Physical and functional cooperation of neural cell adhesion molecule and ß1-integrin in neurite outgrowth induction.

Neural cell adhesion molecule (NCAM) and ß1-integrin are both involved in cell differentiation, with changes in the expression of these two molecules correlating with changes in the malignancy of tumor cells. There is a known functional correlation between NCAM and ß1-integrin in adhesion and also neurite outgrowth in tumor cells. In the present study, we used immunostaining and immunoprecipitation studies to demonstrate that isoform 120 of NCAM associates physically as well as functionally with ß1-integrin in the induction of neurite outgrowth in SH-SY5Y-human neuroblastoma cells. The interaction between these two molecules is mandatory for neurite outgrowth. NCAM blockage completely inhibits the effects of ß1-integrin on neurite outgrowth. These findings further our understanding of the interactions between NCAMs and integrins in malignancy.

Integrin beta-4 signaling plays a key role in mouse embryogenesis.

Integrins, by signaling between extracellular matrix and cell nucleus, serve critical roles in cell proliferation and survival. A knock-in mice was developed by a targeted deletion of the C-terminal segment of the cytoplasmic tail of beta 4-integrin (beta 4-1355T). The beta 4-1355T mice had a longer gestational length, smaller litter sizes, lower fecundity rate, and higher frequency of early pregnancy loss. beta 4-1355T embryos demonstrated a high degree of fragmentation and asymmetry, with fewer surviving to either a morula or blastocyst stage. In wild-type oocytes and embryos, beta1, beta 4, and laminin-5 signals colocalized at the opposing surfaces of blastomeres and between the polar bodies and oocytes. Blastomeres within the beta 4-1355T embryos were less cohesive, with a more diffuse expression of beta 4 and laminin-5 compared with wild type. The alpha 6 beta 4_laminin-5 interaction appears to be vital for maintaining the cohesiveness between the cells of the embryo. Deciphering the role of integrins such as beta 4 in embryogenesis may help explain in vitro fertilization failures.

Targeted deletion of the integrin beta4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kappaB, causing defects in epidermal growth and migration.

The alpha6beta4 integrin-a laminin-5 receptor-mediates assembly of hemidesmosomes and recruitment of Shc and phosphoinositide 3-kinase through the unique cytoplasmic extension of beta4. Mice carrying a targeted deletion of the signaling domain of beta4 develop normally and do not display signs of skin fragility. The epidermis of these mice contains well-structured hemidesmosomes and adheres stably to the basement membrane. However, it is hypoplastic due to reduced proliferation of basal keratinocytes and undergoes wound repair at a reduced rate. Keratinocytes from beta4 mutant mice undergo extensive spreading but fail to proliferate and migrate in response to epidermal growth factor (EGF) on laminin-5. EGF causes significant phosphorylation of extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) and phosphorylation and degradation of IkappaB in beta4 mutant cells adhering to laminin-5. Unexpectedly, however, ERK, JNK, and NF-kappaB remain in the cytoplasm in beta4 mutant cells on laminin-5, whereas they enter effectively into the nucleus in the same cells on fibronectin or in wild-type cells on both matrix proteins. Inhibitor studies indicate that alpha6beta4 promotes keratinocyte proliferation and migration through its effect on NF-kappaB and P-JNK. These findings provide evidence that beta4 signaling promotes epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-kappaB and mitogen-activated protein kinases.

Netrin-integrin signaling in epithelial morphogenesis, axon guidance and vascular patterning.

Netrins and their classical receptors--DCC and neogenin--play key roles in neuronal guidance. Recent developments identify new roles for netrins in epithelial and vascular morphogenesis. Netrins accomplish these effects, at least in part, through binding to integrins and/or activating integrin-associated kinases such as FAK and Fyn. Here we discuss these recent findings and propose that integrins and classical netrin receptors cooperate to regulate multiple aspects of development.

Integrin beta4 signaling promotes tumor angiogenesis.

Mice carrying a targeted deletion of the signaling portion of the integrin beta4 subunit display drastically reduced angiogenesis in response to bFGF in the Matrigel plug assay and to hypoxia in the retinal neovascularization model. Molecular cytology indicates that alpha6beta4 signaling promotes branching of beta4+ medium- and small-size vessels into beta4- microvessels without exerting a direct effect on endothelial cell proliferation or survival. Signaling studies reveal that alpha6beta4 signaling induces endothelial cell migration and invasion by promoting nuclear translocation of P-ERK and NF-kappaB. Upon subcutaneous implantation of various cancer cells, the mutant mice develop smaller and significantly less vascularized tumors than wild-type controls. These results provide genetic evidence that alpha6beta4 signaling promotes the onset of the invasive phase of pathological angiogenesis and hence identify a novel target for antiangiogenic therapy.

Actopaxin is phosphorylated during mitosis and is a substrate for cyclin B1/cdc2 kinase.

Prior to cell division, normal adherent cells adopt a round morphology that is associated with a loss of actin stress fibres and disassembly of focal adhesions. In this study, we investigate the mitotic phosphorylation of the recently described paxillin and actin-binding focal-adhesion protein actopaxin [Nikolopoulos and Turner (2000) J. Cell Biol. 151, 1435-1448]. Actopaxin is comprised of an N-terminus containing six putative cdc2 phosphorylation sites and a C-terminus consisting of tandem calponin homology domains. Here we show that the N-terminus of actopaxin is phosphorylated by cyclin B1/cdc2 kinase in vitro and that this region of actopaxin precipitates cdc2 kinase activity from mitotic lysates. Actopaxin exhibits reduced electrophoretic mobility during mitosis that is dependent on phosphorylation within the first two consensus cdc2 phosphorylation sites. Finally, as cells progress from mitosis to G(1) there is an adhesion-independent dephosphorylation of actopaxin, suggesting that actopaxin dephosphorylation precedes cell spreading and the reformation of focal adhesions. Taken together, these results suggest a role for cyclin B1/cdc2-dependent phosphorylation of actopaxin in regulating actin cytoskeleton reorganization during cell division.

Molecular dissection of actopaxin-integrin-linked kinase-Paxillin interactions and their role in subcellular localization.

Paxillin is a focal adhesion adapter protein involved in integrin signaling. We have recently reported that the paxillin LD1 motif acts as a binding interface for both the actin-binding protein actopaxin and the serine/threonine integrin-linked kinase (ILK). In this report we demonstrate the direct association between actopaxin and ILK and dissect the role of the respective interactions in their subcellular localization. Co-immunoprecipitation experiments were employed to map the binding sites on ILK and actopaxin. ILK binds to the CH2 domain of actopaxin. However, an actopaxin CH2 domain mutant defective for paxillin binding (paxillin binding subdomain mutant) retains the capacity to bind ILK, indicating that paxillin and ILK binding sites on actopaxin are distinct. Actopaxin binds to the C terminus of ILK. Despite the direct binding between actopaxin and ILK, mutation analysis confirmed a primary role for paxillin in their localization to focal adhesions. Interestingly, an ILK mutant (E359K) that was previously reported to act as dominant negative for ILK function was unable to bind actopaxin or paxillin and failed to localize to focal adhesions. This mutant also exhibited in vitro kinase activity comparable with wild-type ILK. Taken together, these data suggest that normal ILK signaling is dependent on efficient localization involving multiple protein interactions.