RESUMO
CDK9 is a prominent member of the transcriptional CDKs subfamily, a group of kinases whose function is to control the primary steps of mRNA synthesis and processing by eukaryotic RNA polymerase II. As a cyclin-dependent kinase, CDK9 activation in vivo depends upon its association with T-type cyclins to assemble the positive transcription elongation factor (P-TEFb). Although CDK9/P-TEFb phosphorylates the C-terminal domain of RNAP II in the same positions targeted by CDK7 (TFIIH) and CDK8 (Mediator), the former does not participate in the transcription initiation, but rather plays a unique role by driving the polymerase to productive elongation. In addition to RNAP II CTD, the negative transcription elongation factors DSIF and NELF also represent major CDK9 substrates, whose phosphorylation is required to overcome the proximal pause of the polymerase. CDK9 is recruited to specific genes through proteins that interact with both P-TEFb and distinct elements in DNA, RNA or chromatin, where it modulates the activity of individual RNAP II transcription complexes. The regulation of CDK9 function is an intricate network that includes post-translational modifications (phosphorylation/dephosphorylation and acetylation/deacetylation of key residues) as well as the association of P-TEFb with various proteins that can stimulate or inhibit its kinase activity. Several cases of CDK9 deregulation have been linked to important human diseases, including various types of cancer and also AIDS (due to its essential role in HIV replication). Not only HIV, but also many other human viruses have been shown to depend strongly on CDK9 activity to be transcribed within host cells. This review summarizes the main advances made on CDK9/P-TEFb field in more than 20 years, introducing the structural, functional and genetic aspects that have been elucidated ever since.
Assuntos
Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Animais , Quinase 9 Dependente de Ciclina/química , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/química , Ligação Proteica , Transcrição Gênica , Vírus/genéticaRESUMO
The human cyclin-dependent kinase 9 (CDK9) protein was expressed in E. coli BL21 using the pET23a vector at 30 degrees C. Several milligrams of protein were purified from soluble fraction using ionic exchange and ATP-affinity chromatography. The structural quality of recombinant CDK9 and the estimation of its secondary structure were obtained by circular dichroism. Structural models of CDK9 presented 26% of helices in agreement with the spectra by circular dichroism analysis. This is the first report on human CDK9 expression in Escherichia coli and structure analysis and provides the first step for the development of CDK9 inhibitors.