Synectin, syndecan-4 cytoplasmic domain binding PDZ protein, inhibits cell migration

Syndecan-4, a member of the syndecan gene family of proteoglycans, is an important regulator of bFGF signaling. In particular, bFGF-dependent regulation of cell growth and migration has been linked to syndecan-4 cytoplasmic domain-mediated interactions. Screening of a yeast two-hybrid library with a cytoplasmic domain of rat syndecan-4 identified a novel binding partner, here termed synectin. Synectin is highly homologous to semaphorin F binding protein semcap1, glucose 1 transporter binding protein glut1cbp, and RGS-GAIP/neuropilin-1 binding protein GIPC. Overexpression of synectin in ECV304 cells in culture led to a dose-dependent inhibition of migration while not affecting cell adhesion or growth rate. We conclude that synectin is involved in syndecan-4-dependent interactions and may play a role in the assembly of syndecan-4 signaling complex.     

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.     

Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site

The transferrin receptor (TR) mediates cellular iron uptake by bringing about the endocytosis of transferrin. We investigated whether the cytoplasmic domain of 65 N-terminal amino acids or phosphorylated sites within this domain constitute a structure that is required for TR endocytosis. To test this hypothesis, we modified the cytoplasmic serine residues or introduced a deletion of 36 amino acids by in vitro mutagenesis of a cDNA expression vector for human TR. Upon expression in transfected mouse Ltk- cells, both the wild-type and phosphorylation site mutant receptors mediated transferrin internalization, whereas the truncated receptor did not. These results provide evidence that the cytoplasmic domain, or part of it, is essential for internalization of the TR, but argue against a role for receptor phosphorylation in endocytosis.     

Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells and keratinocytes. It is demonstrated that HB-EGF is not only a ligand for HER1, as previously reported, but for HER4 as well. HB-EGF binds to NIH 3T3 cells overexpressing either HER1 or HER4 alone, but not HER2 or HER3 alone. Binding to HER4 is independent of HER1. The ability of HB-EGF to bind to two different receptors is in contrast to EGF which binds to HER1, but not to HER4, and heregulin-beta1 which binds to HER4, but not to HER1. Besides binding, HB-EGF activates HER4. For example (i) it induces tyrosine phosphorylation of HER4 in cells overexpressing this receptor and of endogenous HER4 in MDA-MB-453 cells and astrocytes; (ii) it induces association of phosphatidylinositol 3-kinase (PI3-K) activity with HER4; and (iii) it is a potent chemotactic factor for cells overexpressing HER4. Chemotaxis is inhibited by wortmannin, a PI3-K inhibitor, suggesting a possible role for PI3-K in mediating HB-EGF-stimulated chemotaxis. On the other hand, HB-EGF is not a mitogen for cells expressing HER4, in contrast to its ability to stimulate both chemotaxis and proliferation in cells expressing HER1. It was concluded that HER4 is a newly described receptor for HB-EGF and that HB-EGF can activate two EGF receptor subtypes, HER1 and HER4, but with different biological responses.     

SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation-dependent pathway

SPARC, a counteradhesive matricellular protein, inhibits endothelial cell adhesion and proliferation, but the pathways through which these activities are blocked are not known. In this study, we used inhibitors of major signaling proteins to identify mediators through which SPARC exerts its counteradhesive and antiproliferative functions. Pretreatments with the general protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, protected against the inhibitory effect of SPARC on bovine aortic endothelial (BAE) cell spreading by more than 60 %. Similar pretreatments with PTK inhibitors significantly blocked the diminishment of focal adhesions by SPARC in confluent BAE cell monolayers, as determined by the formation of actin stress-fibers and the distribution of vinculin in focal adhesion plaques. Inhibition of endothelial cell cycle progression by SPARC and a calcium-binding SPARC peptide, however, was not affected by PTK inhibitors. Inhibition of DNA synthesis by SPARC was not reversed by inhibitors of the activity of protein kinase C (PKC), or of cAMP-dependent protein kinase (PKA), but was sensitive to pertussis (and to a lesser extent, cholera) toxin. The counteradhesive effect of SPARC on endothelial cells is, therefore, mediated through a tyrosine phosphorylation-dependent pathway, whereas its antiproliferative function is dependent, in part, on signal transduction via a G protein-coupled receptor. J. Cell. Biochem. 70:543–552, 1998. © 1998 Wiley-Liss, Inc.     

A cell regulatory agent, CeReS-18, inhibits mouse 3T6 cell proliferation but not polyomavirus replication

We have purified a cell regulatory sialoglycopeptide, CeReS-18, from intact bovine cerebral cortex cells. This is an 18-kDa molecule that reversibly inhibits cellular DNA synthesis and the proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on mouse 3T6 host cell proliferation and polyomavirus replication was investigated. The results showed that CeReS-18 was able to inhibit 3T6 cell cycling in a concentration-dependent, calcium-sensitive, and reversible manner. Despite the inhibition of cell proliferation, CeReS-18 did not influence polyomavirus infection of 3T6 cells. Indirect immunofluorescent assays revealed that CeReS-18-treated, and cell cycle-arrested, 3T6 cells remained permissive to polyomavirus replication. Electron microscopy and immunogold labeling showed that new viral particles were assembled inside the nuclei of infected cells in the presence of CeReS-18 and during cell cycle arrest. The cellular requirements for the replication of polyomavirus DNA and the synthesis of viral proteins, as well as for the assembly of viral particles, therefore, remained available in CeReS-18-inhibited 3T6 cells. In addition, although polyomavirus infection can be mitogenic, infection of CeReS-18-treated 3T6 cells did not reverse the cell cycle arrest mediated by this cell cycle inhibitor.     

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains

Several bacterial pathogens exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo-angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1-mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1-4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1-4L- and CEACAM1 ΔCT-mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3-mediated internalization. Bacteria-engaged CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1- and GPI-anchored protein-containing portion of the plasma membrane. In addition, interference with cholesterol-rich membrane microdomains severely blocks bacterial uptake via CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI-anchored CEACAM6, both CEACAM1-4L as well as CEACAM1 ΔCT partition into a low-density, Triton-insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1-mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.     

Protein kinase C activation in B cells by indolactam inhibits anti-Ig-mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.     

Interferon-gamma inhibits proliferation, but not commitment, of murine granulocyte-macrophage progenitors.

We investigated the effects of interferon gamma (IFN-gamma) on the growth of murine hematopoietic progenitors. IFN-gamma inhibited granulocyte colony-stimulating factor (G-CSF)- and interleukin-3 (IL-3)-dependent colony growth by granulocyte-macrophage (GM) progenitors derived from the bone marrow cells of normal mice. However, the number of IL-3-dependent GM colonies formed by the bone marrow cells of 5-fluorouracil (5-FU)-treated mice was not influenced by the addition of IFN-gamma. Replating experiments suggested that IFN-gamma suppressed GM colony growth directly and that it exerted an inhibitory effect on the proliferation, but not on the commitment, of GM progenitors. In contrast, IFN-gamma failed to suppress colony growth by mast cell progenitors. Erythroid and megakaryocytic progenitors exhibited different responses to IFN-gamma depending on mouse strains. These results suggest that potent negative regulators are not always inhibitors of hematopoietic progenitors.     

Glioma targeting peptide in combination with the P53 C terminus inhibits glioma cell proliferation in vitro

Glioma is a prevalent malignant primary brain tumor in adults, the treatment for which remains a challenge due to its high infiltration and recurrence. Hence, treatments that lead to the suppression of glioma cell migration and invasion may be used in addition to surgery to increase the therapeutic outcome. In this study, we aimed to construct a multifunctional protein that would exert an effect on glioma cell proliferation and migration. The protein is named GL1-P53C-11R and it consists of the glioma-targeting peptide GL1 (G), the P53 C terminus (Pc) and the cell-penetrating peptide arginine (R). GL1-P53C-R was expressed with the fusion protein ZZ and immunofluorescence analysis showed effective delivery of the fused ZZ-GL1-P53C-R protein represented as ZZ-GPcR. The ZZ-GPcR exhibited an inhibitory effect on the proliferation, migration and invasion of U87ΔEGFR cells. Western blotting results indicated that it caused significant changes in the expression levels of cell cycle and apoptotic proteins. Flow cytometric analysis showed increase apoptosis. Our findings suggest that the P53C in the fusion protein ZZ-GPcR can enter into glioma cells to exert its inhibitory effect.     

The C terminus of c-Src inhibits breast tumor cell growth by a kinase-independent mechanism

Overexpression or increased activity of cellular Src (c-Src) is frequently detected in human breast cancer, implicating involvement of c-Src in the etiology of breast carcinomas. Curiously, overexpression of c-Src in tissue culture cells results in a weakly or non-transforming phenotype, indicating that it alone is not sufficient for oncogenesis. However, the protein has been demonstrated to potentiate mitogenic signals from transmembrane receptors. This report investigates the requirement for c-Src in breast cancer as a transducer and integrator of anchorage-dependent and -independent growth signals by utilizing the Src family pharmacological inhibitors, PP1 and PP2, or stable overexpression of the catalytically inactive c-Src mutant (K- c-Src). Both methods of inhibiting endogenous c-Src diminished formation of soft agar colonies and tumors in nude mice. The majority of the dominant-negative activity of K- c-Src was mapped to the Src homology 2 (SH2) domain and C-terminal half of the molecule, but not to the Unique domain, Src homology 3 (SH3) domain, or the N-terminal half of K- c-Src. Further analysis of the C terminus revealed that its ability to inhibit growth localized to the N-terminal lobe (N-lobe) of the catalytic region. These results underscore the requirement for c-Src to maintain the oncogenic phenotype of breast cancer cells and suggest that c-Src may be manipulated to inhibit cell growth by the direct disruption of its catalytic activity or the introduction of either the SH2 domain or the N-lobe of K- c-Src.     

Verapamil inhibits proliferation but not steroidogenesis of regenerating rat adrenal cortex

The effect of verapamil, a calcium channel antagonist, on proliferation and steroidogenesis was investigated in regenerating rat adrenal cortex. Verapamil was given subcutaneously in two doses (1 and 5 mg/kg) to male Wistar rats subjected to adrenal enucleation combined with contralateral adrenalectomy. It was found that verapamil inhibited the mitotic activity of adrenocortical cells on the 4th and 8th day after surgery in a dose-dependent manner. However, no changes in corticosterone secretion were observed.     

Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation

Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The overexpression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mutants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell membrane localization.     

Nebivolol inhibits vascular smooth muscle cell proliferation by mechanisms involving nitric oxide but not cyclic GMP.

The objective of this study was to elucidate the mechanisms by which nebivolol, a cardio-selective beta-adrenergic receptor antagonist, inhibits rat aortic smooth muscle cell (RASMC) proliferation. Nebivolol was compared with DETA-NO and S-nitroso-N-acetylpenicillamine (SNAP), two nitric oxide (NO) donor agents, and alpha-difluoromethylornithine (DFMO), a known inhibitor of ornithine decarboxylase (ODC). All four test agents inhibited RASMC proliferation in a concentration-dependent manner, with nebivolol being the most potent (IC(50) = 4.5 microM), whereas atenolol, another relatively selective beta(1)-blocker, was inactive. DFMO, nebivolol, and DETA-NO interfered with cell proliferation in a cell-density-dependent manner, the lower the cell density the greater the inhibition of cell proliferation. The cytostatic effects of nebivolol and DETA-NO were completely independent of cyclic GMP, as neither ODQ (cytosolic guanylyl cyclase inhibitor) nor zaprinast (cyclic GMP phosphodiesterase inhibitor) affected the antiproliferative action of nebivolol or DETA-NO. The cytostatic effects of nebivolol, SNAP, and DFMO were largely prevented by the addition of excess putrescine, but not ornithine, to cell cultures. Moreover, nebivolol caused a marked reduction in the intracellular levels of putrescine, spermidine, and spermine. Like DFMO, nebivolol and DETA-NO interfered with the G(1)-phase to S-phase cell cycle transition in RASMC. These observations confirm previous findings that DFMO and NO interfere with RASMC proliferation by inhibiting ODC and polyamine production and provide evidence that nebivolol works by the same mechanism.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Pyridalyl inhibits cellular protein synthesis in insect, but not mammalian, cell lines

To gain insight into the mechanism of action and selectivity of the insecticidal activity of pyridalyl, the cytotoxicity of pyridalyl against various insect and mammalian cell lines was characterized by measuring the inhibition of cellular protein synthesis. When the effect of pyridalyl on the cellular protein synthesis in Sf9 cells was evaluated by measuring the incorporation of [(3)H]leucine, rapid and significant inhibition of protein synthesis was observed. However, pyridalyl did not inhibit protein synthesis in a cell-free protein synthesis system, indicating that pyridalyl does not directly inhibit protein synthesis. No obvious cytotoxicity was observed against any of the mammalian cell lines tested. In the case of insect cell lines, remarkable differences in the cytotoxicity of pyridalyl were observed: the highest cytotoxicity (IC50 mM) was found against Sf9 cells derived from Spodoptera frugiperda, whereas no obvious cytotoxicity was observed against BmN4 cells derived from Bombyx mori. Measurements of the insecticidal activity of pyridalyl against Spodoptera litura and B. mori revealed a correlation between the cytotoxicity against cultured cell lines and the insecticidal activity. From these observations, it was concluded that the selective inhibition of cellular protein synthesis by pyridalyl might contribute significantly to the insecticidal activity and the selectivity of this compound.     

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.     

Rapamycin inhibits mTORC1, but not completely

Rapamycin is widely used as a complete inhibitor of the mTORC1 nutrient-sensitive signaling complex. Using a novel ATP-competitive inhibitor named Torin1, we have found that many mTORC1 functions that regulate cap-dependent translation and autophagy are resistant to inhibition by rapamycin.     

Tunicamycin affects somatic embryogenesis but not cell proliferation of carrot

The antibiotic tunicamycin which specifically blocks the first step in the lipid-linked oligosaccharide pathway is capable of arresting somatic embryogenesis in a reversible way. At the same drug concentration cell proliferation is not affected. The quantitative and qualitative changes induced by tunicamycin in glycolipids and glycoproteins are the same in embryogenic and non-embryogenic conditions and this might therefore indicate some proteins whose glycosylation is essential for development.