Upregulation of cyclin T1/CDK9 complexes during T cell activation

Abstract

Cyclin T1 has been identi®ed recently as a regulatory subunit of CDK9 and as a component of the transcription elongation factor P-TEFb. Cyclin T1/CDK9 complexes phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (RNAP II) in vitro. Here we report that the levels of cyclin T1 are dramatically upregulated by two independent signaling pathways triggered respectively by PMA and PHA in primary human peripheral blood lymphocytes (PBLs). Activation of these two pathways in tandem is su cient for PBLs to enter and progress through the cell cycle. However, the expression of cyclin T1 is not growth and/or cell cycle regulated in other cell types, indicating that regulation of cyclin T1 expression is dependent on tissue-speci®c signaling pathways. Upregulation of cyclin T1 in stimulated PBLs results in induction of the CTD kinase activity of the cyclin T1/CDK9 complex, which in turn correlates directly with phosphorylation of RNAP II in vivo, linking for the ®rst time activation of the cyclin T1/ CDK9 pair with phosphorylation of RNAP II in vivo. In addition, we report here that endogenous CDK9 and cyclin T1 complexes associate with HIV-1 generated Tat in relevant cells and under physiological conditions (HIV-1 infected T cells). This, together with our results showing that HIV-1 replication in stimulated PBLs correlates with the levels of cyclin T1 protein and associated CTD kinase activity, suggests that the cyclin T1/CDK9 pair is one of the HIV-1 required host cellular cofactors generated during T cell activation.

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

“…Similar to CDK7 and CDK8, CTD kinase activity and protein levels of the CDK9-cyclin T complex appear to not signi®cantly¯uctuate during the cell cycle in most eukaryotic cells (Garriga et al, 1998). However, activation of primary human peripheral blood lymphocytes (PBLs) by treatment with the mitogens phorbol 12-myristate 13-acetate (PMA) or phytohemagglutinin (PHA) coincides with a dramatic up-regulation of intracellular cyclin T protein levels and a corresponding increase of RNAP II phosphorylation Garriga et al, 1998). Although cyclin A or cyclin E protein levels can also be induced by treatment with the same mitogens, activation of the corresponding CDK2 complexes in T-cells requires a second mitogenic signal to counteract repression by p27 (Firpo et al, 1994).…”

“…CDK9 can associate with four different cyclin subunits, three different T-type cyclins (cyclin T1, T2a, and T2b) (Peng et al, 1998b) and cyclin K. Interestingly, cyclin K was originally identi®ed in a yeast screen based on its ability to restore cell cycle progression and to rescue the lethality of G1 cyclin gene deletions (Edwards et al, 1998;Fu et al, 1999). Similar to CDK7 and CDK8, CTD kinase activity and protein levels of the CDK9-cyclin T complex appear to not signi®cantly¯uctuate during the cell cycle in most eukaryotic cells (Garriga et al, 1998). However, activation of primary human peripheral blood lymphocytes (PBLs) by treatment with the mitogens phorbol 12-myristate 13-acetate (PMA) or phytohemagglutinin (PHA) coincides with a dramatic up-regulation of intracellular cyclin T protein levels and a corresponding increase of RNAP II phosphorylation Garriga et al, 1998).…”

“…Five out of the seven amino acid residues (YSPTSPS) comprising the CTD repeat sequence are potential targets of protein kinases and CTD phosphorylation has been shown to play an important role in the regulation of RNAP II transcription activity (Dahmus, 1994(Dahmus, , 1996, RNAP II stability (Bregman et al, 1996;Huibregtse et al, 1997;Mitsui and Sharp, 1999), coupling RNAP II transcription to mRNA processing (Bentley, 1999;Hirose and Manley, 2000), and in transcription-coupled DNA repair (Bregman et al, 1996;Ratner et al, 1998;Mitsui and Sharp, 1999). Furthermore, global changes in RNAP II CTD phosphorylation have been observed during the course of the cell cycle (Parsons and Spencer, 1997;Akoulitchev and Reinberg, 1998), in response to growth factors and mitogens (Dubois et al, 1994;Yang et al, 1997;Garriga et al, 1998;Bonnet et al, 1999), upon virus infection (Rangel et al, 1988;Rice et al, 1994), and in response to DNA damage (Ratner et al, 1998;Bregman et al, 2000;Rockx et al, 2000). Consistent with these observations, the major cellular kinase activities involved in CTD phosphorylation have been implicated in cell cycle regulatory events.…”

Cyclin T: Three forms for different roles in physiological and pathological functions

“…Upregulation of cyclin T1 is not linked directly to cell cycle entry and progression, because it is not evoked by serum stimulation even though this treatment is sufficient to stimulate exit from G0 and progression throughout the cell cycle in many cell types (Garriga et al, 1998). In T cells, a single mitogen is sufficient for activation of the CDK9/cyclin T complex.…”

“…In T cells, a single mitogen is sufficient for activation of the CDK9/cyclin T complex. This suggests that upregulation of cyclin T1 is the main mechanism for activation of the CDK9/cyclin T complex in these cells and that cyclin T1 acts as a rate-limiting positive regulatory subunit (Garriga et al, 1998).…”