Biomedical subjects

Jianguo Gu

Publications and source records attributed to Jianguo Gu.

At least 37 records · Page 2Linked to original sources

PubMed2005

Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling.

alpha1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an alpha1,6-linkage to form core fucosylation in mammals. We recently found that disruption of the Fut8 gene induces severe growth retardation and early postnatal death. To investigate the molecular mechanism involved, we have established embryonic fibroblasts of Fut8+/+ and Fut8-/-, derived from wild-type and Fut8-null mice, respectively. Interestingly, the epidermal growth factor (EGF)-induced phosphorylation levels of the EGF receptor (EGFR) were substantially blocked in Fut8-/- cells, compared with Fut8+/+ cells, while there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between two cells. The inhibition of EGFR phosphorylation was completely restored by re-introduction of the Fut8 gene to Fut8-/- cells. Consistent with this, EGFR-mediated JNK or ERK activation was significantly suppressed in Fut8-/- cells. Finally, we found that the core fucosylation of N-glycans is required for the binding of the EGF to its receptor, whereas no effect was observed for the expression levels of EGFR on the cell surface. Collectively, these results strongly suggest that core fucosylation is essential for EGF receptor-mediated biological functions.

Animals↗

PubMed2005

Role of spinal cord glutamate transporter during normal sensory transmission and pathological pain states.

Glutamate is a neurotransmitter critical for spinal excitatory synaptic transmission and for generation and maintenance of spinal states of pain hypersensitivity via activation of glutamate receptors. Understanding the regulation of synaptically and non-synaptically released glutamate associated with pathological pain is important in exploring novel molecular mechanisms and developing therapeutic strategies of pathological pain. The glutamate transporter system is the primary mechanism for the inactivation of synaptically released glutamate and the maintenance of glutamate homeostasis. Recent studies demonstrated that spinal glutamate transporter inhibition relieved pathological pain, suggesting that the spinal glutamate transporter might serve as a therapeutic target for treatment of pathological pain. However, the exact function of glutamate transporter in pathological pain is not completely understood. This report will review the evidence for the role of the spinal glutamate transporter during normal sensory transmission and pathological pain conditions and discuss potential mechanisms by which spinal glutamate transporter is involved in pathological pain.

Amino Acid Transport System X-AG↗

PubMed2005

Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.

The core fucosylation (alpha1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated alpha1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8(-/-) mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8(-/-) mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-beta1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8(-/-) mice have a marked dysregulation of TGF-beta1 receptor activation and signaling, as assessed by TGF-beta1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-beta1 receptor defects found in Fut8(-/-) cells can be rescued by reintroducing Fut8 into Fut8(-/-) cells. Furthermore, exogenous TGF-beta1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8(-/-) lung. We propose that the lack of core fucosylation of TGF-beta1 receptors is crucial for a developmental and progressive/destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.

Activin Receptors, Type I↗

PubMed2005

Bisecting GlcNAc mediates the binding of annexin V to Hsp47.

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.

Acetylglucosamine↗

PubMed2005

Physical interaction and functional coupling between ACDP4 and the intracellular ion chaperone COX11, an implication of the role of ACDP4 in essential metal ion transport and homeostasis.

Divalent metal ions such as copper, manganese, and cobalt are essential for cell development, differentiation, function and survival. These essential metal ions are delivered into intracellular domains as cofactors for enzymes involved in neuropeptide and neurotransmitter synthesis, superoxide metabolism, and other biological functions in a target specific fashion. Altering the homeostasis of these essential metal ions is known to connect to a number of human diseases including Alzheimer disease, amyotrophic lateral sclerosis, and pain. It remains unclear how these essential metal ions are delivered to intracellular targets in mammalian cells. Here we report that rat spinal cord dorsal horn neurons express ACDP4, a member of Ancient Conserved Domain Protein family. By screening a pretransformed human fetal brain cDNA library in a yeast two-hybrid system, we have identified that ACDP4 specifically interacts with COX11, an intracellular metal ion chaperone. Ectopic expression of ACDP4 in HEK293 cells resulted in enhanced toxicity to metal ions including copper, manganese, and cobalt. The metal ion toxicity became more pronounced when ACDP4 and COX11 were co-expressed ectopically in HEK293 cells, suggesting a functional coupling between them. Our results indicate a role of ACDP4 in metal ion homeostasis and toxicity. This is the first report revealing a functional aspect of this ancient conserved domain protein family. We propose that ACDP is a family of transporter protein or chaperone proteins for delivering essential metal ions in different mammalian tissues. The expression of ACDP4 on spinal cord dorsal horn neurons may have implications in sensory neuron functions under physiological and pathological conditions.

Animals↗

PubMed2005

Molecular pain, a new era of pain research and medicine.

Molecular pain is a relatively new and rapidly expanding research field that represents an advanced step from conventional pain research. Molecular pain research addresses physiological and pathological pain at the cellular, subcellular and molecular levels. These studies integrate pain research with molecular biology, genomics, proteomics, modern electrophysiology and neurobiology. The field of molecular pain research has been rapidly expanding in the recent years, and has great promise for the identification of highly specific and effective targets for the treatment of intractable pain. Although several existing journals publish articles on classical pain research, none are specifically dedicated to molecular pain research. Therefore, a new journal focused on molecular pain research is needed. Molecular Pain, an Open Access, peer-reviewed, online journal, will provide a forum for molecular pain scientists to communicate their research findings in a targeted manner to others in this important and growing field.

Biomedical Research↗

PubMed2004

Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal change.

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and the motor cortex. It has been shown that 15-20% of patients with familial ALS (FALS) have defects in the Sod1 gene, which encodes Cu,Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu,Zn-SOD, we examined the issue of whether mutated Cu,Zn-SOD affects the cell cycle. Mouse neuroblastoma Neuro-2a cells were transfected with human wild-type or mutated (G37R, G93A) Cu,Zn-SOD. Mutated, Cu,Zn-SOD-transfected cells exhibited marked retardation in cell growth and G2/M arrest. They also displayed lower reactivity to phalloidin, indicating that the cytoskeleton was disrupted. Immunoprecipitation, two-dimensional gel electrophoresis, and Western blot analysis indicated that mutated Cu,Zn-SOD associates with actin. Similar results were obtained by in vitro incubation experiments with purified actin and mutated Cu,Zn-SOD (G93A). These results suggest that mutated Cu,Zn-SOD in FALS causes cytoskeletal changes by associating with actin, which subsequently causes G2/M arrest and growth retardation.

Actins↗

PubMed2004

Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration.

The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.

Acetylglucosamine↗

PubMed2004

Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8.

Blood vessel formation requires endothelial cell interactions with the extracellular matrix through cell surface receptors, and signaling events that control endothelial cell adhesion, migration, and lumen formation. Laminin-8 (alpha4beta1gamma1) is present in all basement membranes of blood vessels in fetal and adult tissues, but despite its importance in vessel formation, its role in endothelial cell adhesion and migration remains undefined. We examined adhesion and migration of HMEC-1 human microvascular endothelial cells on laminin-8 with an emphasis on the integrin-mediated signaling events, as compared with those on laminin-10/11 and fibronectin. We found that laminin-8 was less potent in HMEC-1 cell adhesion than laminin-1, laminin-10/11, and fibronectin, and mediated cell adhesion through alpha6beta1 integrin. Despite its weak cell-adhesive activity, laminin-8 was as potent as laminin-10/11 in promoting cell migration. Cells adhering to laminin-8 displayed streaks of thin actin filaments and formed lamellipodia at the leading edge of the cells, as observed with cells adhering to laminin-10/11, while cells on fibronectin showed thick actin stress fibers and large focal adhesions. Pull-down assays of GTP-loaded Rho, Rac, and Cdc42 demonstrated that Rac, but not Rho or Cdc42, was preferentially activated on laminin-8 and laminin-10/11, when compared with fibronectin. Furthermore, a dominant-negative mutant of Rac suppressed cell spreading, lamellipodial formation, and migration on laminin-8, but not on fibronectin. These results, taken together, indicate that Rac is activated during endothelial cell adhesion to laminin-8, and is pivotal for alpha6beta1 integrin-mediated cell spreading and migration on laminin-8.

Animals↗

PubMed2004

Regulation of integrin functions by N-glycans.

Integrins are cell surface transmembrane glycoproteins that function as adhesion receptors in cell-ECM interactions and link matrix proteins to the cytoskeleton. Integrins play an important role in cytoskeleton organization and in the transduction of intracellular signals, regulating various processes such as proliferation, differentiation, apoptosis, and cell migration. Although integrin-mediated adhesion is based on the binding of alpha and beta subunits to a defined peptide sequence, the strength of this binding is modulated by various factors including the status of glycosylation of integrin. Glycosylation reactions are catalyzed by the catalytic action of glycosyltransferases, such as N-acetylglucosaminyltransferase III, V and alpha1, 6 fucosyltransferase, etc., which catalyze the formation of glycosidic bonds. This review summarizes effects of the posttranslational modification of N-glycans of alpha3beta1 and alpha5beta1 integrins on their association, activation and biological functions, by using biochemical and genetic approaches.

Animals↗

PubMed2003

N-Acetylglucosaminyltransferase IX acts on the GlcNAc beta 1,2-Man alpha 1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan.

Mammals contain O-linked mannose residues with 2-mono- and 2,6-di-substitutions by GlcNAc in brain glycoproteins. It has been demonstrated that the transfer of GlcNAc to the 2-OH position of the mannose residue is catalyzed by the enzyme, protein O-mannose beta1,2-N-acetylglucosaminyltransferase (POMGnT1), but the enzymatic basis of the transfer to the 6-OH position is unknown. We recently reported on a brain-specific beta1,6-N-acetylglucosaminyltransferase, GnT-IX, that catalyzes the transfer of GlcNAc to the 6-OH position of the mannose residue of GlcNAcbeta1,2-Manalpha on both the alpha1,3- and alpha1,6-linked mannose arms in the core structure of N-glycan (Inamori, K., Endo, T., Ide, Y., Fujii, S., Gu, J., Honke, K., and Taniguchi, N. (2003) J. Biol. Chem. 278, 43102-43109). Here we examined the issue of whether GnT-IX is able to act on the same sequence of the GlcNAcbeta1,2-Manalpha in O-mannosyl glycan. Using three synthetic Ser-linked mannose-containing saccharides, Manalpha1-Ser, GlcNAcbeta1,2-Manalpha1-Ser, and Galbeta1,4-GlcNAcbeta1,2-Manalpha1-Ser as acceptor substrates, the findings show that (14)C-labeled GlcNAc was incorporated only into GlcNAcbeta1,2-Manalpha1-Ser after separation by thin layer chromatography. To simplify the assay, high performance liquid chromatography was employed, using a fluorescence-labeled acceptor substrate GlcNAcbeta1,2-Manalpha1-Ser-pyridylaminoethylsuccinamyl (PAES). Consistent with the above data, GnT-IX generated a new product which was identified as GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1-Ser-PAES by mass spectrometry and (1)H NMR. Furthermore, incorporation of an additional GlcNAc residue into a synthetic mannosyl peptide Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH(2) by GnT-IX was only observed in the presence of POMGnT1. Collectively, these results strongly suggest that GnT-IX may be a novel beta1,6-N-acetylglucosaminyltransferase that is responsible for the formation of the 2,6-branched structure in the brain O-mannosyl glycan.

Brain↗

PubMed2003

Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells.

A rat pheochromocytoma cell line (PC12), when transfected with beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)-mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.

Animals↗

PubMed2003

CD151 regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin cytoskeletal reorganization.

CD151, a member of the tetraspanin family proteins, tightly associates with integrin alpha3beta1 and localizes at basolateral surfaces of epithelial cells. We found that overexpression of CD151 in A431 cells accelerated intercellular adhesion, whereas treatment of cells with anti-CD151 mAb perturbed the integrity of cortical actin filaments and cell polarity. E-Cadherin puncta formation, indicative of filopodia-based adhesion zipper formation, as well as E-cadherin anchorage to detergent-insoluble cytoskeletal matrix, was enhanced in CD151-overexpressing cells. Levels of GTP-bound Cdc42 and Rac were also elevated in CD151-overexpressing cells, suggesting the role of CD151 in E-cadherin-mediated cell-cell adhesion as a modulator of actin cytoskeletal reorganization. Consistent with this possibility, engagement of CD151 by the substrate-adsorbed anti-CD151 mAb induced prominent Cdc42-dependent filopodial extension, which along with E-cadherin puncta formation, was strongly inhibited by calphostin C, a protein kinase C (PKC) inhibitor. Together, these results indicate that CD151 is involved in epithelial cell-cell adhesion as a modulator of PKC- and Cdc42-dependent actin cytoskeletal reorganization.

Actins↗

PubMed2003

Recombinant expression and characterization of a novel fibronectin isoform expressed in cartilaginous tissues.

A novel fibronectin (FN) isoform lacking the segment from IIICS (type III connecting segment) through the I-10 module is expressed predominantly in normal cartilaginous tissues. We expressed and purified recombinant cartilage-type FN using a mammalian expression system and characterized its molecular and biological properties. Although FNs have been shown to be secreted as disulfide-bonded dimers, cartilage-type FN was secreted mainly as a monomer. It was less potent than plasma-type FN in promoting cell adhesion and binding to integrin alpha5beta1, although it was more active than plasma-type FN in binding to chondroitin sulfate E. When added exogenously, cartilage-type FN was poorly assembled into the fibrillar FN matrix, mostly because of its monomeric structure. Given that cartilage is characterized by its non-fibrillar matrix with abundant chondroitin sulfate-containing proteoglycans, it is likely that cartilage-type FN has evolved to adapt itself to the non-fibrillar structure of the cartilage matrix through acquisition of a novel mechanism of alternative pre-mRNA splicing.

Animals↗

PubMed2003

Relationship between elevated FX expression and increased production of GDP-L-fucose, a common donor substrate for fucosylation in human hepatocellular carcinoma and hepatoma cell lines.

The levels of fucosylated glycoproteins in various cancers and inflammatory processes have been a subject of intense study. The level of fucosyltransferases and intracellular GDP-L-fucose, a sugar nucleotide and a common donor substrate for all fucosyltransferases, may regulate the level of fucosylated glycoproteins. This study reports on the determination of GDP-L-fucose levels in human hepatocellular carcinoma (HCC) and surrounding tissues, using a recently established high-throughput assay system. Levels of GDP-L-fucose in HCC tissues were significantly increased compared with adjacent nontumor tissues or normal livers. The mean +/- SD for GDP-L-fucose level was 3.6 +/- 0.2 micro mol/mg in control liver, 4.6 +/- 0.9 micro mol/mg in adjacent noninvolved liver tissues (chronic hepatitis, 4.4 +/- 0.7 micro mol/mg; liver cirrhosis, 4.8 +/- 0.9 micro mol/mg), and 7.1 +/- 2.5 micro mol/mg in HCC tissues. The level of GDP-L-fucose in HCC decreased in proportion with tumor size (r = -0.675, P = 0.0002). When expression of the series of genes responsible for GDP-L-fucose synthesis was investigated, the gene expression of FX was found to be increased in 70% (7 of 10) of the HCC tissues examined compared with that in their surrounding tissues. The levels of GDP-L-fucose were positively correlated with the expression of FX mRNA (r = 0.599, P = 0.0074). The levels of FX gene expression in some human hepatoma and hepatocyte cell lines were determined. FX mRNA production was strongly increased in HepG2 and Chang liver, moderately increased in Hep3B and HLF, and, in HLE, was similar to that of a normal human liver tissue. To investigate the effect of GDP-L-fucose on core fucosylation, FX cDNA was transfected into Hep3B cells, which express a relatively low level of GDP-L-fucose:N-acetyl-beta-D-glucosaminide alpha1-6 fucosyltransferase (alpha1-6 FucT) and FX mRNA. Transfection of this gene caused an increase in GDP-L-fucose levels as well as the extent of fucosylation on glycoproteins, including alpha-fetoprotein, as judged by reactivity to lectins. Collectively, the results herein suggest that the high level of fucosylation in HCC is dependent on a high expression of FX followed by increases in GDP-L-fucose, as well as an enhancement in alpha1-6 FucT expression. Thus, an elevation in GDP-L-fucose levels and the up-regulation of FX expression represent potential markers for HCC.

Carbohydrate Sequence↗

PubMed2003

Molecular cloning and characterization of human GnT-IX, a novel beta1,6-N-acetylglucosaminyltransferase that is specifically expressed in the brain.

A novel beta1,6-N-acetylglucosaminyltransferase (beta1, 6GnT) cDNA was identified by a BLAST search using the amino acid sequence of human GnT-V as a query. The full-length sequence was determined by a combination of 5'-rapid amplification of cDNA end analysis and a further data base search. The open reading frame encodes a 792 amino acid protein with a type II membrane protein structure typical of glycosyltransferases. The entire sequence identity to human GnT-V is 42%. When pyridylaminated (PA) agalacto biantennary N-linked oligosaccharide was used as an acceptor substrate, the recombinant enzyme generated a novel product other than the expected GnT-V product, (GlcNAcbeta1,2-Manalpha1,3-)[GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA. This new product was identified as [GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,3-][Glc-NAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA by mass spectrometry and 1H NMR. Namely, the new GnT (designated as GnT-IX) has beta1,6GnT activity not only to the alpha1,6-linked mannose arm but also to the alpha1,3-linked mannose arm of N-glycan, forming a unique structure that has not been reported to date. Northern blot analysis showed that the GnT-IX gene is exclusively expressed in the brain, whereas the GnT-V gene is expressed ubiquitously. These results suggest that GnT-IX is responsible for the synthesis of a unique oligosaccharide structure in the brain.

Amino Acid Sequence↗

PubMed2003

Csk regulates integrin-mediated signals: involvement of differential activation of ERK and Akt.

Csk phosphorylates Src family tyrosine kinases and down-regulates their activities in vitro and in vivo. To gain insight into the integrin-mediated cellular functions of this negative regulator of the Src family, we examined integrin-mediated signals in Csk-deficient fibroblasts (Csk(-) cells) and their stable transfectants expressing re-introduced Csk (Csk(-)/Csk cells). Integrin-mediated activation of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase in Csk(-)/Csk cells upon adhesion to fibronectin or laminin-10/11 was down-regulated, whereas Akt activation increased. Interestingly, the suppression of ERK-MAP kinase activation in Csk(-)/Csk cells was restored by overexpression of a dominant-negative Akt. In agreement with these results, Csk(-)/Csk cells were more resistant to apoptosis induced by serum depletion, but were less proliferative, compared with Csk(-) cells. These results, taken together, demonstrate that Csk is an important regulator of integrin-mediated signaling and cellular behavior.

Animals↗

PubMed2003

Characterization of the ligand-binding specificities of integrin alpha3beta1 and alpha6beta1 using a panel of purified laminin isoforms containing distinct alpha chains.

Integrins alpha3beta1 and alpha6beta1 are two major laminin receptors expressed on the surface of mammalian cells. Interactions of cells with laminins through these integrins play important roles in cell adhesion, differentiation, motility, and matrix assembly. To determine the binding specificity and affinity of these integrins toward various types of laminins at the level of direct protein-protein interactions, we purified integrins alpha3beta1 and alpha6beta1 from human placenta, and examined their binding to a panel of laminin isoforms, each containing distinct alpha chains (i.e., laminin-1, laminin-2/4, laminin-5, laminin-8, and laminin-10/11). Integrin alpha3beta1 showed clear specificity for laminin-5 and laminin-10/11, with no significant binding to laminin-1, laminin-2/4, and laminin-8. In contrast, integrin alpha6beta1 showed a broad spectrum of specificity, with apparent binding affinity in the following order: laminin-10/11 > laminin-5 > laminin-1 > laminin-2/4 congruent with laminin-8. Integrin titration assays demonstrated that laminin-10/11 was the most preferred ligand among the five distinct laminin isoforms for both alpha3beta1 and alpha6beta1 integrins. Given that laminin-10/11 is the major basement membrane component of many adult tissues, the interaction of laminin-10/11 with these integrins should play a central role in the adhesive interactions of epithelial cells with underlying basement membranes.

Animals↗