Discovery Composition and Biogenesis of PTEFb

Like all other cyclin-dependent kinases (CDKs), CDK9 does not have any catalytic activity unless it complexes with its regulatory partner cyclin T1 (CycTl) to form the CDK9/CycTl heterodimer, which is termed P-TEFb (positive transcription elongation factor b). The 42-kDa CDK9 in complex with CycTl account for -80% of the total P-TEFb in human HeLa cells (Peng et al., 1998; Price, 2000; Wei et al., 1998). Besides CDK9(42), there is also a second isoform of CDK9 called CDK9(55) with a 55-kDa molecular mass and a 117-residue amino terminal extension not present in the 42-kDa form of CDK9 (Shore et al., 2005; Shore et al., 2003). In addition to CycTl, minor CDK9-associated CycT2a, T2b, and K molecules are also present at much lower concentrations than CycTl in many cell types (Price, 2000; Shore et al., 2003). CDK9 was first identified as the cell division cycle 2 (CDC2)-related protein kinase during a cDNA screening intended to isolate novel regulators of the mammalian cell cycle (Grana et al., 1994). Subsequently, it was rediscovered as a component of the P-TEFb complex purified from Drosophila and mammalian cell nuclear extracts (Gold et al., 1998; Herrmann and Rice, 1995; Marshall et al., 1996; Marshall and Price, 1995; Yang et al., 1996; Zhu et al., 1997). Unlike other CDKs with known cell cycle regulatory functions, the expression levels of CDK9 and CycTl as well as the kinase activity of the isolated CDK9/CycTl heterodimer in the absence of any associated factors are fairly constant throughout the cell cycles and also during cell cycle entry from a quiescent state (Garriga et al., 2003; Grana et al., 1994). These observations suggest that the CDK9/CycTl heterodimer of P-TEFb is unlikely to function as a conventional cell cycle regulator, although its activity could still be regulated by the associated factors (see below) for cell cycle progression.

Although a connection of P-TEFb with the cell cycle control is yet to be established, a role for P-TEFb in regulating RNA Pol II transcriptional elongation has become crystal clear. In fact, P-TEFb is required for the expression of most genes in C. elegans and mammals (Chao and Price, 2001; Garriga and Grana, 2004; Shim et al., 2002). When CDK9 in C. elegans early embryo was depleted by RNAi, a technique used to specifically suppress a target gene expression, the CTD phosphorylation on Ser2 but not on Ser5 was found to be dramatically reduced (Shim et al., 2002). These

RNAi experiments further demonstrate that P-TEFb is broadly essential for the expression of early embryonic genes. This result agrees with an earlier observation, which uses a pharmacological inhibitor of CDK9 to show that P-TEFb is required for the expression of most protein-coding genes in mammalian cells (Chao and Price, 2001), indicating that P-TEFb is globally required for the transcription of cellular genes.

In mammalian cells, the formation of the kinase-active CDK9/CycTl heterodimer of P-TEFb involves more than just the combining of CDK9 and CycTl together. Rather, a kinase-specific chaperone pathway is required for the production of the mature CDK9/CycTl heterodimer responsible for P-TEFb-mediated Tat stimulation of HIV-1 transcription (O'Keeffe et al., 2000). Before interacting with CycTl in the nucleus, CDK9 binds to the molecular chaperone Hsp70 and then a kinase-specific chaperone complex, Hsp90/Cdc37, to form two separate chaperone-CDK9 complexes in the cytoplasm. Pharmacological inactivationof Hsp90/Cdc37 function by geldanamycin revealed that these two complexes act sequentially to facilitate CDK9 folding/ stabilization and the subsequent formation of a mature and active CDK9/CycTl heterodimer (O'Keeffe et al., 2000).

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