GSK3ß phosphorylation of the KLF6 tumor suppressor promotes its transactivation of p21.

KLF6, a ubiquitously expressed Krüppel-like transcription factor, is frequently inactivated in human cancer and has significant roles in cellular proliferation, apoptosis, differentiation and development. A key mechanism of KLF6-mediated growth suppression is through p53-independent transactivation of p21. Several cancer-derived KLF6 mutants lead to the loss of p21-mediated growth suppression through an unknown mechanism. Because several colorectal cancer and hepatocellular carcinoma-derived KLF6 mutations affect a glycogen synthase kinase 3ß (GSK3ß) phosphorylation consensus site, we investigated the role of GSK3ß in the regulation of KLF6 function. Based on transient transfection, GSK3ß augments the transactivation of a p21 promoter luciferase by KLF6. Reciprocal co-immunoprecipitation of hemagglutinin (HA)-GSK3ß and Flag-KLF6 validated the interaction between these two proteins. KLF6 phosphorylation is augmented in the presence of GSK3ß based on in vitro and in vivo (32)P incorporation assays. Site-directed mutagenesis of the candidate phosphorylation sites to alanines ('KLF6-4A' phosphomutant) eliminated a higher molecular weight phosphorylated isoform of KLF6 based on western blot. GSK3ß augmented the transactivation by wild-type KLF6, but not KLF6-4A, towards the p21 promoter, and increased p21 protein. Functionally, GSK3ß enhanced KLF6-mediated growth suppression, which was abrogated by the KLF6-4A phosphomutant. These data establish that GSK3ß directly phosphorylates KLF6, which augments its induction of p21 and resultant growth suppression. This interaction may account for the growth-promoting effects of cancer-derived KLF6 mutants that lack tumor suppressor activity.

HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells.

Prior experiments in explants of human lymphoid tissue have demonstrated that human immunodeficiency virus type 1 (HIV-1) productively infects diverse cellular targets including T cells and tissue macrophages. We sought to determine the specific contribution of macrophages and T cells to the overall viral burden within lymphoid tissue. To block infection of macrophages selectively while preserving infection of T cells, we used viruses deficient for viral protein R (Vpr) that exhibit profound replication defects in nondividing cells in vitro. We inoculated tonsil histocultures with matched pairs of congenic viruses that differed only by the presence of a wild-type or truncated vpr gene. Although these viruses exhibited no reduction in the infection or depletion of T cells, the ability of the Vpr-deficient R5 virus to infect tissue macrophages was severely impaired compared with matched wild-type R5 virus. Interestingly, the Vpr-deficient R5 virus also exhibited a 50% reduction in overall virus replication compared with its wild-type counterpart despite the fact that macrophages represent a small fraction of the potential targets of HIV-1 infection in these tissues. Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1.