Calcium Signaling Pathway Dependent Activation Of P-TEFb Transcription Activity By The Cooperative Function Of PP2B And PP1α

In eukaryotes, the transcription of protein-coding genes is performed by RNA polymeraseⅡin a process consisting of several tightly connected stages designated as pre-initiation, initiation, promoter clearance, elongation and termination. The accumulating evidence has substantially demonstrated that the elongation stage, an ignored stage in the past, is, in fact, the most important and complex platform for the regulation of transcription. Among the numerous of transcription related factors, the positive transcription elongation factor b (P-TEFb) has emerged as the one of the most important factor functioning in control of elongation stage. Consisting of CDK.9 and its regulator Cyclin T1, P-TEFb functions as an RNA polymerase (Pol)Ⅱspecific kinase responsible for the phosphorylation of second serine residue (Ser2) of C-terminal domain of PolⅡ. This phosphorylation is key important for stimulation of PolⅡelongation activity to ensure the synthesis of full-length pre-mRNA. As a general transcription factor, P-TEFb not only is required for the transcription of most mRNA-encoding genes, but also is indispensable for the development of embryo, Moreover, P-TEFb also is a cellular co-factor essential for AIDS virus (HIV-1) replication. The abnormal regulation of P-TEFb activity has been demonstrated as a pathogenesis factor of cardiac hypertrophy and tumor. Our previous reports have established the P-TEFb activity regulation model: the nuclear protein HEXIM1 binds to and inhibits P-TEFb activity with 7SK snRNA as scaffold, and the release of P-TEFb from this inactive 7SK snRNP complex means the activation of P-TEFb. However, how the cell releases the P-TEFb from the association of HEXIM1/7SK snRNA to activate its activity is still remaining largely unknown.Reported here, we demonstrate that the activation of cellular P-TEFb activity is accomplished by the cooperative actions of two major signaling pathways. To explore the signal pathway(s) responsible for mediating the disruption of inactive 7SK snRNP complex, we firstly treated cells with different kinds of agents to screen the efficient treating model. Besides UV irradiation and HMBA short term treatment, we found that the other stimulation clues such as DNA damage-inducing agents, hypoxia-inducing agents, etc, could also induce the disruption of the inactive 7SK snRNP complex, suggesting that P-TEFb may serve as an integrating point of different signal pathways.By irradiation with UV and treatment with HMBA, the two classic agents used for disruption the inactive 7SK snRNP complex, we found the strong calcium influx caused by UV and HMBA in HeLa cells. With the pharmacological and molecular approaches, we demonstrated that calcium/PP2B signaling pathway is an essential but not sufficient pathway for mediating the UV- and HMBA-induced disruption of the inactive 7SK snRNP complex. Subsequently, we found that PP1αsignal pathway is also a necessary, however, insufficient pathway for mediating UV- and HMBA-induced disruption of the inactive complex. By using the co-transfection and co-treatment methods, we finally demonstrated that PP2B and PP1αmust function cooperatively and only when PP2B functions prior to PP1αto disrupt the inactive 7SK snRNP complex. With an antibody specifically against phosphor-Thr186 (pT186) of CDK9, we demonstrated that PP1αcould dephosphorylate the pT186 of CDK9, however, only accomplished when PP1αworked together with PP2B or more directly, when the 7SK snRNP was disrupted by RNase treating. Furthermore, this dephosphorylation was proven, both in vitro and in vivo, as key important for releasing P-TEFb from inactive 7SK snRNP complex. Token together, our data suggest a two-stepwise mechanism for the activation of P-TEFb, in which the function of PP2B may only serve as an accessorial factor causing the conformation change of inactive 7SK snRNP complex via the dephosphorylation on unknown phosphor-residue and make the pT186 of CDK9 accessible for PP1αwhich finally dephosphorylates pT186 of CDK9 to release the P-TEFb from inhibitory complex.