Functional inactivation of Cdk9 through oligomerization chain reaction

Abstract

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.

The breast cell growth inhibitor, estrogen down regulated gene 1, modulates a novel functional interaction between estrogen receptor alpha and transcriptional elongation factor cyclin T1

“…Several studies from other laboratories suggest that P-TEFb is a potential therapeutic target. Anchorage-independent colony formation of HeLa cells and H1299 (lung cancer) cells is inhibited though oligomerization chain reaction (OCR)-mediated inactivation of CDK9 (Napolitano et al, 2003). Flavopiridol, a drug currently in clinical trials as a possible cancer therapy agent, is a very potent inhibitor of P-TEFb activity (Chao and Price, 2001).…”

Functional Inactivation of the Transcription Factor Pax8 through Oligomerization Chain Reaction

“…It has been suggested that the oligomerization reaction induced by the CC domain of PML could lead to the formation of aberrant, nonfunctional high-molecular mass complexes, both when this domain is fused to proteins that are able to self-associate (5), as well as when the target of the OCR is a heterooligomer (6). We investigated whether the Pax8/TTF-1 heterocomplex could be targeted by the OCR strategy.…”

“…This property of the CC domain suggested that the presence of this region could be used to modify the function of a target protein. Indeed, two recent reports describe the OCR as an innovative strategy for the inactivation of target proteins that are able to self-associate (5) or that form heterocomplexes (6).…”

The Distal Zinc Finger Domain of AML1/MDS1/EVI1 Is an Oligomerization Domain Involved in Induction of Hematopoietic Differentiation Defects in Primary Cells In vitro

“…In many cases, the loss of the oligomerization domain correlates with the loss of transforming properties by the chimeric proteins (32)(33)(34)(35)(36). Furthermore, the substitution of the self-interaction domain with a heterologous oligomerization module recapitulates the transforming potential of the primary chimeric protein (33) and inactivates known target genes (37,38). Therefore, it is thought that oligomerization of chimeric proteins is required for their oncogenic potential.…”