Neuregulin-heparan-sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle

Neuregulins bind to and activate members of the EGF receptor family of tyrosine kinases that initiate a signaling cascade that induces acetylcholine receptor synthesis in the postsynaptic membrane of neuromuscular synapses. In addition to an EGF-like domain, sufficient for receptor binding and tyrosine auto-phosphorylation, many spliced forms also have an IG-like domain that binds HSPGs and maintains a high concentration of neuregulin at synapses. Here, we show that the IG-like domain functions to keep the EGF-like domain at sufficiently high concentrations for a sufficiently long period of time necessary to induce acetylcholine receptor gene expression in primary chick myotubes. Using recombinant neuregulins with and without the IG-like domain, we found that IG-like domain binding to endogenous HSPGs produces a 4-fold increase in receptor phosphorylation. This enhancement of activity was blocked by soluble heparin or by pretreatment of muscle cells with heparitinase. We show that at least 12-24 h of neuregulin exposure was required to turn on substantial acetylcholine receptor gene expression and that the erbB receptors need to be kept phosphorylated during this time. The need for sustained erbB receptor activation may be the reason why neuregulins are so highly concentrated in the extracellular matrix of synapses.     

Rapid kinetic studies link tetrahydrobiopterin radical formation to heme-dioxy reduction and arginine hydroxylation in inducible nitric-oxide synthase

To understand how heme and (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)B) participate in nitric-oxide synthesis, we followed ferrous-dioxy heme (Fe(II)O(2)) formation and disappearance, H(4)B radical formation, and Arg hydroxylation during a single catalytic turnover by the inducible nitric-oxide synthase oxygenase domain (iNOSoxy). In all cases, prereduced (ferrous) enzyme was rapidly mixed with an O(2)-containing buffer to start the reaction. A ferrous-dioxy intermediate formed quickly (53 s(-1)) and then decayed with concurrent buildup of ferric iNOSoxy. The buildup of the ferrous-dioxy intermediate preceded both H(4)B radical formation and Arg hydroxylation. However, the rate of ferrous-dioxy decay (12 s(-1)) was equivalent to the rate of H(4)B radical formation (11 s(-1)) and the rate of Arg hydroxylation (9 s(-1)). Practically all bound H(4)B was oxidized to a radical during the reaction and was associated with hydroxylation of 0.6 mol of Arg/mol of heme. In dihydrobiopterin-containing iNOSoxy, ferrous-dioxy decay was much slower and was not associated with Arg hydroxylation. These results establish kinetic and quantitative links among ferrous-dioxy disappearance, H(4)B oxidation, and Arg hydroxylation and suggest a mechanism whereby H(4)B transfers an electron to the ferrous-dioxy intermediate to enable the formation of a heme-based oxidant that rapidly hydroxylates Arg.     

Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle

STK15 is an Aurora/Ipl-1 related serine/threonine kinase that is associated with centrosomes and induces aneuploidy when overexpressed in mammalian cells. It is well known that phosphorylation and dephosphorylation of kinases are important for regulation of their activity. But mechanisms by which STK15 activity is regulated have not been elucidated. We report that STK15 contains two functional binding sites for protein phosphatase type 1 (PP1), and the binding of these proteins is cell cycle-regulated peaking at mitosis. Activated STK15 at mitosis phosphorylates PP1 and inhibits PP1 activity in vitro. In vivo, PP1 activity co-immunoprecipitated with STK15 is also reduced. These data indicate that STK15 inhibits PP1 activity during mitosis. Also, PP1 is shown to dephosphorylate active STK15 and abolish its activity in vitro. Furthermore, we show that non-binding mutants of STK15 for PP1 are superphosphorylated, but their kinase activities are markedly reduced. Cells transfected with these non-binding mutants manifest aberrant chromosome alignment during mitosis. Our results suggest that a feedback regulation through phosphorylation/dephosphorylation events between STK15 kinase and PP1 phosphatase operates through the cell cycle. Deregulation of this balance may contribute to anomalous segregation of chromosomes during mitotic progression of cancer cells.     

Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones

The biological activities of transforming growth factor-beta isoforms (TGF-beta(1,2)) are known to be modulated by alpha(2)-macroglobulin (alpha(2)M). alpha(2)M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with alpha(2)M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta(1) molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between (125)I-TGF-beta(1) and activated alpha(2)M (alpha(2)M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta(2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta(1) peptide, whereas the corresponding TGF-beta(3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. (125)I-TGF-beta(3), which lacks Trp-52, bound to alpha(2)M* with an affinity lower than that of (125)I-TGF-beta(1). Furthermore, unlabeled TGF-beta(3) and the mutant TGF-beta(1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-beta(1) in blocking I(125)-TGF-beta(1) binding to alpha(2)M*. TGF-beta(1) and TGF-beta(2) peptides containing Trp-52 were also effective in inhibiting I(125)-nerve growth factor binding to alpha(2)M*. Tauhese results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha(2)M*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with alpha(2)M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha(2)M*.     

Alpha(v) integrin, p38 mitogen-activated protein kinase, and urokinase plasminogen activator are functionally linked in invasive breast cancer cells

We reported previously that endogenous p38 MAPK activity is elevated in invasive breast cancer cells and that constitutive p38 MAPK activity is important for overproduction of uPA in these cells (Huang, S., New, L., Pan, Z., Han, J., and Nemerow, G. R. (2000) J. Biol. Chem. 275, 12266-12272). However, it is unclear how elevated endogenous p38 MAPK activity is maintained in invasive breast cancer cells. In the present study, we found that blocking alpha(v) integrin functionality with a function-blocking monoclonal antibody or down-regulating alpha(v) integrin expression with alpha(v)-specific antisense oligonucleotides significantly decreased the levels of active p38 MAPK and inhibited cell-associated uPA expression in invasive breast cancer MDA-MB-231 cells. These results suggest a function link between alpha(v) integrin, p38 MAPK activity, and uPA expression in invasive tumor cells. We also found that vitronectin/alpha(v) integrin ligation specifically induced p38 MAPK activation and uPA up-regulation in invasive MDA-MB-231 cells but not in non-invasive MCF7 cells. Finally, using a panel of melanoma cells, we demonstrated that the cytoplasmic tail of alpha(v) integrin subunit is required for alpha(v) integrin ligation-induced p38 MAPK activation.     

Overdominant epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice. I. Biomass and grain yield

To understand the genetic basis of inbreeding depression and heterosis in rice, main-effect and epistatic QTL associated with inbreeding depression and heterosis for grain yield and biomass in five related rice mapping populations were investigated using a complete RFLP linkage map of 182 markers, replicated phenotyping experiments, and the mixed model approach. The mapping populations included 254 F(10) recombinant inbred lines derived from a cross between Lemont (japonica) and Teqing (indica) and two BC and two testcross hybrid populations derived from crosses between the RILs and their parents plus two testers (Zhong 413 and IR64). For both BY and GY, there was significant inbreeding depression detected in the RI population and a high level of heterosis in each of the BC and testcross hybrid populations. The mean performance of the BC or testcross hybrids was largely determined by their heterosis measurements. The hybrid breakdown (part of inbreeding depression) values of individual RILs were negatively associated with the heterosis measurements of their BC or testcross hybrids, indicating the partial genetic overlap of genes causing hybrid breakdown and heterosis in rice. A large number of epistatic QTL pairs and a few main-effect QTL were identified, which were responsible for >65% of the phenotypic variation of BY and GY in each of the populations with the former explaining a much greater portion of the variation. Two conclusions concerning the loci associated with inbreeding depression and heterosis in rice were reached from our results. First, most QTL associated with inbreeding depression and heterosis in rice appeared to be involved in epistasis. Second, most ( approximately 90%) QTL contributing to heterosis appeared to be overdominant. These observations tend to implicate epistasis and overdominance, rather than dominance, as the major genetic basis of heterosis in rice. The implications of our results in rice evolution and improvement are discussed.     

Rejection of S-heteroallelic pollen by a dual-specific s-RNase in Solanum chacoense predicts a multimeric SI pollen component

S-heteroallelic pollen (HAP) grains are usually diploid and contain two different S-alleles. Curiously, HAP produced by tetraploids derived from self-incompatible diploids are typically self-compatible. The two different hypotheses previously advanced to explain the compatibility of HAP are the lack of pollen-S expression and the "competition effect" between two pollen-S gene products expressed in a single pollen grain. To distinguish between these two possibilities, we used a previously described dual-specific S(11/13)-RNase, termed HVapb-RNase, which can reject two phenotypically distinct pollen (P(11) and P(13)). Since the HVapb-RNase does not distinguish between the two pollen types (it recognizes both), P(11)P(13) HAP should be incompatible with the HVapb-RNase in spite of the competition effect. We show here that P(11)P(13) HAP is accepted by S(11)S(13) styles, but is rejected by the S(11/13)-RNase, which demonstrates that the pollen-S genes must be expressed in HAP. A model involving tetrameric pollen-S is proposed to explain both the compatibility of P(11)P(13) HAP on S(11)S(13)-containing styles and the incompatibility of P(11)P(13) HAP on styles containing the HVapb-RNase.