Modulation of disease severity in cystic fibrosis transmembrane conductance regulator deficient mice by a secondary genetic factor.

Mice that have been made deficient for the cystic fibrosis transmembrane conductance regulator (Cftr) usually die of intestinal obstruction. We have created Cftr-deficient mice and demonstrate prolonged survival among backcross and intercross progeny with different inbred strains, suggesting that modulation of disease severity is genetically determined. A genome scan showed that the major modifier locus maps near the centromere of mouse chromosome 7. Electrophysiological studies on mice with prolonged survival show that the partial rectification of Cl- and Na+ ion transport abnormalities can be explained in part by up-regulation of a calcium-activated Cl- conductance. Identification of modifier genes in our Cftr(m1HSC)/Cftr(m1HSC) mice should provide important insight into the heterogeneous disease presentation observed among CF patients.

Schizosaccharomyces pombe skp1+ encodes a protein kinase related to mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis mutant.

We report the cloning of the skp1+ gene, a Schizosaccharomyces pombe homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in higher eukaryotes are involved in cell fate determination, nuclear signalling, and hormonal regulation. skp1 is 67% identical to mammalian GSK-3 beta and displays similar biochemical properties in vitro. Like GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and this phosphorylation is required for efficient activity. skp1 is also phosphorylated at a serine which has been identified as S-335. Phosphorylation at this site is likely to inhibit its function. Unlike the mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and can phosphorylate other proteins on tyrosine in bacteria. The skp1+ gene is not essential. However, cells with deletions in skp1+ are sensitive to heat shock and exhibit defects in sporulation. Overexpression of wild-type skp1+ specifically complements cdc14-118, one of several mutations causing a defect in cytokinesis. In addition, certain phosphorylation site mutants induce a delay or block in cytokinesis when overexpressed. Together, these data identify novel interactions of a fission yeast GSK-3 homolog with elements of the cytokinesis machinery.

Constitutive activation of the Ras/MAP kinase pathway and enhanced TCR signaling by targeting the Shc adaptor to membrane rafts.

The Shc adaptor is responsible for coupling receptor tyrosine kinases and tyrosine kinase-associated receptors to the Ras/MAP kinase pathway. Shc is believed to be regulated by a change in subcellular localization from the cytosol to the plasma membrane, where it recruits Grb-2/Sos complexes and hence permits juxtaposition of the guanine nucleotide exchange factor Sos to Ras, resulting in GDP/GTP exchange and Ras activation. Shc has been recently shown to inducibly colocalize in detergent-resistant membrane rafts together with the activated TCR and associated signaling molecules. To understand whether Shc localization in membrane rafts is sufficient to regulate Shc function, we constructed a Shc chimera containing the Ras membrane localization motif at the C-terminus. We show that membrane targeted Shc was constitutively localized in the plasma membrane of T-cells, and was mostly compartmentalized in lipid rafts. Membrane targeted Shc was phosphorylated on tyrosine residues and bound Grb-2/Sos in the absence of TCR engagement. Furthermore, expression of membrane targeted Shc resulted in constitutive downstream signaling, including Erk2 activation and enhancement of TCR dependent activation of the TCR responsive transcription factor NF-AT. Hence localization of Shc in membrane rafts is sufficient for Shc to acquire a signaling competent state. Interestingly, a membrane targeted Shc mutant lacking both Grb-2 binding sites was not only incapable of signaling in the absence of TCR triggering, but transdominantly inhibited endogenous Shc, supporting a non redundant role for Shc in the activation of the Ras/MAP kinase pathway in T-cells.

Glycogen synthase kinase-3: functions in oncogenesis and development.

Study of GSK-3 had an inauspicious beginning rooted in intermediary metabolism. However, owing to the fortuitous convergence of several disparate areas of biology, the enzyme now offers unique opportunities for study of the control of a variety cellular processes. While at first sight a role in transcriptional regulation appears unlikely for a protein first identified as acting on glycogen synthase, it is even more surprising that the same protein should be functionally interchangeable with a fruit fly homeotic gene. Such understandable scepticism, however, is based on teleological bias. Glycogen synthase is a critical enzyme regulating glucose storage. The c-Jun oncoprotein may have the potential to transform cells but this does not excuse it from similar mechanisms of control to glycogen synthase. Likewise, homeotic genes play a crucial role in setting up the body plan of an embryo but must also be subject to control. The main difference is that when such control is lost, the result is rather graphic. It is, therefore, only to be expected that regulatory protein kinases will surface in superficially quite unrelated areas and that many of their targets will be 'housekeeping' proteins. Perhaps the most difficult aspect of protein phosphorylation research is the linking of physiological substrates with particular protein kinases, hence reconstructing pathways. No matter how compelling in vitro data appear, there must be demonstration that the protein is targeted by the specific protein kinase in cells, an extremely difficult process. Most progress in this respect has been made using genetic analysis in lower organisms, especially yeast. Here another problem arises: demonstration of biochemical linkages underlying genetic interactions which requires function to be ascribed to genes identified by a gross effect. The challenge is to co-ordinate these two approaches, a strategy currently being employed to further unravel the biological role of GSK-3.

Structural parameters of the Pf1 gene 5 protein-DNA complex in solution by neutron scattering.

Neutron-scattering experiments have been performed on the intracellular complex formed by the gene 5 protein and single-stranded DNA in cells infected by filamentous bacteriophage Pf1. The contrast matched point of the complex (37% 2H2O) is lower than expected and implies that a substantial fraction of potentially labile hydrogen atoms are unable to exchange with the solvent. The mass/length ratio of the complex (3270 daltons/A) indicates an axial subunit repeat of 5.1 A, a value much larger than the subunit repeat previously determined in fibres. The measured value of the cross-sectional radius of gyration at infinite contrast (Rc = 43.3 A) indicates an outer radius of 60 to 63 A for the complex. The variation in Rc with contrast shows that regions of higher scattering density are located, on average, towards the outside of the complex. The high-angle region of the intensity curve (measured in 2H2O) reveals a clear subsidiary maximum at 0.105 A-1 arising from the 60 A helical pitch of the nucleoprotein complex. The structural parameters of the Pf1 gene 5 protein-DNA complex in solution are compared with those of the fd gene 5 protein-DNA complex.

Thermodynamics of the high-affinity interaction of TCF4 with beta-catenin.

The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.

A Saccharomyces cerevisiae protein-serine kinase related to mammalian glycogen synthase kinase-3 and the Drosophila melanogaster gene shaggy product.

The glycogen synthase kinase-3 (GSK-3) family of protein-serine kinases is implicated in the development and hormonal regulation of higher eukaryotes. GSK-3-related genes have been cloned and characterized in mammals (alpha and beta forms), Drosophila melanogaster (shaggy/zeste-white3) and Saccharomyces cerevisiae (MCK1). Using the polymerase chain reaction and primers designed to hybridize to conserved catalytic domain sequences of this family, a genomic fragment was amplified from budding yeast DNA. Genomic clones encompassing the entire reading frame were subsequently isolated and sequenced. The protein encoded by this gene, termed ScGSK-3, displays high identity with members of the GSK-3 family, sharing several structural features including a regulatory Tyr residue. A phylogenetic analysis of the catalytic domains of these protein kinases suggests that ScGSK-3 represents the bona fide homologue of GSK-3 and the shaggy product, while the related MCK1 protein kinase is encoded by a paralogous gene which originated by a gene duplication event in the yeast lineage.

Identification of a compact DNA-binding domain in the gene 5 protein of Pf1 bacteriophage.

The structure of the gene 5 protein of filamentous bacteriophage Pf1 and its interaction with viral DNA have been investigated by a series of limited proteolysis experiments. The ability of purified proteolytic fragments of the Pf1 gene 5 protein to bind oligonucleotides and polynucleotides was monitored by gel retardation and fluorescence. The results show the presence of a compact DNA-binding "core" domain consisting of residues 1-112 of the protein, which is protected from proteolysis in the nucleoprotein complex. Digestion of the free gene 5 protein with subtilisin produces a smaller fragment (residues 7-102) which can no longer bind DNA. Although the N-terminal "core" domain shows full DNA binding activity by fluorescence, the gel retardation experiments suggest reduced kinetic stability of this domain in complexes with oligonucleotides, resulting from the removal of residues 113-144 from the C-terminus of the protein. The sequence of the C-terminal 32 amino acid residues is unusual, with a high proportion of alanine, glutamine, and proline residues which may be related to the role of this sequence in stabilizing the complex.

The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein.

The 144 amino acid gene 5 protein of bacteriophage Pf1 binds tightly and cooperatively to single-stranded DNA during replication of the phage genome. It has been suggested that aromatic amino acid side chains are important for this interaction, probably through base stacking with the DNA. We have analysed the accessibility of tyrosine residues in the DNA-protein complex, and their importance to the DNA-binding activity of the protein, by chemical modification and protection experiments using tetranitromethane. Tyrosines 21, 30 and 55 are surface accessible in the free protein but are protected from modification in the complex with phage DNA. Moreover, modification of these residues in the free protein abolishes the ability to bind to DNA or oligonucleotides, as judged by fluorescence spectroscopy and gel retardation analysis. Modification of the protein also results in the formation of an intersubunit covalent cross-link between Tyr55 and Phe76, suggesting that Phe76 is located within the DNA-binding cleft of the protein. It is proposed that residues 17-34 of the Pf1 gene 5 protein form a beta-hairpin analogous to the 'DNA-binding wing' of the fd and Ike gene 5 proteins. We suggest the existence of a single-stranded DNA binding motif, in which Tyr30 of the Pf1 protein is equivalent to the functionally important Tyr26 of the fd gene 5 protein.

Nuclear onco-protein targets of signal transduction pathways.

Irradiation of mammalian cells with ultraviolet light (200-400 nm) results in the activation of a number of genes, the so-called "UV response" (Herrlich et al., Rev. Physiol. Biochem. Pharmacol., 119:187-223, 1992). Many of the UV responsive genes are also transcriptionally activated by growth factors and mitogens. Two transcription factors have been demonstrated to acutely respond to these stimuli, namely AP-1 and NF-kappa B. Whereas NF-kappa B proteins are primarily controlled via proteolysis of regulatory domains which prevent nuclear translocation, the AP-1 proteins are regulated at several levels including transcription and posttranslational modification. Here, we discuss progress in the identification of the components of pathways acutely regulating these transcription factors.

Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.

Glycogen synthase kinase-3 (GSK-3) is a protein serine kinase implicated in the cellular response to insulin. The enzyme is the mammalian homologue of the zeste-white3 (shaggy) homeotic gene of Drosophila melanogaster and has been implicated in the regulation of the c-Jun/AP-1 transcription factor. In mammals this protein serine kinase is encoded by two related genes termed GSK-3 alpha and beta. Here, we demonstrate that these two proteins and the fruit fly protein are phosphorylated on tyrosine in vivo. Moreover, GSK-3 beta activity and function are shown to be dependent on tyrosine phosphorylation. The modified tyrosine residue is conserved in all members of the GSK-3 family and is equivalent to that required for activity by mitogen-activated protein (MAP) kinases. However, unlike MAP kinases, GSK-3 is highly phosphorylated on tyrosine and thus active in resting cells.

Glycogen synthase kinase 3 regulates cell fate in Dictyostelium.

Extracellular cyclic AMP (cAMP) induces the formation of prespore cells in Dictyostelium but inhibits stalk cell formation. We have cloned gskA, which encodes the Dictyostelium homolog of glycogen synthase kinase 3 (GSK-3), and discovered that it is required for both cAMP effects. Disruption of gskA creates a mutant that aggregates but forms few spores and an abnormally high number of stalk cells. These stalk cells probably arise from an expanded prestalk B (pstB) cell population, which normally produces the basal disc of the fruiting body. In cultured mutant cells, cAMP neither inhibits pstB cell differentiation nor induces efficient prespore cell differentiation. We propose that cAMP acts through a common pathway that requires GSK-3 and determines the proportion of prespore and pstB cells.

Comparison of Pf1 and Fd gene 5 proteins and their single-stranded DNA complexes by NMR spectroscopy and differential scanning calorimetry.

The Pf1 gene 5 protein forms a large helical nucleoprotein complex (Mr = 3.1 x 10(7)) with single-stranded viral DNA, from which a 32 amino acid sequence rich in alanine, proline, and glutamine residues can be removed from the C-terminus by limited proteolysis. Sharp resonances in the 1H NMR spectrum of the Pf1 nucleoprotein complex indicate that the C-terminal region of the protein subunits enjoys remarkable conformational flexibility in the complex. In contrast, the globular N-terminal domain of the protein subunits is rigidly held and does not contribute to the spectrum. The Fd gene 5 protein lacks this C-terminal flexible domain, and no distinct resonances can be observed in the 1H NMR spectrum when this protein is complexed to single-stranded viral DNA. Differential scanning calorimetry shows that the thermal stability of both the Pf1 and Fd gene 5 protein is increased by 8 degrees C in the complex with DNA, and the transition is highly cooperative. Removal of the C-terminal domain of the Pf1 gene 5 protein subunits has no appreciable effect either on the Tm of the DNA-protein complex or on the cooperative nature of the thermal transition. It is suggested that the C-terminal domain of the Pf1 gene 5 protein acts as a dynamic clamp which kinetically stabilizes the nucleoprotein complex.

Glycogen synthase kinase-3 (GSK-3) is regulated during Dictyostelium development via the serpentine receptor cAR3.

Glycogen synthase kinase-3 (GSK-3) is required during metazoan development to mediate the effects of the extracellular signal wingless/Wnt-1 and hence is necessary for correct cell type specification. GSK-3 also regulates cell fate during Dictyostelium development, but in this case it appears to mediate the effects of extracellular cAMP. By direct measurement of GSK-3 kinase activity during Dictyostelium development, we find that there is a rise in activity at the initiation of multicellular development which can be induced by cAMP. The timing of the rise correlates with the requirement for the Dictyostelium homologue of GSK-3, GSKA, to specify cell fate. We show that loss of the cAMP receptor cAR3 almost completely abolishes the rise in kinase activity and causes a mis-specification of cell fate that is equivalent to that seen in a gskA- mutant. The phenotype of a cAR3(-) mutant however is less severe than loss of gskA and ultimately gives rise to an apparently wild-type fruiting body. These results indicate that in Dictyostelium extracellular cAMP acts via cAR3 to cause a rise in GSKA kinase activity which regulates cell type patterning during the initial stages of multicellularity.

Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA.

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue-specific expression and regulation of CFTR function when animal models are used in gene therapy studies.