Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Chain reversals in model peptides: studies of cystine-containing cyclic peptides. II. Effects of valyl residues and possible i-to-(i + 3) attractive ionic interactions on cyclization of [Cys1], [Cys6] hexapeptides

The synthesis of four N-acetyl N'-methylamide cystine-containing hexapeptides, CVPGVC, CGVVGC, CKPGEC, and CEPGKC, is described. These were used in disulfide-exchange reactions with the peptide CVPGGC as the formal oxidant. The relative propensities for peptide cyclization were thus deduced, and the tendency toward the formation of chain-reversal conformations was established quantitatively. An additional peptide, CVVVVC, was prepared but was never obtained as the cyclic monomer, demonstrating that the formation of chain-reversals in this peptide was of very low probability. Incorporation of pairs of valyl residues decreased the ease of cyclization, but it appeared that conformational flexibility in the cystine-containing hexapeptides may have compensated for substitutions which would have been expected to hinder the adoption of certain beta-turn conformations. The peptides containing ionic residues were cyclized more readily than expected, and this process was relatively insensitive to salt concentration. This observation is discussed with regard to the stabilization of beta-turns by i-to-(i + 3) ionic interactions in peptides and proteins. A method for blocking thiols was introduced as an improvement in the analysis of the equilibrium mixtures.     

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.     

The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.     

Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits. The free catalytic subunit of casein kinase II is not toxic in vivo.

Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme. Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits. Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal. Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099). Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains. Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit. Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit. Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences. Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S. cerevisiae.     

Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters

The yeast glucose transporters Hxt1, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 and Gal2, individually expressed in an hxt1-7 null mutant strain, demonstrate the phenomenon of countertransport. Thus, these transporters, which are the most important glucose transporters in Saccharomyces cerevisiae, are facilitated diffusion transporters. Apparent K(m)-values from high to low affinity, determined from countertransport and initial-uptake experiments, respectively, are: Hxt6 0.9+/-0.2 and 1.4+/-0.1 mM, Hxt7 1.3+/-0.3 and 1.9+/-0.1 mM, Gal2 1.5 and 1.6+/-0.1 mM, Hxt2 2.9+/-0.3 and 4.6+/-0.3 mM, Hxt4 6.2+/-0.5 and 6.2+/-0.3 mM, Hxt3 28.6+/-6.8 and 34.2+/-3.2 mM, and Hxt1 107+/-49 and 129+/-9 mM. From both independent methods, countertransport and initial uptake, the same range of apparent K(m)-values was obtained for each transporter. In contrast to that in human erythrocytes, the facilitated diffusion transport mechanism of glucose in yeast was symmetric. Besides facilitated diffusion there existed in all single glucose transport mutants, except for the HXT1 strain, significant first-order behaviour.     

Insulin-like effects of epidermal growth factor and insulin-like growth factor-I on [3H]2-deoxyglucose uptake, diacylglycerol generation and protein kinase C activation in BC3H-1 myocytes.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were found to provoke increases in [3H]2-deoxyglucose uptake, diacylglycerol (DAG) generation and membrane-bound protein kinase C activity in BC3H-1 myocytes. These effects were similar to those provoked by insulin. The increases in DAG did not appear to be derived from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol, but may have been derived from synthesis of phosphatidic acid de novo, and hydrolysis of phosphatidylcholine, as revealed by studies with [3H]glycerol and [3H]choline respectively. Accordingly, both EGF and IGF-I increased acute [3H]glycerol labelling of DAG (and other lipids) and [3H]choline labelling of phosphocholine. These labelling responses were similar in time course, suggesting that they are closely coupled. Our findings suggest that EGF and IGF-I, like insulin, increase DAG-protein kinase C signalling, apparently by activating co-ordinated lipid-synthesis and -hydrolysis responses, which are distinctly different from the PIP2-hydrolysis response.     

ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC as well as EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse embryonic stem cells

Effect of angiotensin II (ANG II) on mouse embryonic stem (ES) cell proliferation was examined. ANG II increased [(3)H] thymidine incorporation in a time- (>4 h) and dose- (>10(-9) M) dependent manner. The ANG II-induced increase in [(3)H] thymidine incorporation was blocked by inhibition of ANG II type 1 (AT(1)) receptor but not by ANG II type 2 (AT(2)) receptor, and AT(1) receptor was expressed. ANG II increased inositol phosphates formation and [Ca(2+)](i), and translocated PKC alpha, delta, and zeta to the membrane fraction. Consequently, the inhibition of PLC/PKC suppressed ANG II-induced increase in [(3)H] thymidine incorporation. The inhibition of EGF receptor kinase or tyrosine kinase prevented ANG II-induced increase in [(3)H] thymidine incorporation. ANG II phosphorylated EGF receptor and increased Akt, mTOR, and p70S6K1 phosphorylation blocked by AG 1478 (EGF receptor kinase blocker). ANG II-induced increase in [(3)H] thymidine incorporation was blocked by the inhibition of p44/42 MAPKs but not by p38 MAPK inhibition. Indeed, ANG II phosphorylated p44/42 MAPKs, which was prevented by the inhibition of the PKC and AT(1) receptor. ANG II increased c-fos, c-jun, and c-myc levels. ANG II also increased the protein levels of cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 but decreased the p21(cip1/waf1) and p27(kip1), CDK inhibitory proteins. These proteins were blocked by the inhibition of AT(1) receptor, PLC/PKC, p44/42 MAPKs, EGF receptor, or tyrosine kinase. In conclusion, ANG II-stimulated DNA synthesis is mediated by ANG II receptor-dependent Ca(2+)/PKC and EGF receptor-dependent PI3K/Akt/mTOR/p70S6K1 signal pathways in mouse ES cells.