Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase

NF-kappaB is a critical activator of genes involved in inflammation and immunity. Pro-inflammatory cytokines activate the IkappaB kinase (IKK) complex that phosphorylates the NF-kappaB inhibitors, triggering their conjugation with ubiquitin and subsequent degradation. Freed NF-kappaB dimers translocate to the nucleus and induce target genes, including the one for cyclo-oxygenase 2 (COX2), which catalyses the synthesis of pro-inflammatory prostaglandins, in particular PGE. At late stages of inflammatory episodes, however, COX2 directs the synthesis of anti-inflammatory cyclopentenone prostaglandins, suggesting a role for these molecules in the resolution of inflammation. Cyclopentenone prostaglandins have been suggested to exert anti-inflammatory activity through the activation of peroxisome proliferator-activated receptor-gamma. Here we demonstrate a novel mechanism of antiinflammatory activity which is based on the direct inhibition and modification of the IKKbeta subunit of IKK. As IKKbeta is responsible for the activation of NF-kappaB by pro-inflammatory stimuli, our findings explain how cyclopentenone prostaglandins function and can be used to improve the utility of COX2 inhibitors.     

IkappaB kinase-alpha is critical for interferon-alpha production induced by Toll-like receptors 7 and 9

The Toll-like receptor (TLR) family has important roles in microbial recognition and dendritic cell activation. TLRs 7 and 9 can recognize nucleic acids and trigger signalling cascades that activate plasmacytoid dendritic cells to produce interferon-alpha (IFN-alpha) (refs 7, 8). TLR7/9-mediated dendritic cell activation is critical for antiviral immunity but also contributes to the pathogenesis of systemic lupus erythematosus, a disease in which serum IFN-alpha levels are elevated owing to plasmacytoid dendritic cell activation. TLR7/9-induced IFN-alpha induction depends on a molecular complex that contains a TLR adaptor, MyD88, and IFN regulatory factor 7 (IRF-7) (refs 10-14), but the underlying molecular mechanisms are as yet unknown. Here we show that IkappaB kinase-alpha (IKK-alpha) is critically involved in TLR7/9-induced IFN-alpha production. TLR7/9-induced IFN-alpha production was severely impaired in IKK-alpha-deficient plasmacytoid dendritic cells, whereas inflammatory cytokine induction was decreased but still occurred. Kinase-deficient IKK-alpha inhibited the ability of MyD88 to activate the Ifna promoter in synergy with IRF-7. Furthermore, IKK-alpha associated with and phosphorylated IRF-7. Our results identify a role for IKK-alpha in TLR7/9 signalling, and highlight IKK-alpha as a potential target for manipulating TLR-induced IFN-alpha production.     

MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin.

AGONISTS that stimulate protein kinase C (PKC) induce profound changes in cell morphology correlating with the reorganization of submembranous actin, but no direct connection between PKC and actin assembly has been identified. The myristoylated, alanine-rich C kinase substrate (MARCKS) binds calmodulin and is a predominant, specific substrate of PKC which is phosphorylated during macrophage and neutrophil activation , growth factor-dependent mitogenesis and neurosecretion; it is redistributed from plasma membrane to cytoplasm when phosphorylated and is involved in leukocyte motility. Here we report that MARCKS is a filamentous (F) actin crosslinking protein, with activity that is inhibited by PKC-mediated phosphorylation and by binding to calcium-calmodulin. MARCKS may be a regulated crossbridge between actin and the plasma membrane, and modulation of the actin crosslinking activity of the MARCKS protein by calmodulin and phosphorylation represents a potential convergence of the calcium-calmodulin and PKC signal transduction pathways in the regulation of the actin cytoskeleton.     

Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate

cdc2 is a catalytic subunit of a protein kinase complex, called the M-phase promoting factor, that induces entry into mitosis and is universal among eukaryotes. In HeLa cells, cdc2 is shown to be the most abundant phosphotyrosine-containing protein and its phosphotyrosine content is subject to cell-cycle regulation. One site of cdc2 tyrosine phosphorylation in vivo is selectively phosphorylated by pp60c-src in vitro.     

Pyruvate kinase M2 is a phosphotyrosine-binding protein

Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.     

Phosducin is a protein kinase A-regulated G-protein regulator.

Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.     

Phytochrome signalling is mediated through nucleoside diphosphate kinase 2.

Because plants are sessile, they have developed intricate strategies to adapt to changing environmental variables, including light. Their growth and development, from germination to flowering, is critically influenced by light, particularly at red (660 nm) and far-red (730 nm) wavelengths. Higher plants perceive red and far-red light by means of specific light sensors called phytochromes(A-E). However, very little is known about how light signals are transduced to elicit responses in plants. Here we report that nucleoside diphosphate kinase 2 (NDPK2) is an upstream component in the phytochrome signalling pathway in the plant Arabidopsis thaliana. In animal and human cells, NDPK acts as a tumour suppressor. We show that recombinant NDPK2 in Arabidopsis preferentially binds to the red-light-activated form of phytochrome in vitro and that this interaction increases the activity of recombinant NDPK2. Furthermore, a mutant lacking NDPK2 showed a partial defect in responses to both red and farred light, including cotyledon opening and greening. These results indicate that NDPK2 is a positive signalling component of the phytochrome-mediated light-signal-transduction pathway in Arabidopsis.     

TAK1 is a ubiquitin-dependent kinase of MKK and IKK.

TRAF6 is a signal transducer that activates IkappaB kinase (IKK) and Jun amino-terminal kinase (JNK) in response to pro-inflammatory mediators such as interleukin-1 (IL-1) and lipopolysaccharides (LPS). IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2 (ref. 5). TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A (or the functionally equivalent Mms2). This Ubc complex, together with TRAF6, catalyses the formation of a Lys 63 (K63)-linked polyubiquitin chain that mediates IKK activation through a unique proteasome-independent mechanism. Here we report the purification and identification of TRIKA2, which is composed of TAK1, TAB1 and TAB2, a protein kinase complex previously implicated in IKK activation through an unknown mechanism. We find that the TAK1 kinase complex phosphorylates and activates IKK in a manner that depends on TRAF6 and Ubc13-Uev1A. Moreover, the activity of TAK1 to phosphorylate MKK6, which activates the JNK-p38 kinase pathway, is directly regulated by K63-linked polyubiquitination. We also provide evidence that TRAF6 is conjugated by the K63 polyubiquitin chains. These results indicate that ubiquitination has an important regulatory role in stress response pathways, including those of IKK and JNK.     

OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis

Active oxygen species (AOS) generated in response to stimuli and during development can function as signalling molecules in eukaryotes, leading to specific downstream responses. In plants these include such diverse processes as coping with stress (for example pathogen attack, wounding and oxygen deprivation), abscisic-acid-induced guard-cell closure, and cellular development (for example root hair growth). Despite the importance of signalling via AOS in eukaryotes, little is known about the protein components operating downstream of AOS that mediate any of these processes. Here we show that expression of an Arabidopsis thaliana gene (OXI1) encoding a serine/threonine kinase is induced in response to a wide range of H2O2-generating stimuli. OXI1 kinase activity is itself also induced by H2O2 in vivo. OXI1 is required for full activation of the mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 after treatment with AOS or elicitor and is necessary for at least two very different AOS-mediated processes: basal resistance to Peronospora parasitica infection, and root hair growth. Thus, OXI1 is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses.     

IkappaB kinase-alpha acts in the epidermis to control skeletal and craniofacial morphogenesis

IkappaB kinase-alpha (IKK-alpha) exhibits protein-kinase-dependent and -independent functions. Its kinase activity is required for lymphoid organogenesis and mammary gland development, whereas a kinase-independent activity is required for epidermal keratinocyte differentiation. In addition to failed epidermal differentiation, IKK-alpha-deficient mice exhibit abnormal skeletal and craniofacial morphogenesis. As similar defects are not exhibited by mice that experience systemic inhibition of NF-kappaB, we postulated that the morphogenetic defects in IKK-alpha-deficient mice are not caused by reduced NF-kappaB activity but instead are due to failed epidermal differentiation that disrupts proper epidermal-mesodermal interactions. We tested this hypothesis by introducing an epidermal-specific Ikka (also known as Chuk) transgene into IKK-alpha-deficient mice. Mice lacking IKK-alpha in all cell types including bone and cartilage, but not in basal epidermal keratinocytes, exhibit normal epidermal differentiation and skeletal morphology. Thus, epidermal differentiation is required for proper morphogenesis of mesodermally derived skeletal elements. One way by which IKK-alpha controls skeletal and craniofacial morphogenesis is by repressing expression of fibroblast growth factor (FGF) family members, such as FGF8, whose expression is specifically elevated in the limb bud ectoderm of IKK-alpha-deficient mice.     

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase

Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK -- found in guard cells, which control stomatal pores -- has been shown to regulate plasma membrane ion channels. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.     

The wee1 protein kinase is required for radiation-induced mitotic delay.

Cellular feedback or 'checkpoint' mechanisms maintain the order of completion of essential, cell-cycle related functions. In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage. Similarly, in fission yeast, the cdc25 and cdc2 gene products influence the ability of cells to delay mitosis in response to the inhibition of DNA synthesis. Because these two checkpoint controls regulate the same event, mitosis, we observed the effect of gamma-irradiation on cell cycle progression in fission yeast, to test whether the two controls require the same cell-cycle regulatory elements. We show that gamma-radiation-induced mitotic delay requires functional wee1 protein kinase but does not seem to involve the cdc25 pathway. Mitotic delay in response to DNA damage is thus distinct from the delay induced by inhibition of DNA synthesis, which involves cdc25 but is not dependent on wee1.     

Tyrosine kinase receptor RET is a key regulator of Peyer's patch organogenesis

Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7Ralpha-c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFRalpha3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.     

Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors.

Long-term potentiation (LTP) in the hippocampus is thought to contribute to memory formation. In the Ca1 region, LTP requires the NMDA (N-methyl-D-aspartate) receptor-dependent influx of Ca2+ and activation of serine and threonine protein kinases. Because of the high amount of protein tyrosine kinases in hippocampus and cerebellum, two regions implicated in learning and memory, we examined the possible additional requirement of tyrosine kinase activity in LTP. We first examined the specificity in brain of five inhibitors of tyrosine kinase and found that two of them, lavendustin A and genistein, showed substantially greater specificity for tyrosine kinase from hippocampus than for three serine-threonine kinases: protein kinase A, protein kinase C, and Ca2+/calmodulin kinase II. Lavendustin A and genistein selectively blocked the induction of LTP when applied in the bath or injected into the postsynaptic cell. By contrast, the inhibitors had no effect on the established LTP, on normal synaptic transmission, or on the neurotransmitter actions attributable to the actions of protein kinase A or protein kinase C. These data suggest that tyrosine kinase activity could be required postsynaptically for long-term synaptic plasticity in the hippocampus. As Ca2+ calmodulin kinase II or protein kinase C seem also to be required, the tyrosine kinases could participate postsynaptically in a kinase network together with serine and threonine kinases.     

The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID-RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.