Chandrashekhar K. Korgaonkar

Nucleophosmin (B23) Targets ARF to Nucleoli and Inhibits Its Function

ABSTRACT The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARFs p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.

Cell Type-specific Expression of the IκB Kinases Determines the Significance of Phosphatidylinositol 3-Kinase/Akt Signaling to NF-κB Activation

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-κB through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-κB activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-κB DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-κB is retained in the cytoplasm by inhibitory proteins, IκBs, which are phosphorylated and targeted for degradation by IκB kinases (IKKα and IKKβ). Expression and the ratios of IKKα and IKKβ, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKKα (the IKK kinase activated by Akt) to IKKβ were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKKβ diminished the capacity of the inhibitors to block NF-κB DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-κB DNA binding in Ikkβ–/– but not Ikkα–/– or wild-type cells in which the ratio of IKKα to IKKβ is low. Thus, noncoordinate expression of IκB kinases plays a role in determining the cell type-specific role of Akt in NF-κB activation.

Cell type specific expression of the IkB kinases determines the significance of PI 3-kinase/Akt signaling to NF-kB activation

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-κB through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-κB activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-κB DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-κB is retained in the cytoplasm by inhibitory proteins, IκBs, which are phosphorylated and targeted for degradation by IκB kinases (IKKα and IKKβ). Expression and the ratios of IKKα and IKKβ, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKKα (the IKK kinase activated by Akt) to IKKβ were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKKβ diminished the capacity of the inhibitors to block NF-κB DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-κB DNA binding in Ikkβ–/– but not Ikkα–/– or wild-type cells in which the ratio of IKKα to IKKβ is low. Thus, noncoordinate expression of IκB kinases plays a role in determining the cell type-specific role of Akt in NF-κB activation.

ARF function does not require p53 stabilization or Mdm2 relocalization.

ABSTRACT It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal 31RRPR34 did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.

Akt Regulates Basal and Induced Processing of NF-κB2 (p100) to p52

NF-κB is a family of transcription factors important for innate and adaptive immunity. NF-κB is restricted to the cytoplasm by inhibitory proteins that are degraded when specifically phosphorylated, permitting NF-κB to enter the nucleus and activate target genes. Phosphorylation of the inhibitory proteins is mediated by an IκB kinase (IKK) complex, which can be composed of two subunits with enzymatic activity, IKKα and IKKβ. The preferred substrate for IKKβ is IκBα, degradation of which liberates p65 (RelA) to enter the nucleus where it induces genes important to innate immunity. IKKα activates a non-canonical NF-κB pathway in which p100 (NF-κB2) is processed to p52. Once produced, p52 can enter the nucleus and induce genes important to adaptive immunity. This study shows that Akt binds to and increases the activity of IKKα and thereby increases p52 production in cells. Constitutively active Akt augments non-canonical NF-κB activity, whereas kinase dead Akt or inhibition of phosphatidylinositol 3-kinase have the opposite effect. Basal and ligand-induced p52 production is reduced in mouse embryo fibroblasts deficient in Akt1 and Akt2 compared with parental cells. These observations show that Akt plays a role in activation of basal and induced non-canonical NF-κB activity.

ARF Directly Binds DP1: Interaction with DP1 Coincides with the G1 Arrest Function of ARF

ABSTRACT The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARFs ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARFs ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and “culture shock.” Taken together, our results show that DP1 is a critical direct target of ARF.

Tumor Necrosis Factor-α/Receptor Signaling Through the Akt Kinase

Tumor necrosis factor (TNF) is a pleiotropic cytokine that can affect the growth, differentiation, and metabolism of virtually every nucleated cell type in the body. TNF promotes immunity, but its expression is also associated with pathologies, such as rheumatoid arthritis, type II diabetes, and cachexia. Two distinct cell-surface receptors bind TNF, the type I receptor (TNFR1), which contains a conserved motif called a “death domain“ in its C-terminus, and the type II receptor. Binding of TNF to TNFR1 brings the death domains of TNFR1 into physical proximity, thereby promoting their interactions with cytoplasmic proteins that also contain death domains. Thus, a signal transduction cascade is initiated that coincidentally activates caspases that promote cell death and, additionally, anti-apoptotic events. The balance between these arms of the TNFR1 signaling cascade determines whether cells live or die.