jimmunol.org/ FIGURE 1. PHA, PMA, and PMA/PHA up-regulate cyclin T1 protein levels in human PBLs. A, PBLs were activated with PMA (1 ng/ml) and PHA (1 ␮g/ml) or left unactivated and collected at the indicated time points. Cells were lysed under nondenaturing or denaturing conditions, and 20 ␮g of protein lysate was resolved by SDS-PAGE and immunoblotted with anti-cyclin T1 (upper panels). Cyclin T1 protein levels were quantified using MAC BAS. Total ERK1/2 levels were quantified and used to normalize (lower panel). B, Unactivated PBLs (0), unactivated PBLs plus 293 cells (1:1 protein ratio), and 293 cells were collected and lysed under nondenaturing conditions. The indicated amounts of total protein lysate were resolved by 10% SDS-PAGE and immunoblotted with indicated Abs (upper panels). Protein levels were quantified using MAC BAS (lower panel). C, PBLs were activated with PMA, PHA, and PMA/PHA or left unactivated and collected at the indicated time points. Cells were lysed under denaturing lysis conditions, and 20 ␮g of protein lysate was subjected to Western blot (WB) analysis with the indicated Abs. D, PBLs from five different donors were activated with PMA, PHA, and PMA/PHA for 48 h. Protein levels of cyclin T1 were determined by Western blot analysis. by guest on September 27, 2021 clearly up-regulated during T cell activation. Fig. 1A shows up- extent to which each signal induces cyclin T1 protein expression. regulation of cyclin T1 following stimulation of quiescent PBLs Cells were stimulated with PMA (1 or 25 ng/ml), PHA (1 ␮g/ml), with PHA and PMA in whole protein lysates prepared under de- or both signals for 24 and 48 h and then lysed under denaturing naturing conditions immediately after cell harvesting. The samples lysis conditions. Two different PMA concentrations were used to were immediately boiled after resuspending cells in denaturing ensure that the discrepancies between previous studies were not lysis buffer containing 2% SDS and a mixture of protease inhibi- due to differences in the potency of the stimulatory signal (22, 23, tors (see Materials and Methods). Parallel examination of cyclin 35). Stimulation of PBLs led to up-regulation of cyclin T1 steady- T1 levels in cell lysates obtained under nondenaturing conditions state protein levels by 48 h in all the above conditions (Fig. 1C). showed that the levels of cyclin T1 were relatively reduced in CDK9 levels mirrored the increase in cyclin T1 expression, which unstimulated PBLs, suggesting the possibility that a protease could is consistent with CDK9 stabilization dependence on binding to be degrading cyclin T1 in cell lysates prepared under nondenatur- cyclin T1 (43–45). As a control, we measured cyclin A expression, ing conditions. To examine this possibility, we mixed 293 cells, which is only up-regulated by PHA, as well as ERK 1/2 phosphor- which contain stable cyclin T1, with unstimulated quiescent PBLs ylation, which is only induced by PMA. To demonstrate that cyclin and immediately collected them. Whole cell lysates were prepared T1 expression is similarly up-regulated independently of donor under nondenaturing conditions. Fig. 1B shows that significantly variability, we simultaneously analyzed expression in five different less cyclin T1 was detected in the lysates of mixed cells than in donors (Fig. 1D). lysates of 293 cells (cell mixtures were calculated such that ap- proximately half the amount of protein came from each cell type), Cyclin T1 mRNA is induced in PBLs stimulated with PHA but indicating that a protease is present in unstimulated PBLs, which not PMA degrades a portion of cyclin T1 during cell lysis. This protease is Previous studies have suggested that cyclin T1 mRNA levels are inactivated soon after lysis as no further degradation occurs in cell modestly induced by stimulation of PBLs with PMA, which cor- lysates incubated at 37°C (data not shown). Similar results were relates with cyclin T1 protein up-regulation (23). However, it is obtained by mixing nonstimulated (unstable cyclin T1) and stim- not known whether PHA-mediated up-regulation of cyclin T1 is ulated (stable cyclin T1) PBLs (data not shown). Thus, while it is mediated by similar mechanisms. Thus, we measured the effects of clear that cyclin T1 is up-regulated several fold during T cell ac- PMA and/or PHA on cyclin T1 mRNA levels. Northern blot anal- tivation, previous studies slightly overestimated the magnitude of ysis was performed on total RNA isolated from PBLs stimulated cyclin T1 up-regulation (see quantitation; Fig. 1A). for 24 and 48 h, as indicated in Fig. 2A, using a specific cyclin T1 Having established that stimulation of PBLs with both PHA and probe as well as a 7SK small nuclear RNA (snRNA) probe as a PMA leads to increased cyclin T1 expression, we re-examined the loading control and visualized by autoradiography. Interestingly, The Journal of Immunology 6405

that the cyclin T1 gene is not likely a primary response gene candidate. Unexpectedly, Northern blot analysis showed that cyclin T1 mRNA accumulates in PBLs stimulated with PHA in the presence of CHX (Fig. 2B). As expected, the expression of myc, a known primary response gene, was not blocked by the CHX treatment (Fig. 2B). Also, expression of cyclin A, a nonprimary response gene, was completely blocked by the CHX treatment. Of note, cyclin T1 mRNA accumulated with faster kinetics in the presence, rather than in the absence, of CHX, an effect known as superinduction, which is characteristic of primary response genes such as c-myc (46). These results could indicate that cyclin T1 mRNA accumulation in PBLs is a primary response to PHA signaling with unusually slow kinetics. However, previous work has shown that the expression of certain mRNAs is increased by treatment with CHX in the absence of other inducers (47). To test this possibility, PBLs were treated with PHA, CHX, as well as PHA and CHX. Fig. 2C shows that CHX induces cyclin T1 accumulation by itself, suggesting that a short-lived protein negatively reg- Downloaded from ulates cyclin T1 transcription and/or stability.

Cyclin T1 mRNA accumulation is due to cyclin T1 mRNA stabilization rather than cyclin T1 promoter activation

PHA-mediated up-regulation of cyclin T1 mRNA could be a result http://www.jimmunol.org/ of increased RNA transcription, RNA stability, or both. To determine whether PHA stimulation positively regulates the activity of the cyclin T1 promoter, we infected PBLs with a lentivirus expressing a luciferase reporter gene under the control of the cyclin T1 promoter (ϳ2.2 kb upstream of the ATG initiation codon). This promoter region has been reported previously to direct potent reporter activity in a variety of transfected cells (40). As a positive control for regulation of promoter activity, we infected PBLs with a VSV-G-pseudotyped, replication-defective HIV virus expressing by guest on September 27, 2021 a luciferase reporter gene inserted in the NEF gene (41, 42). Thus, luciferase expression is under the control of the HIV LTR. To generate VSV-G-pseudotyped cyclin T1-luciferase lentiviruses, 293T cells were cotransfected with PCEIII.cyclinT1.Luc, Gag-pol, and VSV-G envelope constructs by calcium-phosphate precipitation (Fig. 3A). Viruses were then isolated and concentrated as de- FIGURE 2. Cyclin T1 mRNA is up-regulated in response to PHA, but scribed in Materials and Methods. HIV-luciferase control VSV- not PMA, stimulation of human PBLs. A, PBLs were activated with PMA G-pseudotyped viruses were similarly generated by cotransfecting (1 ng/ml), PHA (1 ␮g/ml), and PMA/PHA and collected at the indicated the NL4-3.Luc.R-E- and VSV-G envelope constructs into 293T time points. Northern blot analysis was performed using the indicated cells. As seen in Fig. 3B, cyclin T1 promoter activity was not probes, and bands were visualized by autoradiography. B and C, PBLs stimulated by PHA. In contrast, and as expected, when PBLs in- were preincubated with CHX (50 ␮g/ml) or vehicle for 1 h and then col- fected with control LTR-luciferase viruses were stimulated with lected or activated with PHA or vector and collected at the indicated time PHA, a 3-fold increase in luciferase activity was detected. These data points. Five micrograms of total RNA was analyzed by Northern blot using show that cyclin T1 promoter activity is not regulated by PHA stim- the indicated probes and visualized by autoradiography. rRNA bands showing equal loading were visualized by ethidium bromide staining. ulation in primary lymphocytes and suggest that the mechanisms leading to cyclin T1 mRNA accumulation are posttranscriptional. To determine whether PHA stimulation of PBLs results in an PHA, but not PMA, resulted in a robust increase in cyclin T1 increase in cyclin T1 mRNA stability, we either activated PBLs ␮ mRNA levels (Fig. 2A). However, stimulation with both mitogens with PHA (1 g/ml) or vehicle for 48 h and applied actinomycin led to a greater increase in cyclin T1 mRNA expression, suggest- D for the last 30 or 90 min before collection. Addition of actino- ␮ ing that additional signaling induced by PMA cooperates with mycin D (1 g/ml) blocks cellular transcription, allowing deter- PHA signaling to induce maximal levels of cyclin T1 mRNA. Of mination of mRNA stability via Northern blot analysis (33). RNA note, 7SK snRNA appears to be down-regulated following PBL was isolated, Northern blot analysis was performed, and bands stimulation with both signals. This down-regulation may, at least were visualized by autoradiography. Fig. 3C shows that PHA ac- partially, result from a decrease in the ratio of 7SK snRNA to total tivation of PBLs results in an increase in cyclin T1 mRNA half- RNA levels due to the general increase in transcription observed life. These data, along with data from Figs. 2A and 3B, demon- following T cell stimulation. strate that PHA activation of PBLs results in increased cyclin T1 As seen in Fig. 2A, the kinetics of cyclin T1 up-regulation fol- RNA stability, which leads to steady accumulation of cyclin T1 lowing stimulation of PBLs with PHA is rather slow, suggesting mRNA by 48 h. 6406 T CELL MITOGEN-SPECIFIC REGULATION OF CYCLIN T1

FIGURE 3. Cyclin T1 mRNA accumulation in PHA-stimulated PBLs is mediated by RNA stabilization, not increased promoter activity. A, Schematic representation of plasmid constructs used to generate VSV-G-pseudotyped viral vectors expressing a Fireﬂy luciferase reporter downstream of the cyclin T1 and LTR promoters, respectively (see text for details). B, PBLs were infected with cyclin T1-Luc (left panel)or LTR-luc (right panel) expressing viruses for 16 h. PBLs were then stimulated with PHA or vehicle and collected 24 and 48 h later. Cells were lysed under nondenaturing conditions, and luciferase activity was measured. C, PBLs were stimulated with vehicle or PHA for 48 h. Acti- nomycin D (1 ␮g/ml) was added for the last 30 or 90 min of activation. RNA was isolated, and 15 ␮g of total RNA was analyzed by Northern blot using a cyclin T1 probe. Bands were visualized by autoradiography and quantiﬁed using Downloaded from MAC BAS. Lower panel, The average of two independent experiments.

JNKs and calcineurin are required for PHA-mediated up- perimental conditions. PBLs were activated with PMA, PHA, or http://www.jimmunol.org/ regulation of cyclin T1 in PBLs both and collected at the indicated time points (Fig. 4A). Cells PHA is a T cell mitogenic plant-lectin, which functions, at least in were lysed under nondenaturing conditions, and lysates were in- part, by binding component chains of the TCR (48). PHA stimu- cubated with GST-c-Jun (1-79) for2hat4°C. JNK assays were performed as described in Materials and Methods. Fig. 4A shows lation of PBLs induces passage through the G0-G1 transition, but entry and progression though S phase requires IL-2 or PMA stim- that JNKs are activated following stimulation of PBLs with PHA and ulation (22, 49). Both calcineurin and JNK are activated by en- PHA/PMA to comparable levels but to a lesser extent with PMA gagement of the TCR, resulting in transduction of signals that me- alone. To demonstrate that c-Jun phosphorylation was indeed cata- diate changes in gene expression (reviewed in Ref. 28). As such, lyzed by JNK, kinase assays were also performed in lysates of PBLs we proceeded to dissect the components of this pathway that are that had been treated with a speciﬁc JNK inhibitor (SP600125) by guest on September 27, 2021 required for cyclin T1 protein and mRNA up-regulation following along with the mitogens. Fig. 4A shows that PHA-induced c-Jun PHA-mediated stimulation of PBLs. We started by performing phosphorylation is inhibited by SP600125. However, the c-Jun JNK assays to determine whether these kinases are activated in phosphorylation induced by PMA appears to be largely resistant to response to PHA and/or PMA stimulation in PBLs under our ex- inhibition. In this assay, it is likely that c-Jun is phosphorylated by

FIGURE 4. PHA-mediated up-regulation of cyclin T1 requires JNK activation. PBLs were incubated with vehicle or SP600125 (10 ␮M), a speciﬁc JNK inhibitor, for 30 min and then collected or activated with PMA, PHA, or PMA/PHA for the indicated times. A, Cells were lysed in nondenaturing kinase assay buffer, JNK assays were performed using c-Jun as exoge- nous substrate, and bands were visualized by autoradiography (see Materials and Methods). B, Total RNA was isolated, and Northern blots were performed. Cyclin T1 mRNA levels were quantiﬁed using MAC BAS (lower panel). C, Cells were lysed under denaturing lysis conditions, and West- ern blots (WB) were performed as in Fig. 1 with the indicated Abs. D, Cells were lysed with nondenaturing kinase assay buffer, and JNK assays were performed as in A. The Journal of Immunology 6407

FIGURE 5. PHA-mediated up-regulation of cyclin T1 requires calcineurin activation. PBLs were incubated with vehicle or FK506 (20 nM), a calcineurin inhibitor, for 30 min and then collected or activated with PHA for the indicated times. Cell lysates were analyzed by Northern blot (A) and Western blot (B). A, Total RNA was isolated, and Northern blots were performed. rRNA is used as loading control. Cy- clin T1 mRNA levels were quantiﬁed using MAC BAS. B, Cells were lysed under denaturing lysis conditions, and 20 ␮g of protein lysate was analyzed by Western blot using the indicated Abs. C, Cells were lysed in nondenaturing kinase assay buffer, and JNK assays were performed as in Fig. 4. Downloaded from

the ERKs, as has been suggested previously (50). To determine the expression of RNAP II CTD kinases and numerous cell cycle http://www.jimmunol.org/ whether JNK activation is important for PHA-mediated up-regu- control proteins as well. lation of cyclin T1, unstimulated PBLs were pretreated for 30 min with vehicle or SP600125 (10 ␮M) and then stimulated with PHA. Calcium-independent PKCs, but not ERKs, are required for As seen in Fig. 4, B and C, up-regulation of cyclin T1 mRNA and PMA-mediated up-regulation of cyclin T1 protein is prevented in the presence of SP600125, which effec- It is known that PMA induces downstream events by activating tively inhibited JNK activity (Fig. 4D), suggesting that JNK acti- PKCs (51, 52). The most abundant PKC in T cells is PKC-␪,a vation controls cyclin T1 expression by stabilizing cyclin T1 calcium-independent PKC, whose activity is up-regulated 30-fold mRNA. CDK9 protein up-regulation mirrored that of cyclin T1 during T cell activation. The activity of PKC-ϰ, a calcium-depen- (Fig. 4C), which is consistent with stabilization of CDK9 by the dent PKC, is up-regulated 4-fold, and all other known PKCs, cal- by guest on September 27, 2021 increased availability of the newly synthesized, limiting, cyclin T1 cium dependent and independent, have either negligible or no in- subunit (43, 44). Up-regulation of cyclin H, the regulatory subunit crease in activity (53). Rottlerin is a calcium-independent PKC of CDK7, which phosphorylates the CTD of RNAP II during transcriptional initiation, was also prevented. Additionally, the expression and phosphorylation status of proteins involved in cell cycle entry was also determined as a control. Fig. 4C shows that PHA also stimulated accumulation of cyclin D2, cyclin D3, and p107, as well as hyperphosphorylation of both p107 and pRB, all of which was prevented by SP600125 treatment, clearly showing that crit- ical events associated with progression through the G1 phase of the cell cycle depend on JNK activity. However, in contrast to cyclin T1, the expression of p107 and cyclins D2 and D3 is regulated at the transcriptional level during cell cycle entry. Next, we examined the dependence of cyclin T1 mRNA and protein up-regulation on the activation of calcineurin. As calcineurin is believed to act upstream of JNK, we anticipated that inhibition of calcineurin using the chemical inhibitor FK506 would block cyclin T1 up-regulation (reviewed in Ref. 28). As anticipated, FK506 blocked cyclin T1 mRNA and protein up-regulation (Fig. 5, A and B). FK506 also prevented accumulation of CDK9, the CTD kinase subunits cyclin H and CDK7, as well as the up- regulation of cell cycle regulators. Consequently, pRB phosphorylation was inhibited. Surprisingly, JNK activity was minimally affected by inhibition of calcineurin to levels that were sufﬁcient to FIGURE 6. PMA-mediated up-regulation of cyclin T1 protein levels re- block the expression of cyclin T1, CDK9, cyclin H, CDK7, and all ϩ quires Ca2 -independent PKC activation but not ERK1/2 activation. PBLs examined cell cycle regulatory proteins (Fig. 5C). Altogether, were incubated with rottlerin (5 ␮M),aCa2ϩ-independent PKC inhibitor (A), these data show that there are two parallel pathways, one JNK or PD98059 (30 ␮M), a MEK1 inhibitor (B), for 30 min and then collected or dependent and the other calcineurin dependent, that are both re- activated with PMA (1 ng/ml) for the indicated times. Cells were lysed under quired for cyclin T1 mRNA stabilization following PHA stimula- denaturing lysis conditions, and cyclin T1, phospho-ERK1/2, and total tion of PBLs. These two pathways appear to independently control ERK1/2 protein levels were analyzed by Western blot (WB). 6408 T CELL MITOGEN-SPECIFIC REGULATION OF CYCLIN T1

FIGURE 8. Signaling pathways and mechanisms responsible for up- regulation of cyclin T1 during T cell activation. PHA-mediated cyclin T1 up-regulation requires JNK and calcineurin activation. The increase in cyclin T1 mRNA and protein is due to cyclin T1 mRNA stabilization. PMA- mediated up-regulation of cyclin T1 requires Ca2ϩ-independent PKCs, but not ERK1/2 activation. The increase in cyclin T1 steady-state protein levels is due to an increase in cyclin T1 protein half-life. Downloaded from

PMA-mediated up-regulation of cyclin T1 is a result of increased protein stability

As demonstrated in Fig. 2A, PMA-mediated up-regulation of the http://www.jimmunol.org/ cyclin T1 protein is not due to an increase in cyclin T1 mRNA levels. Therefore, one possibility is that the increase in cyclin T1 steady-state levels is due to protein stabilization. To test this possibility, we determined whether stimulation of PBLs with PMA results in an increase in cyclin T1 protein half-life. PBLs were stimulated with PMA or vehicle for 48 h, treated with CHX (50 ␮g/ml), and collected 12 and 24 h later. Whole cell lysates obtained under denaturing conditions were analyzed by Western blot FIGURE 7. PMA stimulation increases cyclin T1 half-life through a analysis. A representative experiment is shown in Fig. 7A, and the by guest on September 27, 2021 proteasome-independent pathway. A, PBLs were stimulated with PMA or steady-state levels of cyclin T1 were quantified following densi- vehicle for 48 h and then incubated with CHX (50 ␮g/ml) for the indicated tometry analysis from three separate experiments using three dif- times. Cells were lysed under denaturing lysis conditions, and the levels of cyclin T1 and total ERK1/2 were analyzed by Western blot (WB). B, Cy- ferent donors. Expression values were normalized using total clin T1 levels were quantified using MAC BAS and normalized by total ERK1/2 as a loading control. Cyclin T1 protein half-life was in- ERK1/2 levels. Shown is the average of three independent experiments. C, creased by 12 h in PBLs stimulated with PMA vs control unstimu- PBLs were stimulated with PMA, PHA, or vehicle for 48 h and then in- lated PBLs (Fig. 7, A and B). Because the kinetics of cyclin T1 cubated with CHX (50 ␮g/ml) for the indicated times. Cells were processed up-regulation is slow, this increase in stability is likely sufficient to as in A. D, PBLs were stimulated with PMA or vehicle for 48 h and explain cyclin T1 accumulation over time in the absence of cyclin incubated with lactacystin (10 ␮M), a proteasome inhibitor, for the last 5 h. T1 mRNA up-regulation. Although our data show that PHA in- Cells were lysed under denaturing lysis conditions, and cyclin T1, p53, and duces cyclin T1 mRNA stabilization, it is conceivable that PHA total ERK1/2 expression was analyzed by Western blot. could concomitantly stabilize cyclin T1 protein. Thus, we determined the half-life of cyclin T1 in cells treated with PHA or PMA and in control PBLs. In contrast to PMA, our results clearly show inhibitor that inhibits both PKC-␪ and PKC-␦, with a higher af- that PHA does not result in stabilization of cyclin T1 protein (Fig. finity for PKC-␪. PBLs were pretreated for 30 min with vehicle or 7C). rottlerin (5 ␮M) and then activated with PMA and collected at the We next examined whether the increase in cyclin T1 protein indicated time points. PKC inhibition resulted in a block in cyclin half-life was due to decreased degradation of cyclin T1 by the T1 up-regulation (Fig. 6A). Interestingly, ERK1/2 phosphorylation proteasome in PMA-stimulated cells, as it has been shown previ- was also inhibited, suggesting that PMA-mediated ERK activa- ously that the CDK9/cyclin T1 complex can be found in complexes tion is dependent on activation of PKC-␪ or, less likely, another with the SCFskp2 ubiquitin ligase (44, 54). Our results show no sig- Ca2ϩ-independent PKC. Thus, we asked whether ERK activa- nificant change in cyclin T1 protein levels in either unstimulated or tion was required for PMA-dependent up-regulation of cyclin PMA-stimulated PBLs treated with proteasome inhibitors but dra- T1. Interestingly, pretreating unstimulated PBLs with matic stabilization of a known proteasomal substrate, p53 (Fig. 7D). PD98059, a MEK1 inhibitor, followed by PMA stimulation in These results indicate that cyclin T1 protein stabilization following the presence of PD98059 resulted in dramatic inhibition of PMA stimulation is independent of proteasomal degradation. PMA-induced ERK1/2 phosphorylation but failed to block cyclin T1 up-regulation (Fig. 6B). These data show that although Engagement of the TCR via anti-CD3 stimulation is sufficient to ERK1/2 activation is downstream of a Ca2ϩ-independent PKC, induce cyclin T1 expression most likely PKC-␪, ERK1/2 are not required for PMA-mediated The signaling pathways and the mechanisms involved in up-reg- up-regulation of cyclin T1. ulation of cyclin T1 expression following stimulation of PBLs with The Journal of Immunology 6409

transcriptional initiation (cyclin H and CDK7) and elongation (cyclin T1 and CDK9) through phosphorylation of its RNAP II CTD was observed (Fig. 10 and data not shown). In contrast, the ex-

pression of G1-S cyclins was potently induced by PHA but to a much lesser extent by PMA (Fig. 10). Consistently, no phosphorylation of pocket proteins was induced by PMA (data not shown). As expected, the expression of ERK1/2 was not regulated. These results suggest that both mitogens activate RNAP II CTD CDKs to help mediate the changes in gene expression that are associated with activation of primary T cells.

Discussion We and others (22, 23) have previously reported that cyclin T1 expression is up-regulated following stimulation of human PBLs with PMA and PHA. However, the mechanisms leading to up- regulation of cyclin T1 by independent mitogens have not been studied in detail. In the present study, we report that cyclin T1 FIGURE 9. Stimulation of the TCR via CD3 Abs results in up-regula- expression is regulated independently by two mitogenic pathways tion of cyclin T1. PBLs were costimulated with anti-CD3 and anti-CD28 via distinct mechanisms. Although PHA induces accumulation of Downloaded from Abs as described in Materials and Methods. Cells were collected and pro- both cyclin T1 mRNA and protein, PMA induces cyclin T1 protein cessed for Western blot (WB) analysis as in Fig. 1. up-regulation via stabilization without modulating cyclin T1 mRNA levels. We have also identified the mechanisms and components in each mitogenic signaling pathway that are required for the mitogens PMA and PHA are summarized in Fig. 8. As PHA up-regulation of cyclin T1 (Fig. 8). has been shown to mediate its mitogenic effects through stimula- http://www.jimmunol.org/ tion of the TCR, we determined whether direct engagement of the Cyclin T1 expression is up-regulated during T cell activation TCR via stimulation with anti-CD3 Abs was sufficient to mediate We and others (22, 23) have shown that cyclin T1 expression is cyclin T1 up-regulation. Fig. 9 shows that anti-CD3, but not anti- up-regulated following mitogenic stimulation of human PBLs and CD28, is sufficient to induce cyclin T1 and CDK9 expression. As that this correlates with phosphorylation of RNAP II in vivo and expected, given our results (Figs. 4 and 5), the expression of CDK7 HIV replication (22). However, these studies were later challenged and cyclin H, as well as G -S cell cycle control proteins, was also 1 by a report suggesting that the apparent increase in cyclin T1 ex- up-regulated. pression was due to degradation of cyclin T1 in cell lysates of Expression of RNAP II kinases is coordinated following unstimulated PBLs (35). We have carefully re-examined this issue by guest on September 27, 2021 stimulation with T cell mitogens and found that cyclin T1 protein is clearly up-regulated during costimulation of quiescent PBLs with both PMA and PHA as well Finally, as we have seen a correlation between up-regulation of as by each mitogen singly. Using stringent denaturing conditions, cyclin T1 and other proteins upon stimulation of PBLs with PHA, we typically find that cyclin T1 expression is up-regulated several we performed an experiment with multiple donors to determine fold, with minor variation among donors (Fig. 1). We suggest that whether the expression of cyclins and CDKs involved in the con- the modest increases detected in cyclin T1 protein expression in trol of RNAP II phosphorylation and cell cycle entry was coordi- the Martin-Serrano study (35) may be due to the use of PBMCs, nated following stimulation with PMA and/or PHA. A striking which contain a very significant percentage of monocyte/macro- correlation in the expression and phosphorylation of RNAP II with phages, as well as stimulation with a single mitogen, which leads the expression of the cyclins and CDKs involved in stimulation of to lower induction of cyclin T1 expression than when both mitogens are applied together. In any case, it is clear that lysates should be prepared under denaturing conditions, as there is a protease in quiescent PBLs that apparently targets cyclin T1 for degradation during cell lysis under nondenaturing conditions, even in the presence of protease inhibitors. Unfortunately, this prevents an accu- rate measurement of cyclin T1-associated kinase activity, which can only be measured in nondenaturing lysates. Thus, we conclude that, while previous studies may have slightly overestimated the fold increase in cyclin T1 expression and associated activity, the notion that cyclin T1 expression is up-regulated during T cell activation and its likely relevance in this process remains valid. PHA-mediated regulation of cyclin T1 expression Analysis of cyclin T1 mRNA expression following stimulation with PHA/PMA singly, or in combination, showed that PHA, but not PMA, induces cyclin T1 mRNA. However, costimulation with FIGURE 10. Coordinated expression and phosphorylation of RNAP II PHA and PMA accelerated up-regulation of cyclin T1 mRNA sig- with RNAP II kinases. PBLs from five different donors were stimulated nificantly but not cyclin A mRNA expression, suggesting that simultaneously with PMA/PHA as in Fig. 1 and collected 48 h later. Cells downstream effectors of PMA synergize with PHA signaling in were lysed under denaturing conditions, and the expression of the indicated inducing cyclin T1 mRNA. It is unlikely that the increased ex- proteins was determined by Western blot (WB) analysis. pression of cyclin T1 simply reflects acceleration of steps leading 6410 T CELL MITOGEN-SPECIFIC REGULATION OF CYCLIN T1 to T cell activation because this is not observed for cyclin A the cell cycle (44), the possibility that this ubiquitin ligase regu- mRNA. We also report that the accumulation of cyclin T1 mRNA lates cyclin T1 expression in certain cell types has not been ruled is due to message stabilization and not increased transcription. out. Thus, we tested the possibility that the proteasome could be Interestingly, our data also shows that CHX itself induces cyclin involved in the rapid degradation of cyclin T1 in unstimulated T1 mRNA accumulation, suggesting that cyclin T1 mRNA stabi- PBLs. However, a number of experiments using proteasome in- lization involves depletion of a short-lived protein that stimulates hibitors (lactacystin and MG132) failed to show any significant cyclin T1 mRNA degradation. However, given the effects of CHX stabilization of the cyclin T1 protein in quiescent or PMA stimu- in the absence of mitogens, we cannot conclude that cyclin T1 lated PBLs (Fig. 7 and data not shown). Incubations with protea- mRNA stabilization does not require de novo protein synthesis in some inhibitors were performed for short periods of time (up to PHA-stimulated PBLs. Using PBMCs, as opposed to PBLs, others 5 h) to minimize the possibility of secondary effects caused by have reported that cyclin T1 mRNA levels are up-regulated by stabilization of a protein(s) involved in regulating cyclin T1 sta- PMA (23, 35). Given that PMA does not induce cyclin T1 mRNA bility in unstimulated PBLs. Thus, it appears that the low levels of accumulation in PBLs, the effects observed in PBMCs by others cyclin T1 in unstimulated PBLs are not the result of rapid protea- are likely indirect, possibly due to signaling induced by the mono- somal-dependent degradation. cyte/macrophage fraction present in PBMCs. In agreement with Finally, it is worth noting that, following stimulation of primary this proposal, stimulation with PMA is sufficient to induce T cell T cells with PHA or anti-CD3 Abs, there is a coordinated increase activation in PBMCs (35), but PBLs require costimulation with a in the expression of both cell cycle control proteins and RNAP II second mitogen (22). kinases, coinciding with cell cycle entry into the G1 phase. In

The downstream signaling steps that mediate T cell activation in contrast, in PMA-stimulated PBLs, RNAP II CTD kinases are up- Downloaded from response to engagement of the TCR have been studied in some regulated, but events associated with entry into the G1 phase of the detail (reviewed in Ref. 28). Previous reports have indicated that cell cycle, such as increased expression of G1-S cyclins (Fig. 10) calcineurin is upstream of JNK, whose activation leads to AP1- and pockets protein phosphorylation (data not shown), are poorly dependent stimulation of gene expression (55, 56). Using PBLs, induced. Thus, in PMA-stimulated cells, the up-regulation of we found that PHA stimulation was sufﬁcient in inducing JNK RNAP II CTD kinases is likely in place to keep up with a gene

activation and as potent as costimulation with PHA and PMA (Fig. expression program independent of cell proliferation. It is also http://www.jimmunol.org/ 4). Our experiments demonstrate that the activities of both cal- important to note that the increases in cyclin T1 and CDK9 ex- cineurin and JNK are required for PHA-induced up-regulation of pression are exclusively associated with stimulation of quiescent T cyclin T1 mRNA. Interestingly, we found that an inhibitor of cal- cells, as transformed T cells proliferate in the absence of PMA/ cineurin prevents cyclin T1 mRNA up-regulation without inhibit- PHA mitogens if provided with serum and natively express high ing JNK activation (Fig. 5). This suggests two possibilities: either levels of cyclin T1. Primary T cells are quiescent and respond to JNK is activated by a mechanism independent of calcineurin and TCR/CD28 stimulation by entering the cell cycle and expressing is part of an independent pathway or JNK is upstream of cal- molecules important for T cell function. The coordinated increase cineurin. As calcineurin has been shown to be directly activated by in the expression of cyclin T1 and transcriptional control proteins intracellular Ca2ϩ (reviewed in Ref. 57), we favor the first possi- by both mitogens is likely in place to keep pace with the increased by guest on September 27, 2021 bility (Fig. 9). This study has also revealed that several markers of program of gene expression typical of rapidly proliferating and cell cycle entry and progression through mid-G1 are induced by fully functional activated T cells. PHA. This includes up-regulation of a number of G1-S cyclins and phosphorylation of pRB and p107, which mark passage through Acknowledgments the restriction point. Interestingly, inhibition of either JNK or cal- We thank Qiang Zhou, Scott Shore, Danny Dhanasekaran, Alex Tsyg- ankov, Muneer Hasham, and Clement Lee for cDNA constructs. PNL4- cineurin activities prevents up-regulation of cell cycle markers and 3.Luc.R-E- plasmid was obtained through the National Institutes of Health pocket protein phosphorylation. Up-regulation of RNAP II CTD AIDS Research Reference Reagent Program (National Institute of Allergy kinases (cyclin T1/CDK9 and cyclin H/CDK7) and G1-S cell cycle and Infectious Diseases) from Dr. Nathaniel Landau. We thank Dr. control proteins also occurs via stimulation with anti-CD3 Abs. Gunther Boden, May Truongcao, and the Temple University General Clin- Altogether, this indicates that increased cyclin T1 and CDK9 ex- ical Research Center staff for assisting in the blood collections. We also pression are coordinated with the expression of cell cycle regula- thank Arun L. Jayamaran, Danny Dhanasekaran, and Alex Tsygankov for tors following TCR engagement of quiescent T cells. Of note, cell reading the manuscript and comments. cycle entry in other quiescent cell types, such as primary fibro- Disclosures blasts or T98G cells, does not involve up-regulation of CDK9 and The authors have no financial conflict of interest. cyclin T1 (22, 44). PMA-mediated regulation of cyclin T1 expression References 1. Peng, J., Y. Zhu, J. T. Milton, and D. H. Price. 1998. Identification of multiple Our results show that stimulation of PBLs with PMA does not cyclin subunits of human P-TEFb. Genes Dev. 12: 755–762. result in up-regulation of cyclin T1 mRNA, but cyclin T1 protein 2. Wei, P., M. E. Garber, S. M. Fang, W. H. Fischer, and K. A. Jones. 1998. A novel levels are up-regulated. These effects are mediated by a Ca2ϩ- CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92: 451–462. independent PKC, likely PKC-␪, independently of ERK activation. 3. Fu, T. J., J. Peng, G. Lee, D. H. Price, and O. Flores. 1999. Cyclin K functions In most donors, PMA induces cyclin T1 protein up-regulation to as a CDK9 regulatory subunit and participates in RNA polymerase II transcrip- levels slightly lower than or comparable to those induced by PHA tion. J. Biol. Chem. 274: 34527–34530. 4. Granã, X., A. De Luca, N. Sang, Y. Fu, P. P. Claudio, J. Rosenblatt, (Fig. 1, C and D). Our results show that PMA results in a repro- D. O. Morgan, and A. Giordano. 1994. PITALRE, a nuclear CDC2-related pro- ducible increase in the half-life of the cyclin T1 protein (from 12 tein kinase that phosphorylates the retinoblastoma protein in vitro. Proc. Natl. Acad. Sci. USA 91: 3834–3838. to 24 h). The cyclin T1/CDK9 complex associates with the SCF- 5. Renner, D. B., Y. Yamaguchi, T. Wada, H. Handa, and D. H. Price. 2001. A skp2 E3 ubiquitin ligase, which is involved in targeting multiple highly purified RNA polymerase II elongation control system. J. Biol. Chem. 276: cell cycle regulatory proteins for proteasomal degradation, includ- 42601–42609. 6. Zhu, Y., T. Pe’ery, J. Peng, Y. Ramanathan, N. Marshall, T. Marshall, B. Amendt, ing p27 (58, 59), p130 (34, 60), and myc (61, 62). Although we M. B. Mathews, and D. H. Price. 1997. Transcription elongation factor P-TEFb is have shown that SKP2 does not regulate CDK9 expression during required for HIV-1 tat transactivation in vitro. Genes Dev. 11: 2622–2632. The Journal of Immunology 6411

7. Wada, T., T. Takagi, Y. Yamaguchi, D. Watanabe, and H. Handa. 1998. Evidence 35. Martin-Serrano, J., K. Li, and P. D. Bieniasz. 2002. Cyclin T1 expression is that P-TEFb alleviates the negative effect of DSIF on RNA polymerase II-de- mediated by a complex and constitutively active promoter and does not limit pendent transcription in vitro. EMBO J. 17: 7395–7403. human immunodeficiency virus type 1 Tat function in unstimulated primary lym- 8. Yamaguchi, Y., T. Takagi, T. Wada, K. Yano, A. Furuya, S. Sugimoto, phocytes. J. Virol. 76: 208–219. J. Hasegawa, and H. Handa. 1999. NELF, a multisubunit complex containing RD, 36. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Lab- cooperates with DSIF to repress RNA polymerase II elongation. Cell 97: 41–51. oratory Manual. Cold Spring Harbor Laboratory Press, Plainview. 9. Ivanov, D., Y. T. Kwak, J. Guo, and R. B. Gaynor. 2000. Domains in the SPT5 protein 37. Hibi, M., A. Lin, T. Smeal, A. Minden, and M. Karin. 1993. Identification of an that modulate its transcriptional regulatory properties. Mol. Cell. Biol. 20: 2970–2983. oncoprotein- and UV-responsive protein kinase that binds and potentiates the 10. Kim, S. J., H. D. Lee, P. D. Robbins, K. Busam, M. B. Sporn, and A. B. Roberts. 1991. c-Jun activation domain. Genes Dev. 7: 2135–2148. Regulation of transforming growth factor ␤1 gene expression by the product of the ret- 38. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, inoblastoma-susceptibility gene. Proc. Natl. Acad. Sci. USA 88: 3052–3056. and D. Trono. 1996. In vivo gene delivery and stable transduction of nondividing 11. Kim, J. B., and P. A. Sharp. 2001. Positive transcription elongation factor B phos- cells by a lentiviral vector. Science 272: 263–267. phorylates hSPT5 and RNA polymerase II carboxyl-terminal domain independently 39. Hasham, M. G., and A. Y. Tsygankov. 2004. Tip, an Lck-interacting protein of of cyclin-dependent kinase-activating kinase. J. Biol. Chem. 276: 12317–12323. Herpesvirus saimiri, causes Fas- and Lck-dependent apoptosis of T lymphocytes. 12. Fujinaga, K., D. Irwin, Y. Huang, R. Taube, T. Kurosu, and B. M. Peterlin. 2004. Virology 320: 313–329. Dynamics of human immunodeficiency virus transcription: P-TEFb phosphory- 40. Liu, H., and A. P. Rice. 2000. Isolation and characterization of the human cyclin lates RD and dissociates negative effectors from the transactivation response T1 promoter. Gene 252: 39–49. element. Mol. Cell. Biol. 24: 787–795. 41. He, J., S. Choe, R. Walker, P. Di Marzio, D. O. Morgan, and N. R. Landau. 1995. 13. Ahn, S. H., M. Kim, and S. Buratowski. 2004. Phosphorylation of serine 2 within Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the the RNA polymerase II C-terminal domain couples transcription and 3Ј end pro- G phase of the cell cycle by inhibiting p34cdc2 activity. J. Virol. 69: 6705–6711. cessing. Mol. Cell 13: 67–76. 2 42. Connor, R. I., B. K. Chen, S. Choe, and N. R. Landau. 1995. Vpr is required for 14. Ni, Z., B. E. Schwartz, J. Werner, J. R. Suarez, and J. T. Lis. 2004. Coordination efficient replication of human immunodeficiency virus type-1 in mononuclear of transcription, RNA processing, and surveillance by P-TEFb kinase on heat phagocytes. Virology 206: 935–944. shock genes. Mol. Cell 13: 55–65. 15. Herrmann, C. H., and A. P. Rice. 1993. Specific interaction of the human immuno- 43. O’Keeffe, B., Y. Fong, D. Chen, S. Zhou, and Q. Zhou. 2000. Requirement for deficiency virus Tat proteins with a cellular protein kinase. Virology 197: 601–608. a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 het- Downloaded from 16. Herrmann, C. H., and A. P. Rice. 1995. Lentivirus Tat proteins specifically as- erodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription. sociate with a cellular protein kinase, TAK, that hyperphosphorylates the car- J. Biol. Chem. 275: 279–287. boxyl-terminal domain of the large subunit of RNA polymerase II: candidate for 44. Garriga, J., S. Bhattacharya, J. Calbo, R. M. Marshall, M. Truongcao, a Tat cofactor. J. Virol. 69: 1612–1620. D. S. Haines, and X. Grana. 2003. CDK9 is constitutively expressed throughout 17. Napolitano, G., P. Licciardo, P. Gallo, B. Majello, A. Giordano, and L. Lania. the cell cycle, and its steady-state expression is independent of SKP2. Mol. Cell. 1999. The CDK9-associated cyclins T1 and T2 exert opposite effects on HIV-1 Biol. 23: 5165–5173. Tat activity. AIDS 13: 1453–1459. 45. Chiu, Y. L., H. Cao, J. M. Jacque, M. Stevenson, and T. M. Rana. 2004. Inhi- bition of human immunodeficiency virus type 1 replication by RNA interference 18. Wimmer, J., K. Fujinaga, R. Taube, T. P. Cujec, Y. Zhu, J. Peng, D. H. Price, and http://www.jimmunol.org/ B. M. Peterlin. 1999. Interactions between Tat and TAR and human immunode- directed against human transcription elongation factor P-TEFb (CDK9/cyclin ficiency virus replication are facilitated by human cyclin T1 but not cyclins T2a T1). J. Virol. 78: 2517–2529. or T2b. Virology 255: 182–189. 46. Reed, J. C., J. D. Alpers, P. C. Nowell, and R. G. Hoover. 1986. Sequential 19. Garriga, J., and X. Granã. 2004. Cellular control of gene expression by T-type expression of protooncogenes during lectin-stimulated mitogenesis of normal hu- cyclin/CDK9 complexes. Gene 337: 15–23. man lymphocytes. Proc. Natl. Acad. Sci. USA 83: 3982–3986. 20. Finzi, D., and R. F. Silliciano. 1998. Viral dynamics in HIV-1 infection. Cell 93: 47. Turner, M., D. Chantry, G. Buchan, K. Barrett, and M. Feldmann. 1989. Regulation 665–671. of expression of human IL-1␣ and IL-1␤ genes. J. Immunol. 143: 3556–3561. 21. Yang, X., M. O. Gold, D. N. Tang, D. E. Lewis, E. Aguilar-Cordova, A. P. Rice, 48. Weiss, A., R. Shields, M. Newton, B. Manger, and J. Imboden. 1987. Ligand- and C. H. Herrmann. 1997. TAK, an HIV Tat-associated kinase, is a member of receptor interactions required for commitment to the activation of the interleukin the cyclin-dependent family of protein kinases and is induced by activation of 2 gene. J. Immunol. 138: 2169–2176. peripheral blood lymphocytes and differentiation of promonocytic cell lines. 49. Firpo, E. J., A. Koff, M. J. Solomon, and J. M. Roberts. 1994. Inactivation of a

Proc. Natl. Acad. Sci. USA 94: 12331–12336. Cdk2 inhibitor during interleukin 2-induced proliferation of human T lympho- by guest on September 27, 2021 22. Garriga, J., J. Peng, M. Parreno, D. H. Price, E. E. Henderson, and X. Granã. cytes. Mol. Cell. Biol. 14: 4889–4901. 1998. Up-regulation of cyclin T1/CDK9 complexes during T cell activation. On- 50. Minden, A., A. Lin, T. Smeal, B. Derijard, M. Cobb, R. Davis, and M. Karin. cogene 17: 3093–3102. 1994. c-Jun N-terminal phosphorylation correlates with activation of the JNK 23. Herrmann, C. H., R. G. Carroll, P. Wei, K. A. Jones, and A. P. Rice. 1998. subgroup but not the ERK subgroup of mitogen-activated protein kinases. Mol. Tat-associated kinase, TAK, activity is regulated by distinct mechanisms in pe- Cell. Biol. 14: 6683–6688. ripheral blood lymphocytes and promonocytic cell lines. J. Virol. 72: 9881–9888. 51. Niedel, J. E., L. J. Kuhn, and G. R. Vandenbark. 1983. Phorbol diester receptor 24. Ghose, R., L. Y. Liou, C. H. Herrmann, and A. P. Rice. 2001. Induction of TAK copurifies with protein kinase C. Proc. Natl. Acad. Sci. USA 80: 36–40. ϩ (cyclin T1/P-TEFb) in purified resting CD4 T lymphocytes by combination of 52. Castagna, M., Y. Takai, K. Kaibuchi, K. Sano, U. Kikkawa, and Y. Nishizuka. cytokines. J. Virol. 75: 11336–11343. 1982. Direct activation of calcium-activated, phospholipid-dependent protein ki- 25. Chao, S. H., and D. H. Price. 2001. Flavopiridol inactivates P-TEFb and blocks nase by tumor-promoting phorbol esters. J. Biol. Chem. 257: 7847–7851. most RNA polymerase II transcription in vivo. J. Biol. Chem. 276: 31793–31799. 53. Monks, C. R., H. Kupfer, I. Tamir, A. Barlow, and A. Kupfer. 1997. Selective 26. Flores, O., G. Lee, J. Kessler, M. Miller, W. Schlief, J. Tomassini, and D. Hazuda. 1999. modulation of protein kinase C-␪ during T cell activation. Nature 385: 83–86. Host-cell positive transcription elongation factor b kinase activity is essential and limiting 54. Kiernan, R. E., S. Emiliani, K. Nakayama, A. Castro, J. C. Labbe, T. Lorca, for HIV type 1 replication. Proc. Natl. Acad. Sci. USA 96: 7208–7213. K. Nakayama Ki, and M. Benkirane. 2001. Interaction between cyclin T1 and 27. Chao, S. H., K. Fujinaga, J. E. Marion, R. Taube, E. A. Sausville, SCFSKP2 targets CDK9 for ubiquitination and degradation by the proteasome. A. M. Senderowicz, B. M. Peterlin, and D. H. Price. 2000. Flavopiridol inhibits Mol. Cell. Biol. 21: 7956–7970. P-TEFb and blocks HIV-1 replication. J. Biol. Chem. 275: 28345–28348. 55. Su, B., E. Jacinto, M. Hibi, T. Kallunki, M. Karin, and Y. Ben-Neriah. 1994. JNK is 28. Isakov, N., and A. Altman. 2002. Protein kinase C␪ in T cell activation. Annu. involved in signal integration during costimulation of T lymphocytes. Cell 77: 727–736. Rev. Immunol. 20: 761–794. 56. Avraham, A., S. Jung, Y. Samuels, R. Seger, and Y. Ben-Neriah. 1998. Co- 29. Bennett, B. L., D. T. Sasaki, B. W. Murray, E. C. O’Leary, S. T. Sakata, W. Xu, stimulation-dependent activation of a JNK-kinase in T lymphocytes. Eur. J. Im- J. C. Leisten, A. Motiwala, S. Pierce, Y. Satoh, et al. 2001. SP600125, an anthrapyra- munol. 28: 2320–2330. zolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 98: 13681–13686. 30. Dumont, F. J., M. J. Staruch, P. Fischer, C. DaSilva, and R. Camacho. 1998. 57. Lewis, R. S. 2001. Calcium signaling mechanisms in T lymphocytes. Annu. Rev. Inhibition of T cell activation by pharmacologic disruption of the MEK1/ERK Immunol. 19: 497–521. MAP kinase or calcineurin signaling pathways results in differential modulation 58. Carrano, A. C., E. Eytan, A. Hershko, and M. Pagano. 1999. SKP2 is required for of cytokine production. J. Immunol. 160: 2579–2589. ubiquitin-mediated degradation of the CDK inhibitor p27. Nat. Cell Biol. 1: 193–199. 31. Villalba, M., S. Kasibhatla, L. Genestier, A. Mahboubi, D. R. Green, and A. Altman. 59. Sutterluty, H., E. Chatelain, A. Marti, C. Wirbelauer, M. Senften, U. Muller, and 1999. Protein kinase C␪ cooperates with calcineurin to induce Fas ligand expression W. Krek. 1999. p45SKP2 promotes p27Kip1 degradation and induces S phase in during activation-induced T cell death. J. Immunol. 163: 5813–5819. quiescent cells. Nat. Cell Biol. 1: 207–214. 32. Weinstock, J. V., A. Blum, A. Metwali, D. Elliott, and R. Arsenescu. 2003. IL-18 60. Tedesco, D., J. Lukas, and S. I. Reed. 2002. The pRb-related protein p130 is and IL-12 signal through the NF-␬B pathway to induce NK-1R expression on T regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin li- cells. J. Immunol. 170: 5003–5007. gase SCFSkp2. Genes Dev. 16: 2946–2957. 33. Bickel, M., R. B. Cohen, and D. H. Pluznik. 1990. Post-transcriptional regulation 61. Kim, S. Y., A. Herbst, K. A. Tworkowski, S. E. Salghetti, and W. P. Tansey. of granulocyte-macrophage colony-stimulating factor synthesis in murine T cells. 2003. Skp2 regulates Myc protein stability and activity. Mol. Cell 11: 1177–1188. J. Immunol. 145: 840–845. 62. von der Lehr, N., S. Johansson, S. Wu, F. Bahram, A. Castell, C. Cetinkaya, 34. Bhattacharya, S., J. Garriga, J. Calbo, T. Yong, D. S. Haines, and X. Granã. 2003. P. Hydbring, I. Weidung, K. Nakayama, K. I. Nakayama, et al. 2003. The F-box SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor in human cells. Oncogene 22: 2443–2451. for c-Myc-regulated transcription. Mol. Cell 11: 1189–1200.