Showing papers by "Michael Karin published in 2000"

Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity.

Abstract: NF-κB (nuclear factor-κB) is a collective name for inducible dimeric transcription factors composed of members of the Rel family of DNA-binding proteins that recognize a common sequence motif. NF-κ...

Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IκB kinase

Abstract: NF-κB is a critical activator of genes involved in inflammation and immunity1,2. Pro-inflammatory cytokines activate the IκB kinase (IKK) complex that phosphorylates the NF-κB inhibitors, triggering their conjugation with ubiquitin and subsequent degradation3,4. Freed NF-κB 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 PGE5,6. 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 inflammation7. Cyclopentenone prostaglandins have been suggested to exert anti-inflammatory activity through the activation of peroxisome proliferator-activated receptor-γ (refs 8, 9). Here we demonstrate a novel mechanism of anti-inflammatory activity which is based on the direct inhibition and modification of the IKKβ subunit of IKK. As IKKβ is responsible for the activation of NF-κB by pro-inflammatory stimuli10,11, our findings explain how cyclopentenone prostaglandins function and can be used to improve the utility of COX2 inhibitors.

p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps

Abstract: In the past decade, much has been learned about the molecular mechanisms that govern myogenesis owing mainly to the discovery of two groups of myogenic transcription factors (4, 45, 62). The first group includes the myogenic regulatory factors (MRFs), which belong to the basic helix-loop-helix (bHLH) protein family. This MRF group consists of four members: Myf5, MyoD, myogenin, and MRF4, all of which are specifically expressed in skeletal muscles. One of the unique features of these MRFs is that when they are ectopically expressed in fibroblasts or certain other nonmuscle cells, each has the ability to initiate the myogenic program and convert nonmuscle cells to myogenic derivatives (9, 59). Myogenic bHLH proteins heterodimerize with other ubiquitous bHLH proteins (like the E2A gene products, E12, and E47) to efficiently bind a consensus DNA site: CANNTG (also called the E box) (4, 33). The second group of transcription factors important in muscle differentiation consists of four different myocyte enhancer binding factor 2 (MEF2) proteins, which belong to the MADS box family (7). The MEF2 proteins (MEF2A, MEF2B, MEF2C, and MEF2D) form homo- or heterodimers which bind to a consensus AT-rich sequence (MEF2 site), found in the promoters of many muscle-specific genes. Myogenic bHLH and MEF2 cooperate to synergistically activate muscle-specific transcription through interactions mediated by the basic region and the MADS domain, respectively (44, 45). The study of muscle differentiation has benefited from the availability of several myogenic cell lines which allow biochemical dissection of the myogenic pathway. These myogenic cell lines (e.g., mouse C2C12 and rat L6) can be induced to differentiate by withdrawal of mitogens, such as serum. Many negative regulators of myogenesis (e.g., Id, Twist, oncogenic Ras, and the viral proteins E1A and simian virus 40 T antigen) have been identified (1, 34). However, little is known about the intracellular components that positively regulate the activities of myogenic transcription factors, especially those that are involved in receiving and transducing extracellular cues. One extracellular signal that positively regulates myogenesis is insulin-like growth factor (IGF). IGF activates the phosphatidylinositol-3 kinase (PI3K) signaling pathway, which is required for myogenesis (11, 29, 30). However, how this signaling pathway influences myogenic transcription remains to be defined. In eukaryotic cells, mitogen-activated protein kinases (MAPKs) are components of several important signaling pathways that relay extracellular cues to transcription factors in the nucleus (24, 25, 32, 41, 51). For mammals, three MAPK pathways, including the extracellular signal-regulated kinases (ERK1 and -2), the Jun–N-terminal kinases (JNK1, -2, and -3), and the p38 isoforms (α, β, γ, and δ) have been characterized (references 51 and 43 and references therein). In general, each group of MAPKs is activated by two homologous MAPK kinases (MKKs [also called MAPKKs]), including MEK1 and -2 for the ERKs, JNK kinase 1 and 2 (JNKK1 and -2) (or MKK4 and -7) for the JNKs, and MKK3 and -6 for the p38s (26, 51). Except for JNKK1, which can also activate p38 in vitro (14, 38), all other MKKs specifically activate their MAPK targets and have little activity on members of other groups. The ERK pathway has been implicated in the control of muscle differentiation, although its role remains controversial, with some reports suggesting a positive function (5, 11) and others suggesting a negative function (18). p38 has also been reported to activate certain MEF2 family members (21, 40, 48, 61, 67) and to stimulate muscle differentiation (12, 64). However, the upstream signals which regulate p38 activation at the onset of muscle differentiation and the mechanism(s) by which p38 activates myogenic transcription remain elusive. Using a combination of different approaches, we found that the p38 kinase is rapidly activated in muscle cells induced to differentiate by serum withdrawal, through a pathway distinct from that activated in response to stress and cytokines. Specific inhibition of p38 prevents differentiation of both established muscle cell lines and human primary myoblasts, while deliberate p38 activation stimulates muscle-specific reporters, accelerates myotube formation, and induces the expression of myogenic markers despite the presence of serum, which otherwise inhibits muscle differentiation. p38 exerts its stimulatory effect on myogenesis by enhancing the transcriptional activities of both MyoD and MEF2A and -C through distinct mechanisms. While MEF2 proteins are activated by direct phosphorylation of residues located within the activation domain, p38-mediated activation of MyoD is likely to occur by an indirect mechanism. The p38 pathway is activated independently of the IGF-PI3K pathway, although the integrity of both these pathways is required to stimulate muscle differentiation. Conversely, the ERK pathway plays a dual role during myogenic differentiation, being inhibitory at early stages and stimulatory at late stages.

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation

Abstract: Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.

NAK is an IκB kinase-activating kinase

Abstract: Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration.

Abstract: Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor α, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IκB kinase signaling cascade.

Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells.

Abstract: MyoD inhibits cell proliferation and promotes muscle differentiation. A paradoxical feature of rhabdomyosarcoma (RMS), a tumor arising from muscle precursors, is the block of the differentiation program and the deregulated proliferation despite MyoD expression. A deficiency in RMS of a factor required for MyoD activity has been implicated by previous studies. We report here that p38 MAP kinase (MAPK) activation, which is essential for muscle differentiation, is deficient in RMS cells. Enforced induction of p38 MAPK by an activated MAPK kinase 6 (MKK6EE) restored MyoD function and enhanced MEF2 activity in RMS deficient for p38 MAPK activation, leading to growth arrest and terminal differentiation. Stress and cytokines could activate the p38 MAPK in RMS cells, however, these stimuli did not promote differentiation, possibly because they activated p38 MAPK only transiently and they also activated JNK, which could antagonize differentiation. Thus, the selective and sustained p38 MAPK activation, which is distinct from the stress-activated response, is required for differentiation and can be disrupted in human tumors.

Kinase regulation in inflammatory response

Abstract: The transcription factor NF-κB is a pivotal regulator of innate immune responses, whose activity is rapidly induced by proinflammatory stimuli, most notably the tumour-necrosis factor TNFα and interleukin-1, viruses, and components of bacterial cell walls1. In addition, NF-κB protects cells from the induction of programmed cell death by pro-apoptotic stimuli such as TNFα (refs 2, 3). Another important anti-apoptotic signal-transducing protein is the protein kinase Akt (also known as protein kinase B), whose activity is strongly stimulated by growth factors4. Ozes et al.5 have suggested that Akt is involved in the TNFα-mediated activation of NF-κB5, implying that some of the anti-apoptotic activity of Akt may be mediated through NF-κB. However, we have failed to detect any involvement of Akt in the signalling pathway through which TNFα leads to NF-κB activation.

Synergistic interaction of MEK kinase 2, c-Jun N-terminal kinase (JNK) kinase 2, and JNK1 results in efficient and specific JNK1 activation.

Abstract: Mitogen-activated protein kinases (MAPKs) are activated through cascades or modules consisting of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Investigating the molecular basis of activation of the c-Jun N-terminal kinase (JNK) subgroup of MAPK by the MAPKKK MEKK2, we found that strong and specific JNK1 activation by MEKK2 was mediated by the MAPKK JNK kinase 2 (JNKK2) rather than by JNKK1 through formation of a tripartite complex consisting of MEKK2, JNKK2, and JNK1. No scaffold protein was required for the MEKK2-JNKK2-JNK1 tripartite-complex formation. Expression of JNK1, JNKK2, and MEKK2 significantly augmented the coprecipitation of, respectively, MEKK2-JNKK2, MEKK2-JNK1, and JNKK2-JNK1, indicating that the interaction of MEKK2, JNKK2, and JNK1 is synergistic. Finally, the JNK1 was activated more efficiently in the MEKK2-JNKK2-JNK1 complex than was the JNK1 excluded from the complex. Thus, formation of a signaling complex through synergistic interaction of a MAPKKK, a MAPKK, and a MAPK molecule like MEKK2-JNKK2-JNK1 is likely to be responsible for the efficient, specific flow of information via MAPK cascades.

Signaling pathways leading to nuclear factor-kappa B activation.

Abstract: Publisher Summary Exposure of cells to long wavelength ultraviolet radiation-A (320-400 nm) stimulates signaling pathways that activate transcription factors and lead to induction of specific gene expression. These transcription factors include activator protein (AP)-1, AP-2, and nuclear factor (NF)-ĸB. Singlet oxygen mediates effects of UVA on gene expression and NF-KB activity can be induced on treatment with singlet oxygen generated photochemically by rose bengal or methylene blue plus light. The Rel/NF-ĸB family of transcriptional factors regulates the expression of numerous cellular and viral genes that play important roles in immune and stress responses, in inflammation, and in apoptosis. The NF-ĸB proteins are activated by a variety of stimuli, ranging from cytokines, bacterial and viral infections to various forms of radiation. They are kept in the cytoplasm of nonstimulated cells through interaction with inhibitory proteins, IKBs. This chapter focuses on studying signaling pathways triggered by a stimulus (e.g., UVA) that ultimately lead to NF-KB activation.

IkappaB kinase alpha is essential for development of the mammalian cornea and conjunctiva.

Abstract: PURPOSE To determine the requirement of IkappaB kinase alpha (Ikkalpha) for differentiation of the mammalian cornea and conjunctiva. METHODS Newborn mice or surgically removed embryonic day (E)18 to E19 fetuses of wild-type and IKK:alpha(-/-) mice were analyzed by light microscopy and electron microscopy or immunocytochemistry using anti-keratin (K)12, K4, K5, IkappaB, or nuclear factor (NF)-kappaB (p50) antibody. RESULTS In the IKKalpha(-/-) eyes, the epithelium of the cornea and the conjunctiva consisted of poorly differentiated cells with round nuclei. K5 was much stronger in the conjunctiva of the IKKalpha(-/-) mice. Expression of K12 in the cornea and K4 in the conjunctiva was impaired in the IKKalpha(-/-) mice. IkappaB expression was low in epithelium of the cornea and conjunctiva of the wild type mice but was very strong in that of the IKKalpha(-/-) mice. During normal development of the conjunctiva, nuclear localization of p50 was seen in areas where basal undifferentiated cells give rise to differentiated cell types, marked by expression of cK4. However, in the IKK++alpha(-/-) tissues, no nuclear p50 staining was detected. CONCLUSIONS IKKalpha is specifically required for formation of cornea and conjunctiva. This function may be exerted through an effect on NF-kappaB activity.

[4] Analysis and identification of protein-protein interactions using protein recruitment systems

Abstract: Publisher Summary The immense output of various genome-sequencing projects has provided the scientific community with numerous proteins with no assigned function. There is no single protein that functions in isolation within the cell, and protein–protein interactions serve as a general means to provide functional specificity and to control the activity of enzymes and signaling molecules. To achieve a better functional understanding of known and novel gene products, much current research activity in molecular cell biology is focused on the identification of interacting proteins and characterization of these interactions. To date, multiple methods exist to identify and characterize protein–protein interactions; however, few of them employ a genetic selection system in vivo such as the yeast two-hybrid system. The two-hybrid system is an excellent research tool and is commonly used to identify and characterize novel and known interaction partners for proteins of interest. Although powerful, the two-hybrid system, which is based on a transcriptional readout, exhibits several limitations and inherent problems. To overcome these problems and limitations, a cytoplasmic protein recruitment system designated the son of sevenless “(Sos) recruitment system” (SRS) has been developed. This and the related Ras recruitment system (RRS) take advantage of a general phenomenon in signal transduction—namely, that the generation of local high concentrations of a signaling intermediate can result in a dramatic increase in signaling activity.