Microthrombomodulin. Residues 310-486 from the epidermal growth factor precursor homology domain of thrombomodulin will accelerate protein C activation

Thrombomodulin is the endothelial cell cofactor for thrombin-catalyzed activation of protein C. Recently, we isolated a 10-kDa thrombin binding fragment, CB3, from the epidermal growth factor precursor homology domain (epidermal growth factor (EGF)-like regions) of thrombomodulin (Kurasawa, S., Stearns, D. J., Jackson, K.W., and Esmon, C.T. (1988) J. Biol. Chem. 263, 5993-5996). The CB3 fragment did not, however, support protein C activation. A 29-kDa fragment, called CB23, has now been isolated and corresponds to residues 310-486 in the EGF-like region of thrombomodulin. The CB23 fragment bound thrombin and accelerated thrombin-catalyzed protein C activation. With two separate preparations of CB23, the Km for protein C was 1.6 and 1.9 microM and the Kd for thrombin was 8.9 and 13.2 nM. The carboxyl terminus of CB23 and CB3 was identified by isolation and sequence analysis of a tryptic peptide from CB3. The sequence of this peptide corresponded to Asn457-Ser486, indicating that the carboxyl terminus of these fragments is 6 residues beyond the sixth EGF-like region of thrombomodulin. In addition, although CB3 cannot accelerate protein C activation, CB3 did inhibit the rate of thrombin-catalyzed fibrinopeptide release from fibrinogen. Thus, like native thrombomodulin, CB3 will alter thrombin's substrate specificity, but protein C activation requires additional information all of which can be provided by other regions of the EGF-like domain.     

Epidermal growth factor increases c-myc mRNA without eliciting phosphoinositide turnover, protein kinase C activation, or calcium ion mobilization in Swiss 3T3 fibroblasts

Incubation of quiescent cultures of Swiss 3T3 cells with epidermal growth factor (EGF) caused an increase in c-myc mRNA. Under these conditions, EGF did not induce phosphoinositide turnover, formation of diacylglycerol, formation of inositol tris-, bis-, and monophosphates, protein kinase C activation, or Ca2+ mobilization. Although it has been reported that both protein kinase C and Ca2+ may be responsible for the platelet-derived growth factor- and fibroblast growth factor-induced increases in c-myc mRNA in Swiss 3T3 cells (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., & Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192), these results indicate that neither protein kinase C nor Ca2+ is involved in the EGF-induced increase in c-myc mRNA, and that an unidentified system may be involved in this reaction.     

Plakins in development and disease

Plakins are large multi-domain molecules that have various functions to link cytoskeletal elements together and to connect them to junctional complexes. Plakins were first identified in epithelial cells where they were found to connect the intermediate filaments to desmosomes and hemidesmosomes [Ruhrberg, C., and Watt, F.M. (1997). The plakin family: versatile organizers of cytoskeletal architecture. Curr Opin Genet Dev 7, 392-397.]. They were subsequently found to be important for the integrity of muscle cells. Most recently, they have been found in the nervous system, where their functions appear to be more complex, including cross-linking of microtubules (MTs) and actin filaments [Leung, C.L., Zheng, M., Prater, S.M., and Liem, R.K. (2001). The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles. J Cell Biol 154, 691-697., Leung, C.L., Sun, D., Zheng, M., Knowles, D.R., and Liem, R.K. (1999). Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J Cell Biol 147, 1275-1286.]. These plakins have also indicated their relationship to the spectrin superfamily of proteins and the plakins appear to be evolutionarily related to the spectrins, but have diverged to perform different specialized functions. In invertebrates, a single plakin is present in both Drosophila melanogaster and Caenorhabditis elegans, which resemble the more complex plakins found in mammals [Roper, K., Gregory, S.L., and Brown, N.H. (2002). The 'spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families. J Cell Sci 115, 4215-4225.]. In contrast, there are seven plakins found in mammals and most of them have alternatively spliced forms leading to a very complex group of proteins with potential tissue specific functions [Jefferson, J.J., Leung, C.L., and Liem, R.K. (2004). Plakins: goliaths that link cell junctions and the cytoskeleton. Nat Rev Mol Cell Biol 5, 542-553.]. In this review, we will first describe the plakins, desmoplakin, plectin, envoplakin and periplakin and then describe two other mammalian plakins, Bullous pemphigoid antigen 1 (BPAG1) and microtubule actin cross-linking factor 1 (MACF1), that are expressed in multiple isoforms in different tissues. We will also describe the relationship of these two proteins to the invertebrate plakins, shortstop (shot) in Drosophila and VAB-10 in C. elegans. Finally, we will describe an unusual mammalian plakin, called epiplakin.     

Targeting cellular memory to reprogram the epigenome, restore potential, and improve somatic cell nuclear transfer

Successful cloning by somatic cell nuclear transfer (SCNT) is thought to require reprogramming of a somatic nucleus to a state of restored totipotentiality [Dean, W., Santos, F., Reik, W., 2003. Epigenetic programming in early mammalian development and following somatic cell nuclear transfer. Semin. Cell. Dev. Biol. 14, 93-100; Jouneau, A., Renard, J.P., 2003. Reprogramming in nuclear transfer. Curr. Opin. Genet. Dev. 13, 486-491; ]. Though SCNT-induced reprogramming is reminiscent of the reprogramming that occurs after fertilization, reprogramming a differentiated nucleus to an embryonic state is delayed and incomplete in comparison (for review, see ). This is likely due to the existence of an epigenetic-based cellular memory, or program, that serves to regulate global patterns of gene expression, and is the basis of a genome defense mechanism that silences viruses and transposons. The mechanisms of this memory include CpG methylation and modification of histones. Recent evidence by Feng et al. [Feng, Y.-Q., Desprat, R., Fu, H., Olivier, E., Lin, C.M., Lobell, A., Gowda, S.N., Aladjem, M.I., Bouhasira, E.E., 2006. DNA methylation supports intrinsic epigenetic memory in mammalian cells. PLOS Genet. 2, 0461-0470], using a transgenic experimental system, indicates that these marks may be acquired in more than one order and thus, silent heterochromatic structure can be initiated by either methylation of CpG dinucleotides or by histone modifications. In this system, however, CpG methylation appears to differ from histone modifications because it bestows a persistent epigenetic, or cellular, memory. In other words, CpG methylation can independently confer cellular memory, whereas histone modifications appear to be limited in this capacity. Therefore, in the context of genomic reprogramming induced by SCNT, efficient demethylation is likely a key (if not the only) rate-limiting step to improving the efficiency and outcomes of SCNT cloning. This review discusses the possibility of targeting cellular memory, and in particular inducing demethylation of a somatic nucleus prior to nuclear transfer, to enable reprogramming events typically carried out by oocyte factors and thereby improve developmental competence of SCNT-reconstructed embryos. Several recent published reviews of SCNT, cellular reprogramming and genomic demethylation served as valuable sources for the authors and are recommended as supplemental reading. These include the following: Bird, A., 2002. DNA methylation patterns and epigenetic memory. Gen. Dev. 16, 6-21; Grafi, G., 2004. How cells dedifferentiate: a lesson from plants. Dev. Biol. 268, 1-6; Latham, K.E., 2005. Early and delayed aspects of nuclear reprogramming during cloning. Biol. Cell 97, 119-132; Lyko, F., Brown, R., 2005. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J.Natl. Cancer Inst. 97, 1498-1506; Morgan, H.D., Santos, F., Green, K., Dean, W., Reik, W., 2005. Epigenetic reprogramming in mammals. Hum. Mol. Gen. 14, R47-R58; Szyf, M., 2005. DNA methylation and demethylation as targets for anticancer therapy. Biochemistry 70, 533-549; Buszczak, M., Spradling, A.C., 2006. Searching chromatin for stem cell identity. Cell 125, 233-236; Gurdon, J.B., 2006. From nuclear transfer to nuclear reprogramming: the reversal of cell differentiation. Annu. Rev. Cell. Dev. Biol. 22, 1-22; Yoo, C.B., Jones, P.A., 2006. Epigenetic therapy of cancer: past, present and future. Nat. Rev. 5, 37-50.     

Transcytosis of epidermal growth factor. The epidermal growth factor receptor mediates uptake but not transcytosis

Madin-Darby canine kidney (MDCK) cells polarize and generate distinct apical and basolateral membrane domains when grown on permeable filter supports. Under these conditions, they transcytose fluid-phase markers. Recently, receptor-mediated transcytosis of epidermal growth factor (EGF) across MDCK cells has been reported (Maratos-Flier, E., Kao, C.-Y. Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595-1601). We examined the role of the EGF receptor in this process. Transcytosis of EGF occurred only in the basolateral-to-apical direction, was time-dependent, and inhibited by the addition of unlabeled EGF in a concentration-dependent manner. In contrast to previous work, we found that only about 5% of basolaterally bound EGF was transported to the apical chamber. The half-time of transport was 90 min. A mutant cell line of MDCK, MDCKII-RCAr, was used to study the expression of the EGF receptor. Cell surface glycoproteins of these mutant cells can be efficiently labeled with [3H]galactose by exogalactosylation. The EGF receptor was found to be expressed only on the basolateral surface. Addition of EGF to the basolateral medium resulted in rapid internalization and degradation of the receptor. Testing directly for transcytosis of basolateral glycoproteins, we detected several proteins transported across the cell. The EGF receptor, however, was not among this group of proteins. Taking these results together, we suggest the following model. Internalization of EGF on the basolateral surface is mediated by the EGF receptor. EGF dissociates from the receptor in an endocytic compartment. A fraction of the EGF is then diverted nonselectively to the transcytotic pathway, as found for other fluid-phase markers previously (Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., and Simons, K. (1989) J. Cell Biol. 109, 3243-3258.     

Synergism among lysophosphatidic acid, beta1A integrins, and epidermal growth factor or platelet-derived growth factor in mediation of cell migration.

GD25 cells lacking the beta1 integrin subunit or expressing beta1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), ligands of receptor tyrosine kinases, or to lysophosphatidic acid (LPA), a ligand of G-protein-coupled receptors (Sakai, T., Zhang, Q., F?ssler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538 and Sakai, T., Peyruchaud, O., F?ssler, R., and Mosher, D. F. (1998) J. Biol. Chem. 273, 19378-19382). We demonstrate here that LPA synergizes with signals induced by beta1A integrins and ligated EGF or PDGF receptors to modulate migration. When LPA was mixed with EGF or PDGF, migration was greater than with EGF or PDGF alone. The enhancement was greater for beta1A-expressing cells than for beta1-null cells. Cells expressing beta1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs had blunted migratory responses to mixtures of LPA and EGF or PDGF. The major effects on beta1A-expressing cells of LPA when combined with EGF or PDGF were to sensitize cells so that maximal responses were obtained with >10-fold lower concentrations of growth factor and increase the chemokinetic component of migration. Sensitization by LPA was lost when cells were preincubated with pertussis toxin or C3 exotransferase. There was no evidence for transactivation or sensitization of receptors for EGF or PDGF by LPA. EGF or PDGF and LPA caused activation of mitogen-activated protein kinase by pertussis toxin-insensitive and -sensitive pathways respectively, but activation was not additive. These findings indicate that signaling pathways initiated by the cytoplasmic domains of ligated beta1A integrins and tyrosine kinase receptors interact with signaling pathways initiated by LPA to facilitate directed cell migration.     

GON-1 and fibulin have antagonistic roles in control of organ shape

Most developing organs are surrounded by an extracellular matrix (ECM), which must be remodeled to accommodate growth and morphogenesis. In C. elegans, the GON-1 ADAMTS metalloprotease regulates both elongation and shape of the developing gonad . Here, we report that either human ADAMTS-4 or ADAMTS-9 can substitute for GON-1 in transgenic worms, suggesting functional conservation between human and nematode homologs. We further identify fibulin (FBL-1), a widely conserved ECM component , as critical for gonadal morphogenesis. FBL-1 is expressed in nongonadal tissues but is present at the surface of the elongating gonad. A fibulin deletion mutant has a wider than normal gonad as well as body size defects. We find that GON-1 and fibulin have antagonistic roles in controlling gonadal shape. Depletion of fbl-1, but not other ECM components, rescues gon-1 elongation defects, and removal of gon-1 rescues fbl-1 width defects. Therefore, the GON-1 protease normally promotes tissue elongation and expansion, whereas the fibulin ECM protein blocks these key morphogenetic processes. We suggest that control of organ shape by GON-1 and fibulin in C. elegans may provide a model for similar cellular processes, including vasculogenesis, in humans.     

FGF and EGF act synergistically to induce proliferation in BC3H1 myoblasts

BC3H1 muscle cells proliferate when grown in high concentrations of FBS (20%). Lowering the FBS concentration to 0.5% causes the cells to stop proliferating and is permissive for the morphological and biochemical differentiation of BC3H1 cells. Exposure of differentiated BC3H1 myocytes to high concentrations of serum or to the purified growth factors FGF or TGF-b induced a shutdown of this differentiation program but did not induce cell proliferation (Olson et al., J. Cell Biol., 103:1799-1805, 1986; Lathrop et al., J. Cell Biol., 100:1540-1547, 1985, and J. Cell Biol., 101:2194-2198, 1985). We explored the possibility that BC3H1 cells require factors to act synergistically to induce proliferation. We found that EGF and FGF function in a synergistic fashion to stimulate BC3H1 proliferation. Moreover, the temporal requirement for these growth factors suggest that they are functioning as competence and progression factors for BC3H1 cell proliferation.     

BMP7 inhibits branching morphogenesis in the prostate gland and interferes with Notch signaling

The mouse prostate gland develops by branching morphogenesis from the urogenital epithelium and mesenchyme. Androgens and developmental factors, including FGF10 and SHH, promote prostate growth (Berman, D.M., Desai, N., Wang, X., Karhadkar, S.S., Reynon, M., Abate-Shen, C., Beachy, P.A., Shen, M.M., 2004. Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis. Dev. Biol. 267, 387-398; Donjacour, A.A., Thomson, A.A., Cunha, G.R., 2003. FGF-10 plays an essential role in the growth of the fetal prostate. Dev. Biol. 261, 39-54), while BMP4 signaling from the mesenchyme has been shown to suppresses prostate branching (Lamm, M.L., Podlasek, C.A., Barnett, D.H., Lee, J., Clemens, J.Q., Hebner, C.M., Bushman, W., 2001. Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate. Dev. Biol. 232, 301-314). Here, we show that Bone Morphogenetic Protein 7 (BMP7) restricts branching of the prostate epithelium. BMP7 is expressed in the periurethral urogenital mesenchyme prior to formation of the prostate buds and, subsequently, in the prostate epithelium. We show that BMP7(lacZ/lacZ) null prostates show a two-fold increase in prostate branching, while recombinant BMP7 inhibits prostate morphogenesis in organ culture in a concentration-dependent manner. We further explore the mechanisms by which the developmental signals may be interpreted in the urogenital epithelium to regulate branching morphogenesis. We show that Notch1 activity is associated with the formation of the prostate buds, and that Notch1 signaling is derepressed in BMP7 null urogenital epithelium. Based on our studies, we propose a model that BMP7 inhibits branching morphogenesis in the prostate and limits the number of domains with high Notch1/Hes1 activity.     

A mitogen-activated protein (MAP) kinase activating factor in mammalian mitogen-stimulated cells is homologous to Xenopus M phase MAP kinase activator.

The mitogen-activated protein (MAP) kinases, a family of 40-45-kDa kinases whose activation requires both tyrosine and threonine/serine phosphorylations, are suggested to play key roles in various phosphorylation cascades. A previous study of Krebs and co-workers (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) detected an activity in epidermal growth factor (EGF)-stimulated 3T3 cells that can stimulate inactive MAP kinases. We observed this activity in rat 3Y1 cells treated with various mitogenic factors and in PC12 cells treated with nerve growth factor (NGF). Its kinetics of activation and deactivation following EGF or NGF stimulation roughly paralleled that of MAP kinase. The MAP kinase activator required the presence of ATP and a divalent cation such as Mn2+ and Mg2+ and was inactivated by phosphatase 2A treatment in vitro. This activator has been isolated from EGF-stimulated 3Y1 cells by sequential chromatography and identified as a 45-kDa monomeric protein. It was able to activate mammalian and Xenopus MAP kinases in vitro and was very similar to Xenopus M phase MAP kinase activating factor, which was purified previously from mature oocytes (Matsuda, S., Kosako, H., Takenaka, K., Moriyama, K., Sakai, H., Akiyama, T., Gotoh, Y., and Nishida, E. (1992) EMBO J. 11, 973-982), in terms of its functional, immunological, and physicochemical properties. Thus, the same or a similar upstream activating factor may function in mitogen-induced and M phase-promoting factor-induced MAP kinase activation pathways.     

O-linked fucose is present in the first epidermal growth factor domain of factor XII but not protein C.

Epidermal growth factor (EGF) domains are found in many proteins, particularly those of the coagulation/fibrinolytic system. We and others have demonstrated that tissue plasminogen activator (t-PA) and prourokinase are modified by the attachment of fucose to equivalent threonine residues within their EGF domains. Factor XII and protein C each contain two EGF domains; in both proteins, the EGF domain nearest the N terminus has a threonine residue in a position homologous to that which is fucosylated in t-PA. In protein C, this site is 3 residues from the position of another post-translational modification, beta-hydroxylation of Asp-71. We isolated peptides containing these sites to determine, primarily by mass spectrometric analysis, the presence of O-linked fucose and/or beta-hydroxyaspartate. We found that factor XII is fully fucosylated at Thr-90. Protein C is unmodified at the equivalent site (Thr-68) and is completely beta-hydroxylated at Asp-71. It has been recently reported that the first EGF domain of human factor VII has O-linked fucose at the equivalent position (Ser-60) (Bjoern, S., Foster, D. C., Thim, L., Wiberg, F. C., Christensen, M., Komiyama, Y., Pedersen, A. H., and Kisiel, W. (1991) J. Biol. Chem. 266, 11051-11057), while it is unmodified at Asp-63 despite having the consensus sequence for beta-hydroxylation at the latter site. These observations raise the possibility that O-linked fucosylation and beta-hydroxylation of EGF domains are mutually exclusive post-translational modifications.     

A specific enhancing effect of N,N-dimethylsphingosine on epidermal growth factor receptor autophosphorylation. Demonstration of its endogenous occurrence (and the virtual absence of unsubstituted sphingosine) in human epidermoid carcinoma A431 cells

Our previous study suggested that N,N-dimethylsphingosine, but not unsubstituted sphingosine, could be a modulator of protein kinase C in epidermoid carcinoma A431 cells, since N,N-dimethyl-D-erythrosphingenine showed a stronger stereospecific effect on protein kinase C activity in comparison with N,N-dimethyl-L-erythrosphingenine, unsubstituted D- or L-erythrosphingenine, and gangliosides (Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) Biochemistry 28, 6796-6800). Other studies also indicated that commercial sphingosine preparation has an enhancing effect on epidermal growth factor (EGF) receptor kinase activity in A431 cells (Davis, R. J., Girones, N., and Faucher, M. F. (1988) J. Biol. Chem. 263, 5373-5379; Faucher, M. F., Girones, N., Hannun, Y. A., Bell, R. M., and Davis, R. J. (1988) J. Biol. Chem. 263, 5319-5327). In the present paper, we report (i) the effect of N,N-dimethylsphingosine as compared with lyso-glycosphingolipids and other sphingolipid breakdown products on EGF receptor autophosphorylation and (ii) demonstration of endogenous N,N-dimethylsphingosine synthesis and the virtual absence of unsubstituted sphingosine in A431 cells. The autophosphorylation of EGF receptor in the absence of detergent was strongly enhanced by N,N-dimethyl-D-erythrosphingenine; this effect was even obvious in the absence of EGF and synergistic in the presence of EGF. Similar enhancing activity was not produced by N,N-dimethyl-L-erythrosphingenine, D- and L-erythrosphingenine, N-monomethyl-D-erythrosphingenine, N-acetyl-D-erythrosphingenine, or the five lyso-glycosphingolipids tested. Labeling of sphingosine in A431 cells by culturing in medium containing [3H]Ser for various durations, followed by extraction and isolation of sphingolipids by standard procedures, resulted in clear bands corresponding to N,N-dimethylsphingosine and ceramide, whereas the band corresponding to sphingosine was virtually absent. The bands corresponding to N,N-dimethylsphingosine and ceramide intensified when cells were treated with metabolic inhibitor for UDP-Glc:Cer beta-Glc transferase (which causes accumulation of ceramide). These results indicate that N,N-dimethylsphingosine acts as a stereospecific enhancer for EGF receptor kinase and is able to produce EGF-like activity in vitro even in the absence of EGF and detergent. Under physiological conditions, N,N-dimethylsphingosine is the major catabolite resulting from ceramide breakdown.     

Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis.

Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways. In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis [Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We have cloned a 3.1-kb genomic S. pombe fragment which can functionally complement a thi2 mutant. The fragment maps genetically at the thi2 site, indicating that it carries thi2. As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole. The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine [Maundrell, J. Biol. Chem. 265 (1990) 10857-10864; Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 [Schweingruber et al., Genetics (1992) in press]. Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3. This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S. pombe.     

The DNA-binding protein Hdf1p (a putative Ku homologue) is required for maintaining normal telomere length in Saccharomyces cerevisiae.

In mammalian cells, the Ku autoantigen is an end- binding DNA protein required for the repair of DNA breaks [Troelstra, C. and Jaspers, N.G.J. (1994) Curr. Biol., 4, 1149- 1151]. A yeast gene (HDF1) encoding a putative homologue of the 70 kDa subunit of Ku has recently been identified [Feldmann, H. and Winnacker, E. L. (1993) J. Biol. Chem., 268, 12895- 12900]. We find that hdf1 mutant strains have substantially shorter telomeres than wild-type strains. We speculate that Hdf1p may bind the natural ends of the chromosome, in addition to binding to the ends of broken DNA molecules. Strains with both an hdf1 mutation and a mutation in TEL 1 (a gene related to the human ataxia telangiectasia gene) have extremely short telomeres and grow slowly.     

Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474–7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.     

Biosynthesis of the epidermal growth factor receptor: post-translational glycosylation-independent acquisition of tyrosine kinase autophosphorylation activity

We previously showed that the epidermal growth factor (EGF) receptor in human A431 epidermoid carcinoma cells undergoes a slow post-translational modification whereby it acquires (t1/2 = 30-40 min) EGF binding capacity (Slieker, L.J., et. al. (1986) J. Biol. Chem., 261, 15233-15241). This activation occurs in the endoplasmic reticulum and requires core N-linked glycosylation. By employing both anti-EGF receptor and anti-phosphotyrosine antibodies to immunoprecipitate receptor pulse-labeled with [35S]methionine, we demonstrate here that the EGF receptor also acquires tyrosine kinase autophosphorylation activity post-translationally (t1/2 = 10-15 min). The acquisition of tyrosine kinase activity is independent of the acquisition of EGF binding capacity, since it precedes the latter process and does not require N-linked glycosylation.     

Microtubules are involved in anterior-posterior axis formation in C. elegans embryos

Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O'Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55-70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89-92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673-684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92-96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527-3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.     

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)