cdk11p58 regulation of cell cycle progression in cancer development

The Cdk11 protein kinases are members of the cyclin-dependent kinase superfamily. There exist two distinct but closely related human CDK11 genes (p110 and p58). Cdk11p58 is a Ser/Thr kinase involved in cell cycle progression, promoting the centrosome maturation; apoptotic signaling and tumorigenesis.

Using immunoprecipitation and mass spectrometry approaches we demonstrate that GADD45, PDEF and cdk11p58 interact in a complex that might be involved in cell cycle regulation in cancer. GADD45 protein family members (α, β, and γ) are involved in the induction or prevention of apoptosis, cell cycle arrest and participate in the DNA repair machinery. Arrest at the G2/M phase mediated by GADD45 have been identified to rely on its capacity to inhibit the kinase activities such as the CDK1/cyclinB1 complex and the Aurora A, a serine/threonine kinase that play critical roles in the regulation of mitotic events. PDEF (prostate-derived ETS factor) is a transcriptional factor that plays a major role in cancer development and metastasis.

In this study, the kinase activity of cdk11p58 over PDEF as well as the importance of the cdk11p58 binding to the GADD45 family members were evaluated and we demonstate that cdk11p58 phosphorilate PDEF and the phosphorilation process is inhibited by GADD45.

Co-expression of PDEF and cdk11p58 in 293T cells leads to PDEF ubiquitination and degradation, which can be reverted by MG132, a proteasome inhibitor. Corroborating this data, confocal microscope analysis revealed that cdk11p58 co-localized with PDEF and ubiquitin into the nucleus. However, the process is partially blockage by the presence of GADD45 gene, which seems to prevent PDEF degradation mediated by cdk11p58. Deregulation of the ETS factors leads to altered expression of key genes required for cancer progression. PDEF acts as a negative regulator of tumor progression by modulating the expression of growth and migration promoting genes. The degradation of PDEF by cdk11p58 may be a critical step in cancer cell tumorigenesis and more experiments are under way to unravel the importance of this event in cancer cell migration and invasion.

In order to gain further insights about the role of cdk11p58 and GADD45 in cell cycle regulation of cancer cells, we developed DU145 cell lines overexpressing the GADD45α (DU145-GADD45+) and the cdk11p58 (DU145-p58+) proteins. Additionally, DU145 with the cdk11p58 knocked-down (DU145-p58si) was obtained by transduction of lentivirus containing siRNA against the cdk11p58 gene sequence (Sigma).

The biological effect of overexpression or know-down of the cdk11p58 gene in the cell cycle phase was assessed by flow cytometry (FACS) analysis of propidium iodide stained cells. FACS analysis revealed that DU145-p58+ cells progress faster when compared to a control DU145 cell line, while DU145-GADD45+ and DU145-p58si induced cell cycle arrest at the G2/M phase. Furthemore, overexpression of GADD45 in the DU145-p58+ cell line induced an arrest at the G2/M phase, comparable to the control group. Preliminary result of transient transfection of PDEF indicates that ovexpression of PDEF in 293T cells leads to arrest at the G1/S phase.

Transient tranfections of cdk11p58 leads to induction of proliferation when compared with control cells while cells expressing the GADD45 α and PDEF genes demonstrate slower proliferation rates.

In conclusion, PDEF was identified to be a novel target for the kinase activity of cdk11p58. Cdk11p58 induced cell cycle progression and cell proliferation of a prostate cancer cell line that can be inhibited by GADD45 contributing to cell cycle arrest at the G2/M phase.