Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.

A human model for multigenic inheritance: Phenotypic expression in Hirschsprung disease requires both the RET gene and a new 9q31 locus

Reduced penetrance in genetic disorders may be either dependent or independent of the genetic background of gene carriers. Hirschsprung disease (HSCR) demonstrates a complex pattern of inheritance with ≈50% of familial cases being heterozygous for mutations in the receptor tyrosine kinase RET. Even when identified, the penetrance of RET mutations is only 50–70%, gender-dependent, and varies with the extent of aganglionosis. We searched for additional susceptibility genes which, in conjunction with RET, lead to phenotypic expression by studying 12 multiplex HSCR families. Haplotype analysis and extensive mutation screening demonstrated three types of families: six families harboring severe RET mutations (group I); and the six remaining families, five of which are RET-linked families with no sequence alterations and one RET-unlinked family (group II). Although the presence of RET mutations in group I families is sufficient to explain HSCR inheritance, a genome scan reveals a new susceptibility locus on 9q31 exclusively in group II families. As such, the gene at 9q31 is a modifier of HSCR penetrance. These observations imply that identification of new susceptibility factors in a complex disease may depend on classification of families by mutational type at known susceptibility genes.

In vitro hematopoietic and endothelial potential of flk-1−/− embryonic stem cells and embryos

Mice deficient in the Flk-1 receptor tyrosine kinase are known to die in utero because of defective vascular and hematopoietic development. Here, we show that flk-1−/− embryonic stem cells are nevertheless able to differentiate into hematopoietic and endothelial cells in vitro, although they give rise to a greatly reduced number of blast colonies, a measure of hemangioblast potential. Furthermore, normal numbers of hematopoietic progenitors are found in 7.5-day postcoitum flk-1−/− embryos, even though 8.5-day postcoitum flk-1−/− embryos are known to be deficient in such cells. Our results suggest that hematopoietic/endothelial progenitors arise independently of Flk-1, but that their subsequent migration and expansion require a Flk-1-mediated signal.

p27 and Rb are on overlapping pathways suppressing tumorigenesis in mice

The commitment of cells to replicate and divide correlates with the activation of cyclin-dependent kinases and the inactivation of Rb, the product of the retinoblastoma tumor suppressor gene. Rb is a target of the cyclin-dependent kinases and, when phosphorylated, is inactivated. Biochemical studies exploring the nature of the relationship between cyclin-dependent kinase inhibitors and Rb have supported the hypothesis that these proteins are on a linear pathway regulating commitment. We have been able to study this relationship by genetic means by examining the phenotype of Rb+/−p27−/− mice. Tumors arise from the intermediate lobe cells of the pituitary gland in p27−/− mice, as well as in Rb+/− mice after loss of the remaining wild-type allele of Rb. Using these mouse models, we examined the genetic interaction between Rb and p27. We found that the development of pituitary tumors in Rb+/− mice correlated with a reduction in p27 mRNA and protein expression. To determine whether the loss of p27 was an indirect consequence of tumor formation or a contributing factor to the development of this tumor, we analyzed the phenotype of Rb+/−p27−/− mice. We found that these mice developed pituitary adenocarcinoma with loss of the remaining wild-type allele of Rb and a high-grade thyroid C cell carcinoma that was more aggressive than the disease in either Rb+/− or p27−/− mice. Importantly, we detected both pituitary and thyroid tumors earlier in the Rb+/−p27−/− mice. We therefore propose that Rb and p27 cooperate to suppress tumor development by integrating different regulatory signals.

Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins

Aβ1–42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer’s pathogenesis. Neurotoxicity of amyloid β protein (Aβ) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Aβ oligomers (referred to as ADDLs, for Aβ-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer’s disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer’s disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Aβ-derived diffusible ligands acting upon particular neural signal transduction pathways.

Vascular endothelial growth factor: Direct neuroprotective effect in in vitro ischemia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an in vitro model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR-2 receptors and neuropilin-1, but not VEGFR-1 receptors. The neuropilin-1 ligand placenta growth factor-2 failed to reproduce the protective effect of VEGF, pointing to VEGFR-2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3′-kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3′-kinase/Akt signal transduction system in VEGF-mediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxic-ischemic injury.

Mode matches and their locations in the hydrophobic free energy sequences of peptide ligands and their receptor eigenfunctions

Patterns in sequences of amino acid hydrophobic free energies predict secondary structures in proteins. In protein folding, matches in hydrophobic free energy statistical wavelengths appear to contribute to selective aggregation of secondary structures in “hydrophobic zippers.” In a similar setting, the use of Fourier analysis to characterize the dominant statistical wavelengths of peptide ligands’ and receptor proteins’ hydrophobic modes to predict such matches has been limited by the aliasing and end effects of short peptide lengths, as well as the broad-band, mode multiplicity of many of their frequency (power) spectra. In addition, the sequence locations of the matching modes are lost in this transformation. We make new use of three techniques to address these difficulties: (i) eigenfunction construction from the linear decomposition of the lagged covariance matrices of the ligands and receptors as hydrophobic free energy sequences; (ii) maximum entropy, complex poles power spectra, which select the dominant modes of the hydrophobic free energy sequences or their eigenfunctions; and (iii) discrete, best bases, trigonometric wavelet transformations, which confirm the dominant spectral frequencies of the eigenfunctions and locate them as (absolute valued) moduli in the peptide or receptor sequence. The leading eigenfunction of the covariance matrix of a transmembrane receptor sequence locates the same transmembrane segments seen in n-block-averaged hydropathy plots while leaving the remaining hydrophobic modes unsmoothed and available for further analyses as secondary eigenfunctions. In these receptor eigenfunctions, we find a set of statistical wavelength matches between peptide ligands and their G-protein and tyrosine kinase coupled receptors, ranging across examples from 13.10 amino acids in acid fibroblast growth factor to 2.18 residues in corticotropin releasing factor. We find that the wavelet-located receptor modes in the extracellular loops are compatible with studies of receptor chimeric exchanges and point mutations. A nonbinding corticotropin-releasing factor receptor mutant is shown to have lost the signatory mode common to the normal receptor and its ligand. Hydrophobic free energy eigenfunctions and their transformations offer new quantitative physical homologies in database searches for peptide-receptor matches.

A point mutation in the MET oncogene abrogates metastasis without affecting transformation

The MET oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF), known to stimulate invasive growth of epithelial cells. MET is overexpressed in a significant percentage of human cancers and is amplified during the transition between primary tumors and metastasis. To investigate whether this oncogene is directly responsible for the acquisition of the metastatic phenotype, we exploited a single-hit oncogenic version of MET, able to transform and to confer invasive and metastatic properties to nontumorigenic cells, both in vitro and in nude mice. We mutagenized the signal transducer docking site of Met (Y1349VHVX3Y1356VNV), which has the uncommon property of binding and activating multiple src homology region 2 (SH2)-containing intracellular effectors. Notably, a point mutation (H1351 → N) increased the transforming ability of the oncogene but abolished its metastatic potential. This mutation duplicates the Grb2 binding site, super-activating the Ras pathway and preventing the binding of the other intracellular transducers. Complementation in trans with another nonmetastatic mutant (N1358 → H), recruiting all the transducers downstream to Met except Grb2, rescued the invasive–metastatic phenotype. It is concluded that the metastatic potential of the MET oncogene relies on the properties of its multifunctional docking site, and that a single point mutation affecting signal transduction can dissociate neoplastic transformation from metastasis.

ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.

Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling

The high affinity receptor for IgE, FcɛRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcɛRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcɛRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcɛRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcɛRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcɛRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcɛRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor β receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Trans-spliced leader addition to mRNAs in a cnidarian

A search of databases with the sequence from the 5′ untranslated region of a Hydra cDNA clone encoding a receptor protein-tyrosine kinase revealed that a number of Hydra cDNAs contain one of two different sequences at their 5′ ends. This finding suggested the possibility that mRNAs in Hydra receive leader sequences by trans-splicing. This hypothesis was confirmed by the finding that the leader sequences are transcribed as parts of small RNAs encoded by genes located in the 5S rRNA clusters of Hydra. The two spliced leader (SL) RNAs (SL-A and -B) contain splice donor dinucleotides at the predicted positions, and genes that receive SLs contain splice acceptor dinucleotides at the predicted positions. Both of the SL RNAs are bound by antibody against trimethylguanosine, suggesting that they contain a trimethylguanosine cap. The predicted secondary structures of the Hydra SL RNAs show significant differences from the structures predicted for the SLs of other organisms. Messenger RNAs have been identified that can receive either SL-A or -B, although the impact of the two different SLs on the function of the mRNA is unknown. The presence and features of SL addition in the phylum Cnidaria raise interesting questions regarding the evolution of this process.

Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair

Nerve growth factor (NGF) is a polypeptide which, in addition to its effect on nerve cells, is believed to play a role in inflammatory responses and in tissue repair. Because fibroblasts represent the main target and effector cells in these processes, to investigate whether NGF is involved in lung and skin tissue repair, we studied the effect of NGF on fibroblast migration, proliferation, collagen metabolism, modulation into myofibroblasts, and contraction of collagen gel. Both skin and lung fibroblasts were found to produce NGF and to express tyrosine kinase receptor (trkA) under basal conditions, whereas the low-affinity p75 receptor was expressed only after prolonged NGF exposure. NGF significantly induced skin and lung fibroblast migration in an in vitro model of wounded fibroblast and skin migration in Boyden chambers. Nevertheless NGF did not influence either skin or lung fibroblast proliferation, collagen production, or metalloproteinase production or activation. In contrast, culture of both lung and skin fibroblasts with NGF modulated their phenotype into myofibroblasts. Moreover, addition of NGF to both fibroblast types embedded in collagen gel increased their contraction. Fibrotic human lung or skin tissues displayed immunoreactivity for NGF, trkA, and p75. These data show a direct pro-fibrogenic effect of NGF on skin and lung fibroblasts and therefore indicate a role for NGF in tissue repair and fibrosis.

Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-d-aspartate receptors

The N-methyl-d-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

Macrophage Stimulating Protein Is a Novel Neurotrophic Factor

Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are α/β heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration.