The Arg-Gly-Asp Motif in the Cell Adhesion Molecule L1 Promotes Neurite Outgrowth via Interaction with the αvβ3 Integrin

The cell adhesion molecule L1 is a potent inducer of neurite outgrowth and it has been implicated in X-linked hydrocephalus and related neurological disorders. To investigate the mechanisms of neurite outgrowth stimulated by L1, attempts were made to identify the neuritogenic sites in L1. Fusion proteins containing different segments of the extracellular region of L1 were prepared and different neuronal cells were assayed on substrate-coated fusion proteins. Interestingly, both immunoglobulin (Ig)-like domains 2 and 6 (Ig2, Ig6) promoted neurite outgrowth from dorsal root ganglion cells, whereas neural retinal cells responded only to Ig2. L1 Ig2 contains a previously identified homophilic binding site, whereas L1 Ig6 contains an Arg-Gly-Asp (RGD) sequence. The neuritogenic activity of Ig6 was abrogated by mutations in the RGD site. The addition of RGD-containing peptides also inhibited the promotion of neurite outgrowth from dorsal root ganglion cells by glutathione S-transferase-Ig6, implicating the involvement of an integrin. The monoclonal antibody LM609 against αvβ3 integrin, but not an anti-β1 antibody, inhibited the neuritogenic effects of Ig6. These data thus provide the first evidence that the RGD motif in L1 Ig6 is capable of promoting neurite outgrowth via interaction with the αvβ3 integrin on neuronal cells.

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C2-Man-)Trp.

RhoA-Dependent Phosphorylation and Relocalization of ERM Proteins into Apical Membrane/Actin Protrusions in Fibroblasts

The ERM proteins (ezrin, radixin, and moesin) are a group of band 4.1-related proteins that are proposed to function as membrane/cytoskeletal linkers. Previous biochemical studies have implicated RhoA in regulating the association of ERM proteins with their membrane targets. However, the specific effect and mechanism of action of this regulation is unclear. We show that lysophosphatidic acid stimulation of serum-starved NIH3T3 cells resulted in relocalization of radixin into apical membrane/actin protrusions, which was blocked by inactivation of Rho by C3 transferase. An activated allele of RhoA, but not Rac or CDC42Hs, was sufficient to induce apical membrane/actin protrusions and localize radixin or moesin into these structures in both Rat1 and NIH3T3 cells. Lysophosphatidic acid treatment led to phosphorylation of radixin preceding its redistribution into apical protrusions. Significantly, cotransfection of RhoAV14 or C3 transferase with radixin and moesin revealed that RhoA activity is necessary and sufficient for their phosphorylation. These findings reveal a novel function of RhoA in reorganizing the apical actin cytoskeleton and suggest that this function may be mediated through phosphorylation of ERM proteins.

B-50/GAP-43-induced Formation of Filopodia Depends on Rho-GTPase

In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50–induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177–226). Cotransfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50–induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.

Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.

RhoA GTPase and Serum Response Factor Control Selectively the Expression of MyoD without Affecting Myf5 in Mouse Myoblasts

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.

Na–H Exchange Acts Downstream of RhoA to Regulate Integrin-induced Cell Adhesion and Spreading

The ubiquitously expressed Na–H exchanger NHE1 functions in regulating intracellular pH and cell volume. NHE1 activity is stimulated by hormones, growth factors, and activation of integrin receptors. We recently determined that NHE1 activity is also stimulated by activation of the low molecular weight GTPase RhoA and that increases in NHE1 activity are necessary for RhoA-induced formation of actin stress fibers. We now show that NHE1 acts downstream of RhoA to modulate initial steps in integrin signaling for the assembly of focal adhesions. Adhesion of CCL39 fibroblasts on fibronectin was markedly delayed in the presence of the NHE inhibitor ethylisopropylamiloride. In mutant PS120 cells, derived from CCL39 fibroblasts but lacking NHE1, adhesion was also delayed but was rescued in PS120 cells stably expressing NHE1. In the absence of NHE1 activity, cell spreading was inhibited, and the accumulation of integrins, paxillin, and vinculin at focal contacts was impaired. Additionally, tyrosine phosphorylation of p125FAK induced by integrin clustering was also impaired. Inactivation of RhoA with C3 transferase and inhibition of the Rho-kinase p160ROCK with the pyridine derivative Y-27632 completely abolished activation of NHE1 by integrins but not by platelet-derived growth factor. These findings indicate that NHE1 acts downstream of RhoA to contribute a previously unrecognized critical signal to proximal events in integrin-induced cytoskeletal reorganization.

αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-αvβ3 antibodies prevent cell adhesion to FGF-2. Also, purified human αvβ3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-αvβ3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with αvβ3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.

Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EMSAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903–10907]. All cell-free extracts formed high-molecular weight protein–DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein–DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA–protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a “mycoparasitic” protein–protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein–DNA complex.

Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.

Cyclin E, a redundant cyclin in breast cancer

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.

Interferon γ expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity

Interferon γ (IFN-γ) has pleiotropic biological effects, including intrinsic antiviral activity as well as stimulation and regulation of immune responses. An infectious recombinant human respiratory syncytial virus (rRSV/mIFN-γ) was constructed that encodes murine (m) IFN-γ as a separate gene inserted into the G-F intergenic region. Cultured cells infected with rRSV/mIFN-γ secreted 22 μg mIFN-γ per 106 cells. The replication of rRSV/mIFN-γ, but not that of a control chimeric rRSV containing the chloramphenicol acetyl transferase (CAT) gene as an additional gene, was 63- and 20-fold lower than that of wild-type (wt) RSV in the upper and lower respiratory tract, respectively, of mice. Thus, the attenuation of rRSV/mIFN-γ in vivo could be attributed to the activity of mIFN-γ and not to the presence of the additional gene per se. The mice were completely resistant to subsequent challenge with wt RSV. Despite its growth restriction, infection of mice with rRSV/mIFN-γ induced a level of RSV-specific antibodies that, on day 56, was comparable to or greater than that induced by infection with wt RSV. Mice infected with rRSV/mIFN-γ developed a high level of IFN-γ mRNA and an increased amount of interleukin 12 p40 mRNA in their lungs, whereas other cytokine mRNAs tested were unchanged compared with those induced by wt RSV. Because attenuation of RSV typically is accompanied by a reduction in immunogenicity, expression of IFN-γ by an rRSV represents a method of attenuation in which immunogenicity can be maintained rather than be reduced.

Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation

Spinal cord neuronal restricted progenitor (NRP) cells, when transplanted into the neonatal anterior forebrain subventricular zone, migrate to distinct regions throughout the forebrain including the olfactory bulb, frontal cortex, and occipital cortex but not to the hippocampus. Their migration pattern and differentiation potential is distinct from anterior forebrain subventricular zone NRPs. Irrespective of their final destination, NRP cells do not differentiate into glia. Rather they synthesize neurotransmitters, acquire region-specific phenotypes, and receive synapses from host neurons after transplantation. Spinal cord NRPs express choline acetyl transferase even in regions where host neurons do not express this marker. The restricted distribution of transplanted spinal cord NRP cells and their acquisition of varied region-specific phenotypes suggest that their ultimate fate and phenotype is dictated by a combination of intrinsic properties and extrinsic cues from the host.

Huntingtin aggregation monitored by dynamic light scattering

An initial stage of fibrillogenesis in solutions of glutathione S-transferase-huntingtin (GST-HD) fusion proteins has been studied by using dynamic light scattering. Two GST-HD systems with poly-l-glutamine (polyGln) extensions of different lengths (20 and 51 residues) have been examined. For both systems, kinetics of z-average translation diffusion coefficients (Dapp) and their angular dependence have been obtained. Our data reveal that aggregation does occur in both GST-HD51 and GST-HD20 solutions, but that it is much more pronounced in the former. Thus, our approach provides a powerful tool for the quantitative assay of GST-HD fibrillogenesis in vitro.

Characterization of a calmodulin kinase II inhibitor protein in brain

Ca2+/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII α and β and potently inhibited kinase activity with an IC50 of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i.e., in the presence of Ca2+/CaM or after autophosphorylation) by using glutathione S-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.