Functional inactivation of Cdk9 through oligomerization chain reaction.
The oligomerization chain reaction (OCR) strategy is a recently described technique for inactivation of target proteins that function as homoassociate complexes. This novel strategy is based on the fusion of self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins. Here, we present the successful application of the OCR strategy for inactivation of the heterodimeric Cdk9/cyclin T1 complex. Cyclin T1/Cdk9 (P-TEFb) complex is a positive regulator of gene transcription, whose function is underlined by the ability to phosphorylate the carboxyl-terminal domain (CTD) of the RNA polymerase II conferring productive transcript elongation. Fusion of the CC domain to Cdk9 leads to the formation of high molecular complexes to which the endogenous cyclin T1 is recruited. The CC-Cdk9 chimera effectively inhibits HIV-1 Tat activation, whose transcription activity is exquisitely dependent upon cyclin T1/Cdk9 function. Furthermore, expression of CC-Cdk9 protein inhibits cell proliferation, as shown by colony-formation assay. Collectively, our findings add further support to the OCR strategy for functional inactivation of hetero-associated factors such as the Cdk9/cyclin T1 complex, and highlight a putative function of Cdk9 in cell growth control.
Napolitano
Giuliana
G
Department of Genetics, Molecular and General Biology, University of Naples Federico II, Naples, Italy.
Mazzocco
Alberto
A
Fraldi
Alessandro
A
Majello
Barbara
B
Lania
Luigi
L
D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D003114:Colony-Forming Units Assay; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016213:Cyclins / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D015696:Gene Products, tat / Q000037:antagonists & inhibitors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D009363:Neoplasm Proteins / Q000737:chemistry*; D009687:Nuclear Proteins; D042925:Positive Transcriptional Elongation Factor B; D000071156:Promyelocytic Leukemia Protein; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012342:RNA, Small Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic; D014162:Transfection; D025521:Tumor Suppressor Proteins
C503684:CCNT1 protein, human; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases