Figure 6.

Silencing of Jade-1 increases cell proliferation and promotes AKT-dependent anchorage-independent growth. A. Cell numbers were counted with a hemocytometer for seven consecutive days. Error bars indicate SD. B. Growing cells were labeled with BrdU solution (10 μM, 1.5 hours). Incorporated BrdU was detected with anti-BrdU-POD solution (Roche) and visualized with 3, 3’-diaminobenzidine (DAB) substrate (Vector Laboratories). Error bars indicate SD. C. Jade-1-silenced HK-2 cell lines formed colonies in soft agarose. HK-2 cell lines were grown in an agarose suspension. Formation of colonies was monitored microscopically. D. Reintroduction of Jade-1 into Jade-1sh2 cells inhibited anchorage-independent growth. E. LY294002 inhibited anchorage-independent growth of Jade-1sh2 cells. F. Anchorage-independent growth of Jade-1 silenced HK-2 cells is AKT-dependent. HK-2 stable cell lines were infected with adenovirus expressing either β-gal control, dominant-negative AKT (dnAKT), or constitutively active AKT (myr-AKT) before they were suspended in soft agarose. Error bars indicate SD. G. A model of AKT inhibition by Jade-1/pVHL. Starting on the lower left, serine/threonine kinase AKT is activated by phosphorylation on T308 and S473. Binding of ATP in the catalytic cleft promotes formation of a stabilized form of AKT in which the phosphorylated residues are protected in a “phosphatase shielding cage”. With AKT phosphorylation of a substrate protein and ATP hydrolysis, AKT adopts an “uncaged” conformation that is more susceptible to dephosphorylation and inactivation. Short-lived protein Jade-1 is stabilized by pVHL. During AKT activation, increased Jade-1 preferentially binds “uncaged” AKT, shifting the balance toward this form of phospho-AKT that is more susceptible to inactivation by phosphatases. Conversely, in renal cancer, loss of Jade-1/pVHL would shift the balance toward the “caged” form of AKT, resulting in higher phospho-AKT levels and increased AKT kinase activity.