Frank S. Lee

Activation of the IκBα Kinase Complex by MEKK1, a Kinase of the JNK Pathway

Both NF-kappaB and c-Jun are activated by cytokines such as TNF-alpha and by stresses such as UV irradiation. A key step in the activation of NF-kappaB is the phosphorylation of its inhibitor, IkappaB alpha, by a ubiquitination-inducible multiprotein kinase complex (IkappaB alpha kinase). A central kinase in the c-Jun activation pathway is mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1). Here, we show that MEKK1 induces the site-specific phosphorylation of IkappaB alpha in vivo and, most strikingly, can directly activate the IkappaB alpha kinase complex in vitro. Thus, MEKK1 is a critical component of both the c-Jun and NF-kappaB stress response pathways. Since the IkappaB alpha kinase complex can be independently activated by ubiquitination or MEKK1-dependent phosphorylation, it may be an integrator of multiple signal transduction pathways leading to the activation of NF-kappaB.

Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson disease.

The mechanism by which dopaminergic neurons are selectively lost in Parkinson disease (PD) is unknown. Here we show that accumulation of α-synuclein in cultured human dopaminergic neurons results in apoptosis that requires endogenous dopamine production and is mediated by reactive oxygen species. In contrast, α-synuclein is not toxic in non-dopaminergic human cortical neurons, but rather exhibits neuroprotective activity. Dopamine-dependent neurotoxicity is mediated by 54–83-kD soluble protein complexes that contain α-synuclein and 14-3-3 protein, which are elevated selectively in the substantia nigra in PD. Thus, accumulation of soluble α-synuclein protein complexes can render endogenous dopamine toxic, suggesting a potential mechanism for the selectivity of neuronal loss in PD.

HIF-1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation

Hypoxia-inducible factor-1α (HIF-1α)1 is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1α is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction thereby promotes the rapid degradation of HIF-1α. Under hypoxic conditions, HIF-1α is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1α, and that this regulation depends on a posttranslational modification of HIF-1α. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1α in this region, as well as MS and in vitro binding assays, we now provide evidence that this modification, which occurs under normoxic conditions, is hydroxylation of Pro-564 of HIF-1α. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.

MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta.

A critical step in the signal-induced activation of the transcription factor NF-kappaB is the site-specific phosphorylation of its inhibitor, IkappaB, that targets the latter for degradation by the ubiquitin-proteasome pathway. We have previously shown that mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IkappaB alpha at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight IkappaB kinase complex in vitro. Subsequently, others have identified two proteins, IkappaB kinase alpha (IKK-alpha) and IkappaB kinase beta (IKK-beta), that are present in a tumor necrosis factor alpha-inducible, high molecular weight IkappaB kinase complex. These kinases are believed to directly phosphorylate IkappaB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. We show herein that recombinant IKK-alpha and IKK-beta can, in fact, directly phosphorylate IkappaB alpha at Ser-32 and Ser-36, as well as homologous residues in IkappaB beta in vitro, and thus are bona fide IkappaB kinases. We also show that MEKK1 can induce the activation of both IKK-alpha and IKK-beta in vivo. Finally, we show that IKK-alpha is present in the MEKK1-inducible, high molecular weight IkappaB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-alpha in vitro. We conclude that IKK-alpha and IKK-beta can mediate the NF-kappaB-inducing activity of MEKK1.

Mitogen-activated Protein Kinase/ERK Kinase Kinases 2 and 3 Activate Nuclear Factor-κB through IκB Kinase-α and IκB Kinase-β

Recent evidence indicates that nuclear factor-κB (NF-κB), a transcription factor critically important for immune and inflammatory responses, is activated by a protein kinase cascade. The essential features of this cascade are that a mitogen-activated protein kinase kinase kinase (MAP3K) activates an IκB kinase (IKK) that site-specifically phosphorylates IκB. The IκB protein, which ordinarily sequesters NF-κB in the cytoplasm, is subsequently degraded by the ubiquitin-proteasome pathway, thereby allowing the nuclear translocation of NF-κB. Thus far, only two MAP3Ks, NIK and MEKK1, have been identified that can activate this pathway. We now show that MEKK2 and MEKK3 can in vivoactivate IKK-α and IKK-β, induce site-specific IκBα phosphorylation, and, relatively modestly, activate an NF-κB reporter gene. In addition, dominant negative versions of either IKK-α or IKK-β abolish NF-κB activation induced by MEKK2 or MEKK3, thereby providing evidence that these IKKs mediate the NF-κB-inducing activities of these MEKKs. In contrast, other MAP3Ks, including MEKK4, ASK1, and MLK3, fail to show evidence of activation of the NF-κB pathway. We conclude that a distinct subset of MAP3Ks can activate NF-κB.

Sequence Determinants in Hypoxia-inducible Factor-1α for Hydroxylation by the Prolyl Hydroxylases PHD1, PHD2, and PHD3

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor induced by hypoxia. Under normoxic conditions, site-specific proline hydroxylation of the α subunits of HIF allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets these subunits for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this hydroxylation is inhibited, allowing the α subunits of HIF to escape VHL-mediated degradation. Three enzymes, prolyl hydroxylase domain-containing proteins 1, 2, and 3 (PHD1, -2, and -3; also known as HIF prolyl hydroxylase 3, 2, and 1, respectively), have recently been identified that catalyze proline hydroxylation of HIF α subunits. These enzymes hydroxylate specific prolines in HIF α subunits in the context of a strongly conserved LXXLAP sequence motif (where Xindicates any amino acid and P indicates the hydroxylacceptor proline). We report here that PHD2 has the highest specific activity toward the primary hydroxylation site of HIF-1α. Furthermore, and unexpectedly, mutations can be tolerated at the −5, −2, and −1 positions (relative to proline) of the LXXLAP motif. Thus, these results provide evidence that the only obligatory residue for proline hydroxylation in HIF-1α is the hydroxylacceptor proline itself.

Expressing a human proinsulin cDNA in a mouse ACTH-secreting cell. Intracellular storage, proteolytic processing, and secretion on stimulation

The AtT-20 cell line, derived from the mouse anterior pituitary, synthesizes an adrenocorticotropic hormone (ACTH) precursor, proteolytically processes it to mature ACTH, stores it in secretory granules, and releases mature ACTH on stimulation with a secretagogue. A cDNA for human proinsulin inserted downstream from the SV40 early promoter in an SV40-pBR322 recombinant vector was introduced into AtT-20 cells. The stably transformed cell line, AtT-20ins4b/1, stores immunoreactive insulin, proteolytically processes proinsulin to smaller fragments, and on stimulation with secretagogues releases insulin-like material, not proinsulin, into the medium. Similarly transformed fibroblast L-cells secrete only proinsulin; they do not store it, and their secretion rate is unaffected by secretagogues. The transport mechanism for precursor ACTH thus appears to recognize other prohormones.

A Gain-of-Function Mutation in the HIF2A Gene in Familial Erythrocytosis

Hypoxia-inducible factor (HIF) alpha, which has three isoforms, is central to the continuous balancing of the supply and demand of oxygen throughout the body. HIF-alpha is a transcription factor that modulates a wide range of processes, including erythropoiesis, angiogenesis, and cellular metabolism. We describe a family with erythrocytosis and a mutation in the HIF2A gene, which encodes the HIF-2alpha protein. Our functional studies indicate that this mutation leads to stabilization of the HIF-2alpha protein and suggest that wild-type HIF-2alpha regulates erythropoietin production in adults.

Molecular Biology of Interleukin 4 and Interleukin 5 Genes and Biology of their Products that Stimulate B Cells, T Cells and Hemopoietic Cells

Lymphokines produced by helper T cells activated by antigen mediate numerous efTector functions of T cells. Unlike immunoglobulin or the T cell receptor, lymphokines do not bear antigen specificity and stimulate proliferation and differentiation of lymphocytes and hemopoietic cells (Arai et al. 1986). Since many lymphokines are composed of single polypeptide chains, their coding sequences can be isolated by functional expression in appropriate host cells. Using a pcD cDNA expression vector (Okayama & Berg 1983), we have developed a screening procedure employing transfection of plasmid DNAs into mammalian cells followed by assay of transfected cell supernatants for lymphokine activities of interest (Yokota et al. 1984. 1985, 1987c). Based on this expression cloning protocol, many T cell lymphokine genes have been isolated and their primary structures determined. These studies have revealed a regulatory network formed between lymphoid cells and hemopoietic cells through the action of multiple lymphokines produced by activated T cells. For example, interleukin 2 (IL-2) stimulates predominantly T cells whereas interleukin 3 (lL-3) and granulocytemacrophage colony stimulating factor (GM-CSF) stimulate hemopoietic cells. A number of molecules stimulate B cells. Among them, interleukin 4 (IL-4), interleukin 5 (IL-5) and B cell stimulatory factor 2 (BSF-2) have been chemically defined by molecular cloning (Lee et al. 1986, Noma et al. 1986, Yokota et al. !986,

Regulation of Dopamine D1 Receptor Function by Physical Interaction with the NMDA Receptors

Functional interactions between dopamine D1-like receptors and NMDA subtype glutamate receptors have been implicated in the maintenance of normal brain activity and neurological dysfunction. Although modulation of NMDA receptor functions by D1 receptor activation has been the subject of extensive investigation, little is known as to how the activation of NMDA receptors alters D1 function. Here we report that NMDA receptors regulate D1 receptor function via a direct protein–protein interaction mediated by the carboxyl tail regions of both receptors. In both cotransfected cells and cultured hippocampal neurons the activation of NMDA receptors increases the number of D1 receptors on the plasma membrane surface and enhances D1 receptor-mediated cAMP accumulation via a SNARE-dependent mechanism. Furthermore, overexpression of mini-genes encoding either NR1 or D1 carboxyl tail fragments disrupts the D1–NR1 direct protein–protein interaction and abolishes NMDA-induced changes in both D1 cell surface expression and D1-mediated cAMP accumulation. Our results demonstrate that the D1–NR1 physical interaction enables NMDA receptors to increase plasma membrane insertion of D1 receptors and provides a novel mechanism by which the activation of NMDA receptors upregulates D1 receptor function. Understanding the molecular mechanisms by which D1 and NMDA receptors functionally interact may provide insight toward elucidating the molecular neurobiological mechanisms involved in many neuropsychiatric illnesses, such as schizophrenia.