CCN3 (NOV) Interacts with Connexin43 in C6 Glioma Cells

Fu, Christine; Bechberger, John F.; Ozog, Mark A.; Perbal, Bernard; Naus, Christian C.

doi:10.1074/jbc.m403952200

Cited by 148 publications

(43 citation statements)

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“…For example, the T1 site may become more available or exposed for ␣ 6 ␤ 1 binding if the CT domain of CCN1 interacts with another protein or is removed by proteolysis. Existing evidence for proteolytic processing in CCN2 (35,36) and protein-protein interaction through the CT domain in CCN3 (37)(38)(39) are consistent with these possibilities.…”

Section: Discussionsupporting

confidence: 71%

Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61)

Leu¹,

Chen²,

Chen³

et al. 2004

Journal of Biological Chemistry

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The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin ␣ 6 ␤ 1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel ␣ 6 ␤ 1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support ␣ 6 ␤ 1 -and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with ␣ 6 ␤ 1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished ␣ 6 ␤ 1 -HSPGmediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain ␣ v ␤ 3 -mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in ␣ 6 ␤ 1 -HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for ␣ 6 ␤ 1 -HSPG-dependent CCN1 activities and provide a strategy to investigate these ␣ 6 ␤ 1 -HSPG-specific activities in vivo.

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Section: Discussionsupporting

confidence: 71%

Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61)

Leu¹,

Chen²,

Chen³

et al. 2004

Journal of Biological Chemistry

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“…(1) full-length Cx43 driven by the cytomegalovirus (CMV) promoter (CMV-Cx43) (Fu et al, 2004); (2) C-terminally truncated Cx43 tagged with GFP [Cx43⌬244 -382GFP (Cx43t-GFP)] (Bates et al, 2007); (3) U6 -Cx43siRNA [sequence, CAATTCCTCCTGCCGCAAT (Iacobas et al, 2008); gift from Dr. Eliana Scemes, Albert Einstein College of Medicine, Bronx, NY]; and (4) U6-control-siRNA (sequence, CTC-CTTTTTT; gift from Dr. Timothy O'Connor, University of British Columbia, British Columbia, Canada). The introduction of DNA by in utero electroporation was conducted as described previously (Tabata and Nakajima, 2002;Barnabé-Heider et al, 2005).…”

Section: Methodsmentioning

confidence: 99%

“…Many proteins have been shown to interact with the C-terminal domain of Cx43, linking it to cytoskeletal organization. These proteins include CCN3/NOV (McLeod et al, 2001;Fu et al, 2004), zona occludens-1 (ZO-1) (Giepmans and Moolenaar, 1998), and N-cadherins (Xu et al, 2001), and each represents a key component of a different cellular structure or function, any one of which could assist in regulating migration. Scaffolding proteins such as actin are linked to tight junctions and adherence junctions via members of the membraneassociated guanylate kinase family of proteins such as ZO-1 (Itoh et al, 1997;Perez-Moreno et al, 2003).…”

Section: The Cytoplasmic C-terminal Tail Of Cx43 Is Crucial In Cell Mmentioning

confidence: 99%

Involvement of the Cytoplasmic C-Terminal Domain of Connexin43 in Neuronal Migration

Cina

Maass²,

Theis³

et al. 2009

J. Neurosci.

142

146

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During brain development, young neurons closely associate with radial glial while migrating from the ventricular zone (VZ) to the cortical plate (CP) of the neocortex. It has been shown previously that gap junctions are needed for this migration to occur properly, but the precise mechanism responsible is still in question. Here, we used Cre recombinase, driven by the nestin promoter, to conditionally knock-out a floxed coding DNA of the connexin43 (Cx43) gene in mice. Radial glia in the VZ normally express connexin43. They undergo divisions that produce neurons and astrocytes and serve as migratory guides for the daughter cells that they produce. Based on histological analysis, we suggest that removing Cx43 from radial glia alters the normal lamination of the mouse neocortex. To monitor newborn neurons during development, we introduced a plasmid containing green fluorescent protein driven by a neuronal (T␣1 tubulin) promoter into the embryonic neocortex using in utero electroporation. The transfected migrating neurons remain in the VZ/intermediate zone (IZ) of the Cx43 conditional knock-out (Cx43cKO) animals, whereas in Cx43fl/fl mice, neurons migrate through the IZ into the CP, indicating that deletion of Cx43 from nestin-positive cells disrupts neuronal migration. We were able to rescue migration of Cx43cKO neurons by electroporating a cytomegalovirus-Cx43 expression plasmid into the embryonic cortex. In contrast, a C-terminal truncated form of Cx43 failed to rescue neuronal migration. In addition, Cx43K258stop mice, in which Cx43 lacks the last 125 amino acid residues of the cytoplasmic C-terminal domain, gave results similar to those seen with the Cx43cKO mice. This study illustrates that deletion of the C-terminal domain of Cx43 alters neuronal migration in the neocortex.

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“…In melanocytes, the discoidin domain receptor 1 mediates CCN3-dependent adhesion (11). CCN3 has also been observed to associate with Notch1 (12), fibulin 1C (13), S100A4 (14), and the gap junction protein Cx43 3 (15,16), suggesting that CCN3 may also modulate cell growth via non-integrin signaling pathways.…”

mentioning

confidence: 99%

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

Sin

Tse

Planque

et al. 2009

Journal of Biological Chemistry

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CCN3 (NOV), a putative ligand for integrin receptors, is tightly associated with the extracellular matrix and mediates diverse cellular functions, including cell adhesion and proliferation. CCN3 has been shown to negatively regulate growth although it promotes migration in a cell type-specific manner. In this study, overexpression of CCN3 reduces growth and increases intercellular adhesion of breast cancer cells. Interestingly, CCN3 overexpression also led to the formation of multiple pseudopodia that are enriched in actin, CCN3, and vinculin. Breast cancer cells preincubated with exogenous CCN3 protein also induced the same phenotype, indicating that secreted CCN3 is sufficient to induce changes in cell morphology. Surprisingly, extracellular CCN3 is internalized to the early endosomes but not to the membrane protrusions, suggesting pseudopodia-enriched CCN3 may derive from a different source. The presence of an intracellular variant of CCN3 will be consistent with our finding that the cytoplasmic tail of the gap junction protein connexin43 (Cx43) associates with CCN3. Cx43 is a channel protein permitting intercellular communication to occur. However, neither the channel properties nor the protein levels of Cx43 are affected by the CCN3 protein. In contrast, CCN3 proteins are down-regulated in the absence of Cx43. Finally, we showed that overexpression of CCN3 increases the activity of the small GTPase Rac1, thereby revealing a pathway that links Cx43 directly to actin reorganization.

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CCN3 (NOV) Interacts with Connexin43 in C6 Glioma Cells

Cited by 148 publications

References 50 publications

Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61)

Targeted Mutagenesis of the Angiogenic Protein CCN1 (CYR61)

Involvement of the Cytoplasmic C-Terminal Domain of Connexin43 in Neuronal Migration

Matricellular Protein CCN3 (NOV) Regulates Actin Cytoskeleton Reorganization

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