The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.     

Integrin-linked kinase controls neurite outgrowth in N1E-115 neuroblastoma cells

Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against beta(1) integrin inhibits neurite outgrowth. Thus, beta(1) integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of beta(1) integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinase kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK-expressed cells. The time course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells.     

Phospholipase D2 activation by p38 MAP kinase is involved in neurite outgrowth

p38 mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth. However, the underlying molecular mechanism(s) remains unclear. Here, we demonstrate that phospholipase D2 (PLD2) mediates p38 signaling in neurite outgrowth. Stimulation of rat pheochromocytoma PC12 cells with nerve growth factor activated PLD2 and augmented neurite outgrowth, both of which were inhibited by pharmacological suppression of p38. Overexpression of constitutively active MAP kinase kinase 6 (MKK6-CA) activated coexpressed PLD2 in PC12 and mouse neuroblastoma N1E-115 cells. Overexpression of wild-type PLD2 in these cells strongly augmented the neurite outgrowth induced by MKK6-CA, whereas lipase-deficient PLD2 suppressed it. These findings provide evidence that PLD2 functions as a downstream molecule of p38 in the neurite outgrowth signaling cascade.     

Regulation of neurite outgrowth in N1E-115 cells through PDZ-mediated recruitment of diacylglycerol kinase zeta

Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase zeta (DGK-zeta), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-zeta and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-zeta in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-zeta kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-zeta mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-zeta promotes neurite outgrowth through association with the GTPase Rac1. DGK-zeta colocalized with Rac1 in neuronal processes and DGK-zeta-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-zeta directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-zeta mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1(V12)) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-zeta with Rac1(V12), suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-zeta binding to active Rac1. Collectively, these findings suggest DGK-zeta, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.     

Wnt-3a and Dvl induce neurite retraction by activating Rho-associated kinase

Dvl is a key protein that transmits the Wnt signal to the canonical beta-catenin pathway and the noncanonical planar cell polarity (PCP) pathway. We studied the roles of Rho-associated kinase (Rho-kinase), which is activated by Dvl in the PCP pathway of mammalian cells. The expression of Dvl-1, Wnt-1, or Wnt-3a activated Rho-kinase in COS cells, and this activation was inhibited by the Rho-binding domain of Rho-kinase. The expression of Dvl-1 in PC12 cells activated Rho and inhibited nerve growth factor (NGF)-induced neurite outgrowth. This inhibition was reversed by a Rho-kinase inhibitor but not by a c-Jun N-terminal kinase inhibitor. Dvl-1 also inhibited serum starvation-dependent neurite outgrowth of N1E-115 cells, and expression of the Rho-binding domain of Rho-kinase reversed this inhibitory activity of Dvl-1. Dvl-1 mutants that did not activate Rho-kinase did not inhibit the neurite outgrowth of N1E-115 cells. Furthermore, the purified Wnt-3a protein activated Rho-kinase and inhibited the NGF-dependent neurite outgrowth of PC12 cells. Wnt-3a-dependent neurite retraction was also prevented by a Rho-kinase inhibitor and a Dvl-1 mutant that suppresses Wnt-3a-dependent activation of Rho-kinase. These results suggest that Wnt-3a and Dvl regulate neurite formation through Rho-kinase and that PC12 and N1E-115 cells are useful for analyzing the PCP pathway.     

Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin-binding proteins by the yeast two-hybrid assay. One of the positive clones contained cDNA encoding a carboxyl-terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix-associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full-length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin-binding site of hnRNP U was located near a carboxyl-terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure.     

Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in differentiation.     

Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells

The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.     

Characterization of a brain-specific Rho GTPase-activating protein,p200RhoGAP

The Rho GTPase-activating proteins (RhoGAPs) are a family of multifunctional molecules that transduce diverse intracellular signals by regulating Rho GTPase activities. A novel RhoGAP family member, p200RhoGAP, is cloned in human and mouse. The murine p200RhoGAP shares 86% sequence identity with the human homolog. In addition to a conserved RhoGAP domain at the N terminus, multiple proline-rich motifs are found in the C-terminal region of the molecules. Northern blot analysis revealed a brain-specific expression pattern of p200RhoGAP. The RhoGAP domain of p200RhoGAP stimulated the GTPase activities of Rac1 and RhoA in vitro and in vivo, and the conserved catalytic arginine residue (Arg-58) contributed to the GAP activity. Expression of the RhoGAP domain of p200RhoGAP in Swiss 3T3 fibroblasts inhibited actin stress fiber formation stimulated by lysophosphatidic acid and platelet-derived growth factor-induced membrane ruffling but not Bradykinin-induced filopodia formation. Endogenous p200RhoGAP was localized to cortical actin in naive N1E-115 neuroblastoma cells and to the edges of extended neurites of differentiated N1E-115 cells. Transient expression of the RhoGAP domain and the full-length molecule, but not the catalytic arginine mutants, readily induced a differentiation phenotype in N1E-115 cells. Finally, p200RhoGAP was capable of binding to the Src homology 3 domains of Src, Crk, and phospholipase Cgamma in vitro and became tyrosine-phosphorylated upon association with activated Src in cells. These results suggest that p200RhoGAP is involved in the regulation of neurite outgrowth by exerting its RhoGAP activity and that its cellular activity may be regulated through interaction with Src-like tyrosine kinases.     

A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line

We have examined the phosphorylation of cellular microtubule proteins during differentiation and neurite outgrowth in N115 mouse neuroblastoma cells. N115 differentiation, induced by serum withdrawal, is accompanied by a fourfold increase in phosphorylation of a 54,000-mol-wt protein identified as a specific isoform of beta-tubulin by SDS PAGE, two-dimensional isoelectric focusing/SDS PAGE, and immunoprecipitation with a specific monoclonal antiserum. Isoelectric focusing/SDS PAGE of [35S]methionine-labeled cell extracts revealed that the phosphorylated isoform of beta-tubulin, termed beta 2, is one of three isoforms detected in differentiated N115 cells, and is diminished in amounts in the undifferentiated cells. Taxol, a drug which promotes microtubule assembly, stimulates phosphorylation of beta-tubulin in both differentiated and undifferentiated N115 cells. In contrast, treatment of differentiated cells with either colcemid or nocodazole causes a rapid decrease in beta-tubulin phosphorylation. Thus, the phosphorylation of beta-tubulin in N115 cells is coupled to the levels of cellular microtubules. The observed increase in beta-tubulin phosphorylation during differentiation then reflects developmental regulation of microtubule assembly during neurite outgrowth, rather than developmental regulation of a tubulin kinase activity.     

Identification of functional cAMP-dependent protein kinase in a 'neurite minus' mouse neuroblastoma cell line

We have characterized and quantitated the level of cAMP-dependent protein kinase in the NS-20, N1E-115, N-18 and N1A-103 mouse neuroblastoma clonal cell lines, and we have correlated the occurrence of functional cAMP-dependent protein kinase with the dibutyryl cAMP-induced differentiated functions in these cells. Our results demonstrate the presence of functional cAMP-dependent protein kinase in extracts of all four cell lines examined, including the 'neurite minus' N1A-103 cell line. Dibutyryl cAMP induced neurite outgrowth and acetylcholinesterase activity in the NS-20, N1E-115 and N-18 neuroblastoma cell lines, but not in the N1A-103 cell line. However, dibutyryl cAMP caused a 2-3-fold increase in the R1 regulatory subunit protein and cAMP-phosphodiesterase activity in the 'neurite minus' N1A-103 cells in a manner similar to that of the other three 'neurite positive' cell lines. These results suggest that the biochemical lesion(s) subserving the neurite-minus phenotype of the N1A-103 cells may be distal to the activation of cAMP-dependent protein kinase and is in a biochemical pathway distinct from the induction of R1 regulatory subunit protein and cAMP-phosphodiesterase activity.     

Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin

Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein-protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.