Selective degradation of the IκB kinase （IKK） by autophagy

Proteasome-mediated degradation and autophagy are the two major pathways mediating the turnover of cellular proteins. The proteasomal pathway is known to be a highly specific and regulated process mediating the degradation of short-lived proteins such as many important factors involved in cellular signaling. In contrast, it is generally thought that autophagy is rather nonselective as it is responsible for the bulk degradation of long-lived proteins and organelles. Challenging this general view, in this issue of Cell Research, Qing et al. report that selective degradation of the IκB kinase （IKK） triggered by the loss of Hsp90 function is mediated by autophagy [1].     

3-D structure and dynamics of protein kinase B—new mechanism for the allosteric regulation of an AGC kinase

New developments regarding the structure and in vivo dynamics of protein kinase B (PKB/Akt) have been recently exposed. Here, we specifically review how the use of multi-disciplinary approaches has resulted in reaching the recent progress made to relate the quaternary structure of PKB to its in vivo function. Using X-ray crystallography, the structure of PKB pleckstrin homology (PH) and kinase domains was determined separately. The molecular mechanisms involved in (a) the binding of the phosphoinositides to the PH domain and (b) the activation of the kinase with the rearrangement of the catalytic site and substrate binding were determined. In vitro, nuclear magnetic resonance and circular dychroism studies gave complementary information on the interaction of the PH domain with the phosphoinositides. However, the molecular nature and the function of the interactions between the PKB domains could not be deduced from the X-ray data since the full-length PKB has not been crystallised. In vitro, dynamic information on the inter-domain conformational changes related to PKB activation states emerged with the use of tandem mass spectrometry. Cell imaging and Förster resonance energy transfer provided in vivo dynamics. Molecular modelling and dynamic simulations in conjunction with mutagenesis and biochemical analysis were used to investigate the complex interactions between the PKB domains in vivo and understand at the molecular level how it linked to its activity. The compilation of the information obtained on the 3-D structure and the spatiotemporal dynamics of this widely studied oncogene could be applied to the study of other proteins. This inter-disciplinary approach led to a more profound understanding of PKB complex activation mechanism in vivo that will shed light onto new ideas and possibilities for modulating its activity.     

Roles of IκB kinase ε in the innate immune defense and beyond

IκB kinase ε(IKKε) is a non-canonical IκB kinase that is extensively studied in the context of innate immune response. Recently, significant progress has been made in understanding the role of IKKεin interferon(IFN) signaling. In addition to its roles in innate immunity, recent studies also demonstrate that IKKε is a key regulator of the adaptive immune response. Specifically, IKKεfunctions as a negative feedback kinase to curtail CD8 T cell response, implying that it can be a potential therapeutic target to boost antiviral and antitumor T cell immunity. In this review, we highlight the roles of IKKε in regulating IFN signaling and T cell immunity, and discuss a few imminent questions that remain to be answered.     

Diacylglycerol kinase θ counteracts protein kinase C-mediated inactivation of the EGF receptor

Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC) signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGKθ isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGKθ physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGKθ attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGKθ downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGKθ translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGKθ attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling.     

Probing the determinants of diacylglycerol binding affinity in the C1B domain of protein kinase Cα

C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of signaling response and the selectivity of this response among DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG binding affinities. In this work, we characterized the C1B domain of protein kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or the sub-nanosecond dynamics of the protein backbone, but resulted in a > 100-fold increase in DAG binding affinity and a substantial change in microsecond timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between wild type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue (Gln128) in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan side chain into the water-lipid interface are important factors that modulate the DAG binding properties of the C1 domains.     

The MC159 protein from the molluscum contagiosum poxvirus inhibits NF-κB activation by interacting with the IκB kinase complex

Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immunoevasion proteins. For example, the MCV MC159 protein inhibits TNF-R1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show in this article that, in human embryonic kidney 293 T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 overexpression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IκB kinase (IKK) complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, coimmunoprecipitated with IKKγ. MC159 coimmunoprecipitated with IKKγ when using mouse embryonic fibroblasts that lack either IKKα or IKKβ, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ coimmunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159's inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.     

Role of inhibitory κB kinase and c-Jun NH2-terminal kinase in the development of hepatic insulin resistance in critical illness diabetes

Hyperglycemia and insulin resistance induced by acute injuries or critical illness are associated with increased mortality and morbidity, as well as later development of type 2 diabetes. The molecular mechanisms underlying the acute onset of insulin resistance following critical illness remain poorly understood. In the present studies, the roles of serine kinases, inhibitory κB kinase (IKK) and c-Jun NH(2)-terminal kinase (JNK), in the acute development of hepatic insulin resistance were investigated. In our animal model of critical illness diabetes, activation of hepatic IKK and JNK was observed as early as 15 min, concomitant with the rapid impairment of hepatic insulin signaling and increased serine phosphorylation of insulin receptor substrate 1. Inhibition of IKKα or IKKβ, or both, by adenovirus vector-mediated expression of dominant-negative IKKα or IKKβ in liver partially restored insulin signaling. Similarly, inhibition of JNK1 kinase by expression of dominant-negative JNK1 also resulted in improved hepatic insulin signaling, indicating that IKK and JNK1 kinases contribute to critical illness-induced insulin resistance in liver.     

Nitric oxide-induced activation of the AMP-activated protein kinase α2 subunit attenuates IκB kinase activity and inflammatory responses in endothelial cells

Background

In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO).

Methodology/Principal Findings

Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2^-/- mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2^-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2^+/+ versus AMPKα2^-/- mice.

Conclusions

These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.     

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.     

12-O-tetradecanoylphorbol-1, 3-acetate induces the negative regulation of protein kinase B by protein kinase Cα during gastric cancer cell apoptosis

The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCα, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCα into the nucleus, where PKCα exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCα in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.     

Is the subunit the minimal function unit of creatine kinase?

The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M'M') and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M'B), as isolated by PAGE, has the same Km for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB') has half the activity of the native muscle enzyme. The M'B, MB' and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M"B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide and the native brain enzyme submit. The above results suggest strongly the independent catalytic function of the subunit of creatine kinase.     

NFκB regulates expression of Polo-like kinase 4

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulated NFκB activity in cancer can lead to increased genomic instability and uncontrolled proliferation.