Negative Regulation of NF-κB by the ING4 Tumor Suppressor in Breast Cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.     

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.     

Regulation of IκB kinase by GβL through recruitment of the protein phosphatases

G protein β-like (GβL) is a member of WD repeat-containing family which are involved in various intracellular signaling events. In our previous report, we demonstrated that GβL regulates TNFα-stimulated NF-κB signaling by interacting with and inhibiting phosphorylation of IκB kinase. However, GβL itself does not seem to regulate IKK directly, because it contains no functional domains except WD domains. Here, using immunoprecipitation and proteomic analyses, we identified protein phosphatase 4 as a new binding partner of GβL. We also found that GβL interacts with PP2A and PP6, other members of the same phosphatase family. By interacting with protein phosphatases, which do not directly bind to IKKβ, GβL mediates the association of phosphatases with IKKβ. Overexpression of protein phosphatases inhibited TNFκ-induced activation of NF-κB signaling, which is an effect similar to that of GβL overexpression. Down-regulation of GβL by small interfering RNA diminished the inhibitory effect of phosphatases, resulting in restoration of NF-κB signaling. Thus, we propose that GβL functions as a negative regulator of NF-κB signaling by recruiting protein phosphatases to the IKK complex.     

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.     

CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans

The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-κB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-κB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-κB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-κB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-κB, indicating that Hyphae-induced NF-κB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-κB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-κB activation, they regulate different signaling events in which CARD9 mediates IκBα kinase ubiquitination, whereas Syk regulates IκBα kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-κB activation in response to C. albicans hyphae challenging.     

Casein Kinase 2 (CK2)-mediated Phosphorylation of Hsp90β as a Novel Mechanism of Rifampin-induced MDR1 Expression

The P-glycoprotein (P-gp) encoded by the MDR1 gene is a drug-exporting transporter located in the cellular membrane. P-gp induction is regarded as one of the main mechanisms underlying drug-induced resistance. Although there is great interest in the regulation of P-gp expression, little is known about its underlying regulatory mechanisms. In this study, we demonstrate that casein kinase 2 (CK2)-mediated phosphorylation of heat shock protein 90β (Hsp90β) and subsequent stabilization of PXR is a key mechanism in the regulation of MDR1 expression. Furthermore, we show that CK2 is directly activated by rifampin. Upon exposure to rifampin, CK2 catalyzes the phosphorylation of Hsp90β at the Ser-225/254 residues. Phosphorylated Hsp90β then interacts with PXR, causing a subsequent increase in its stability, leading to the induction of P-gp expression. In addition, inhibition of CK2 and Hsp90β enhances the down-regulation of PXR and P-gp expression. The results of this study may facilitate the development of new strategies to prevent multidrug resistance and provide a plausible mechanism for acquired drug resistance by CK2-mediated regulation of P-gp expression.     

Regulation of the Src Homology 2 Domain-containing Inositol 5��-Phosphatase (SHIP1) by the Cyclic AMP-dependent Protein Kinase

Many agents that activate hematopoietic cells use phos pha tidyl ino si tol 3,4,5-trisphosphate (PtdIns 3,4,5-P₃) to initiate signaling cascades. The SH2 domain-containing inositol 5′ phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phos pha tidyl ino si tol 3-kinase and reducing the levels of PtdIns 3,4,5-P₃. Activation of the cyclic AMP-de pend ent protein kinase (PKA) also opposes many of the pro-inflammatory responses of hematopoietic cells. We tested to see whether the activity of SHIP1 was regulated via phos pho ryl a tion with PKA. We prepared pure recombinant SHIP1 from HEK-293 cells and found it can be rapidly phos pho ryl a ted by PKA to a stoichiometry of 0.6 mol of PO₄/mol of SHIP1. In ³²P-labeled HEK-293 cells transfected with SHIP1, stimulation with Sp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate (Sp-cAMPS) or activation of the β-adrenergic receptor increased the phos pho ryl a tion state of SHIP1. Inhibition of protein phosphatase activity with okadaic acid also increased the phos pho ryl a tion of SHIP1. Phosphorylation of SHIP1 in vitro or in cells by PKA increased the 5′ phosphatase activity of SHIP1 by 2–3-fold. Elevation of Ca²⁺ in DT40 cells in response to B cell receptor cross-linking, an indicator of PtdIns 3,4,5-P₃ levels, was markedly blunted by pretreatment with Sp-cAMPS. This effect was absent in SHIP^−/− DT40 cells showing that the effect of Sp-cAMPS in DT40 cells is SHIP1-de pend ent. Sp-cAMPS also blunted the ability of the B cell receptor to increase the phos pho ryl a tion of Akt in DT40 and A20 cells. Overall, activation of G protein-coupled receptors that raise cyclic AMP cause SHIP1 to be phos pho ryl a ted and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 regulation.Activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase)² is central to regulation of multiple cell functions including cell shape changes, cell migration, cell activation, and proliferation (1). PtdIns 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate in the inner leaflet of the plasma membrane to generate phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P₃) (2). PtdIns 3,4,5-P₃ then activates downstream signaling pathways by interacting with pleckstrin homology domain-containing proteins, such as phosphoinositide-dependent kinase 1 and the serine-threonine kinase Akt (3). The finding of abnormal activation of the PtdIns 3-kinase pathway in cancer cells has led to interest in the development of inhibitors for PtdIns 3-kinase (4).The level of PtdIns 3,4,5-P₃ is stimulated by multiple members of the PtdIns 3-kinase family (2) and is opposed by two phosphatidylinositol phosphatases: the Src homology 2 (SH2) domain-containing inositol 5′ phosphatase (SHIP) and the 3′ inositol phosphatase, phosphatase and tensin homolog (PTEN) (5). PTEN removes phosphate from the 3′ position in the inositol ring of PtdIns 3,4,5-P₃ and converts it to phosphatidylinositol 4,5-bisphosphate (6). PTEN has a C2 domain, a PDZ-binding motif, and a N-terminal phosphatidylinositol 4,5-bisphosphate binding motif essential for translocation to the membrane and interaction with other regulatory proteins (7). There are serine and threonine residues in PTEN that have been found to be phosphorylated, but their role in regulating the activity of the enzyme is not clear (8). Mutations in the PTEN protein have been observed in many tumors, suggesting a role for this enzyme in cancer (9).In contrast, SHIP dephosphorylates the 5′ position on the inositol ring and produces phosphatidylinositol 3,4-bisphosphate (10). There are three isoforms of SHIP: the 145-kDa hematopoietic cell restricted SHIP (also known as SHIP1); the 104-kDa stem cell-restricted SHIP, sSHIP; and the more widely expressed 150-kDa SHIP2 (11). SHIP1 is the major inositol phosphatase regulating PtdIns 3,4,5-P₃ in monocytes, macrophages, B cells, and T cells (11). SHIP1 has three known structural features: the N-terminal SH2 domain, the central inositol 5′ phosphatase domain, and two NPXY sequences in the C-terminal region. The currently accepted model for regulation of PtdIns 3,4,5-P₃ levels by SHIP1 envisions translocation of SHIP1 from the cytosol to the membrane. Upon stimulation by growth factors, cytokine receptors, or immunoreceptors, SHIP1 is recruited via its N-terminal SH2 domain to phosphorylated tyrosine residues in receptor kinases and degrades the elevated levels of PtdIns 3,4,5-P₃ near the activated receptor (12). During this translocation process, SHIP1 is not thought to change its 5′ phosphatase activity (13). Although it is known that SHIP1 can be phosphorylated on tyrosine residues by the lyn cytoplasmic kinase (12) or following the activation of the T cell receptor (14), neither event appears to influence the 5′ phosphatase activity. To date, direct regulation of SHIP1 activity by serine/threonine kinases has not been studied.Activation of G protein-coupled receptors that raise cAMP (i.e. β-adrenergic receptors or adenosine A_2a receptors) is known to blunt the pro-inflammatory responses generated by receptors that raise the level of PtdIns 3,4,5-P₃ (15). Therefore, we investigated the possibility that phosphorylation of SHIP1 by cyclic AMP-dependent protein kinase (PKA) might regulate the activity of SHIP1. We found that SHIP1 can be phosphorylated by PKA both in vitro and in cells leading to a stimulation of SHIP1 activity. Activation of PKA in DT40 and A20 cells blunted indicators of the PtdIns 3,4,5-P₃ response to B cell receptor stimulation. These results indicate that SHIP1 activity can be regulated both in vitro and in cells by activation of the cyclic AMP-dependent protein kinase and highlight a new mode of SHIP regulation by G protein-coupled receptors.