PP2B and PP1alpha cooperatively disrupt 7SK snRNP to release P-TEFb for transcription in response to Ca2+ signaling

Abstract

The positive transcription elongation factor b (P-TEFb), consisting of Cdk9 and cyclin T, stimulates RNA polymerase II elongation and cotranscriptional pre-mRNA processing. To accommodate different growth conditions and transcriptional demands, a reservoir of P-TEFb is kept in an inactive state in the multisubunit 7SK snRNP. Under certain stress or disease conditions, P-TEFb is released to activate transcription, although the signaling pathway(s) that controls this is largely unknown. Here, through analyzing the UV- or hexamethylene bisacetamide (HMBA)-induced release of P-TEFb from 7SK snRNP, an essential role for the calcium ion (Ca2+)-calmodulin-protein phosphatase 2B (PP2B) signaling pathway is revealed. However, Ca2+ signaling alone is insufficient, and PP2B must act sequentially and cooperatively with protein phosphatase 1alpha (PP1alpha) to disrupt 7SK snRNP. Activated by UV/HMBA and facilitated by a PP2B-induced conformational change in 7SK snRNP, PP1alpha releases P-TEFb through dephosphorylating phospho-Thr186 in the Cdk9 T-loop. This event is also necessary for the subsequent recruitment of P-TEFb by the bromodomain protein Brd4 to the preinitiation complex, where Cdk9 remains unphosphorylated and inactive until after the synthesis of a short RNA. Thus, through cooperatively dephosphorylating Cdk9 in response to Ca2+ signaling, PP2B and PP1alpha alter the P-TEFb functional equilibrium through releasing P-TEFb from 7SK snRNP for transcription.

Figure 1.

Ca²⁺ influx is key for UV/HMBA to dissociate HEXIM1 from P-TEFb and activate P-TEFb-dependent transcription. (A) HeLa cells loaded with Fluo-3/AM, a Ca²⁺-specific fluorescent indicator, were preincubated with either buffer or Nifedipine (Nife), treated with HMBA or UV, and then analyzed by fluorescence microscopy with untreated cells as a control. (B) F1C2 cells were pretreated with the indicated amounts of nifedipine and then exposed to UV (left) or HMBA (right). Anti-Flag immunoprecipitates derived from NE were analyzed by Western blotting (WB) for the indicated proteins. The levels of HEXIM1 bound to P-TEFb were quantified, with those in untreated cells set to 100%. (C) F1C2 cells were treated with the indicated amounts of HMBA and/or ionomycin. Anti-Flag immunoprecipitates from NE were subjected to WB and quantification to determine the amounts of HEXIM1 bound to P-TEFb. (D) HeLa cells with an integrated HIV-1 LTR-luciferase gene were treated with the indicated amounts of HMBA and/or ionomycin. Cell lysates were obtained to measure luciferase activity. (E) F1C2 cells grown in either DMEM (+Ca²⁺) or S-MEM (−Ca²⁺) were treated with UV or HMBA. Anti-Flag immunoprecipitates from NE were analyzed by WB. The amounts of P-TEFb-bound HEXIM1 were quantified as in B. (F) F1C2 cells cultured in Ca²⁺/phosphate-free modified DMEM supplemented with the indicated compounds were treated with UV or HMBA. Anti-flag immunoprecipitates were analyzed and quantified as in B. (G,H) HeLa cells with an integrated HIV-1 LTR-luciferase gene were incubated with the indicated compounds. Luciferase activities were analyzed as in D.

Figure 2.

PP2B and calmodulin are required for UV/HMBA-induced dissociation of HEXIM1 from P-TEFb. (A,B) F1C2 cells were preincubated with the indicated amounts of CsA(A) or FK506 (B) and then treated with UV or HMBA. Anti-Flag immunoprecipitates from NE were subjected to Western blotting (WB) and quantification to determine the levels of P-TEFb-bound HEXIM1. (C) HeLa cells cotransfected with the Cdk9-f cDNA and the indicated HA-PP2B-expressing plasmids (CA or IN) or an empty vector (V) were treated with UV or HMBA. Anti-Flag immunoprecipitates from NE were analyzed by WB to determine the levels of P-TEFb-bound HEXIM1. (D) F1C2 cells were preincubated with the indicated amounts of W-7 and then treated with UV or HMBA. Anti-Flag immunoprecipitates were analyzed and quantified to determine the levels of P-TEFb-bound HEXIM1.

Figure 3.

PP1α plays a key role in UV/HMBA-induced disruption of 7SK snRNP. (A,B) F1C2 cells preincubated with the indicated amounts of CalyA(A) or MCLR(B) were treated with UV or HMBA. Anti-Flag immunoprecipitates from NE were analyzed by Western blotting (WB), and the levels of P-TEFb-bound HEXIM1 are quantified below. (C) HeLa cells were cotransfected with cDNA constructs expressing Cdk9-f and the indicated isoforms of PP1 as EGFP fusions. Anti-Flag immunoprecipitates from NE were analyzed by WB and quantification as in A. The levels of the various EGFP-PP1 isoforms in NE were determined by anti-GFP WB. (D) 7SK snRNP immobilized on anti-Flag beads via Cdk9-f was incubated with either buffer alone (−) or PP1α (+). The reaction supernatant (right) and the materials bound to the beads after extensive washes (left) were analyzed by WB and Northern blotting (NB) for the indicated components.

Figure 4.

PP2B and PP1α sequentially and cooperatively dissociate HEXIM1 from P-TEFb and stimulate P-TEFb-dependent transcription. (A) HeLa cells were cotransfected with plasmids expressing Cdk9-f and the indicated HA-tagged PP1α and/or PP2B. Anti-Flag immunoprecipitates from NE were analyzed by Western blotting (WB). and the amounts of HEXIM1 bound to P-TEFb were quantified. The levels of HA-PP1α and HA-PP2B in NE were examined by anti-HA WB. (B) Lysates of HeLa cells transfected with the indicated cDNA constructs or an empty vector were analyzed by glycerol gradient (10%–30%) sedimentation. The distributions of Cdk9 and HEXIM1 in gradient fractions were examined by WB. (C) HeLa cells containing the HIV-1 LTR-luciferase reporter gene were cotransfected with the indicated expression plasmids. Luciferase activities were measured 48 h later. The levels of HA-PP1α and HA-PP2B in transfected cells were examined by anti-HA WB. (D) 7SK snRNP attached to anti-Flag beads via Cdk9-f was incubated with the indicated amounts of recombinant PP1α and/or PP2B. After washing, the levels of HEXIM1 bound to Cdk9-f were determined by WB and quantification. (E) Immobilized 7SK snRNP from F1C2 NE was incubated sequentially with the indicated phosphatases. Extensive washes separated the two incubations. The amounts of HEXIM1 bound to P-TEFb were determined by WB and quantification.

Figure 5.

PP2B induces a more relaxed conformation in 7SK snRNP to facilitate PP1α dephosphorylation of Cdk9 at T186. (A) Anti-Flag immunoprecipitates from NE of HeLa cells expressing wild-type or various mutant Cdk9-f proteins were analyzed by Western blotting (WB) with the indicated antibodies. The levels of HEXIM1 in NE serve as loading controls. (B) Affinity-purified 7SK snRNP was incubated first with RNase A or RNasin and then the indicated amounts of PP1α. The reaction mixtures were analyzed by WB and quantification to determine the levels of bulk and pT186 form of Cdk9. (C) 7SK snRNP was incubated first with RNase A and then buffer or the indicated amounts of PP1α or PP2B. The reaction mixtures were analyzed as in B. (D) 7SK snRNP was incubated with the indicated amounts of PP1α and PP2B in the presence of RNasin. The reaction mixtures were analyzed as in B. (E) HeLa cells were cotransfected with plasmids expressing Cdk9-f and the indicated HA-tagged phosphatases. Anti-Flag immunoprecipitates from NE were analyzed by WB to determine the levels of pT186 and bulk Cdk9. Anti-HA WB was used to monitor the levels of transfected phosphatases in NE. (F) 7SK snRNP was pretreated with (lanes 5,6) or without (lanes 1–4) RNase A and then incubated in reactions containing the indicated components. The reaction mixtures were analyzed by WB with anti-CycT1 antibody. (FL) Full-length; (*) partially degraded CycT1 in untreated 7SK snRNP preparation.

Figure 6.

PP1α-mediated dephosphorylation of Cdk9 at T186 is required for UV/HMBA to disrupt 7SK snRNP and release P-TEFb that can be recruited by Brd4. (A,B) F1C2 cells were treated with HMBA (A) or UV (B) and then harvested at the indicated time points in hours. Anti-Flag immunoprecipitates from NE were analyzed by Western blotting (WB) and quantification to determine the levels of pT186 and P-TEFb-bound HEXIM1. (C,D) F1C2 cells were pretreated with the indicated chemical inhibitors and then subjected to UV irradiation. Anti-Flag immunoprecipitates from NE were analyzed as in A. (E) HeLa cells were cotransfected with the Cdk9-f cDNA and either an empty vector (−) or a mixture of two shPP1α-expressing plasmids (shPP1α #1 and #2) and then treated with UV or HMBA. Anti-Flag immunoprecipitated from NE were analyzed as in A. The abilities of shPP1α #1 and #2 to deplete HA-PP1α but not CycT1 or α-tubulin were confirmed by WB. (F) NEs (right) or anti-Flag immunoprecipitates from NEs (left) of the indicated cell lines were analyzed by WB for the indicated components or pT186 levels. (G) HeLa cells were cotransfected with plasmids expressing Cdk9-f and the indicated HA-tagged phosphatases. Anti-Flag immunoprecipitates from NEs were analyzed by WB to determine the levels of pT186 and the indicated Cdk9-f-associated factors.

Figure 7.

A model depicting the cooperation between PP2B and PP1α to dephosphorylate Cdk9 T-loop and release P-TEFb from 7SK snRNP for transcription in response to Ca²⁺ signaling. UV/HMBA triggers Ca²⁺ influx through the L-type Ca²⁺ channels (LTCC) in the plasma membrane. Coupled to Ca²⁺ entry, activation of calmodulin (CaM) results in the nuclear import of activated PP2B, which targets a yet-to-be identified component of 7SK snRNP (indicated by a question mark [?]; the depiction of HEXIM1 as a target is purely hypothetical) and alters the conformation of the complex. This facilitates the dephosphorylation of Cdk9 at T186 by PP1α, which is activated by UV/HMBA via a separate, as-yet-undefined signaling pathway. As a result of the sequential and cooperative actions of PP2B and PP1α, P-TEFb is released from 7SK snRNP. This process is blocked at various stages by the indicated pharmacological inhibitors. The released, unphosphorylated P-TEFb is transferred to Brd4, which recruits it to the PIC. After the synthesis of a short RNA, Cdk9 is rephosphorylated at T186 by an unknown kinase to become transcriptionally active. Upon completion of transcription, the excess amount of active, pT186-containing P-TEFb is reassembled into 7SK snRNP to maintain a functional equilibrium for optimal growth.