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W H Moolenaar

Publications and source records attributed to W H Moolenaar.

At least 37 records · Page 2Linked to original sources
PubMed1999

Activation of RhoA by lysophosphatidic acid and Galpha12/13 subunits in neuronal cells: induction of neurite retraction.

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein-coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Galpha12 and Galpha13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.

Animals↗
PubMed1998

Regulation of astrocyte morphology by RhoA and lysophosphatidic acid.

Astrocytes in the CNS undergo morphological changes and start to proliferate after breakdown of the blood-brain barrier. In culture, proliferating astrocytes have a flat, polygonal shape. When treated with cAMP-raising agents, astrocytes adopt a stellate, process-bearing morphology resembling their in vivo appearance. Stellation is accompanied by loss of actin stress fibers and focal adhesions. Lysophosphatidic acid (LPA), a blood-borne mitogen that signals through its cognate G protein-coupled receptor, stimulates DNA synthesis in astrocytes and causes rapid reversal of cAMP-induced stellation. LPA reversal of stellation is initiated by f-actin reassembly and tyrosine phosphorylation of focal adhesion proteins such as paxillin. Botulinum C3 toxin, which inactivates the Rho GTPase, mimics cAMP-raising agents in inducing stellation, f-actin disassembly, paxillin dephosphorylation, and growth arrest. However, unlike cAMP-induced stellation, C3-induced stellation cannot be reversed by LPA. Conversely, astrocytes expressing activated RhoA fail to undergo cAMP-induced stellation. Thus, RhoA controls astrocyte morphology in that active RhoA directs LPA reversal of stellation, while inactivation of RhoA is sufficient to induce stellation.

ADP Ribose Transferases↗
PubMed1998

Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling.

Rho-like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T-lymphoma cells into a fibroblast monolayer is induced by Tiam1, an activator of the Rho-like GTPase Rac, and by constitutively active V12Rac1. Here we show that activated V12Cdc42 can also induce invasion of T-lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho-inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T-lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum-borne lipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) (10(-7)-10(-6) M), which act on distinct G protein-linked receptors to activate RhoA and phospholipase C (PLC)-Ca2+ signaling. LPA- and S1P-induced invasion is preceded by Rho-dependent F-actin redistribution and pseudopodia formation. However, expression of both V14RhoA and V12Rac1 does not bypass the LPA/S1P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U-73122, but not the inactive analog U-73343, abolishes invasion. Our results indicate that T-lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation, and is dependent on LPA/S1P receptor-mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.

ADP Ribose Transferases↗
PubMed1998

Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells.

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho-ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.

3T3 Cells↗
PubMed1998

Exogenous phospholipase D generates lysophosphatidic acid and activates Ras, Rho and Ca2+ signaling pathways.

BACKGROUND: Phospholipase D (PLD) hydrolyzes phospholipids to generate phosphatidic acid (PA) and a free headgroup. PLDs occur as both intracellular and secreted forms; the latter can act as potent virulence factors. Exogenous PLD has growth-factor-like properties, in that it induces proto-oncogene transcription, mitogenesis and cytoskeletal changes in target cells. The underlying mechanism is unknown, although it is generally assumed that PLD action is mediated by PA serving as a putative second messenger. RESULTS: In quiescent fibroblasts, exogenous PLD (from Streptomyces chromofuscus) stimulated accumulation of the GTP-bound form of Ras, activation of mitogen-activated protein (MAP) kinase and DNA synthesis, through the pertussis-toxin-sensitive inhibitory G protein Gi. Furthermore, PLD mimicked bioactive lysophospholipids (but not PA) in inducing Ca2+ mobilization, membrane depolarization and Rho-mediated neurite retraction. PLD action was mediated by Iysophosphatidic acid (LPA) derived from Iysophosphatidylcholine acting on cognate G-protein-coupled LPA receptor(s). There was no evidence for the involvement of PA in mediating the effects of exogenous PLD. CONCLUSIONS: Our results provide a molecular explanation for the multiple cellular responses to exogenous PLDs. These PLDs generate bioactive LPA from pre-existing Iysophosphatidylcholine in the outer membrane leaflet, resulting in activation of G-protein-coupled LPA receptors and consequent activation of Ras, Rho and Ca2+ signaling pathways. Unscheduled activation of LPA receptors may underlie, at least in part, the known pathogenic effects of exogenous PLDs.

Animals↗
PubMed1998

Acute loss of cell-cell communication caused by G protein-coupled receptors: a critical role for c-Src.

Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.

Animals↗
PubMed1998

Sphingosine 1-phosphate signalling through the G-protein-coupled receptor Edg-1.

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are structurally related lipid mediators that act on distinct G-protein-coupled receptors to evoke similar responses, including Ca2+ mobilization, adenylate cyclase inhibition, and mitogen-activated protein (MAP) kinase activation. However, little is still known about the respective receptors. A recently cloned putative LPA receptor (Vzg-1/Edg-2) is similar to an orphan Gi-coupled receptor termed Edg-1. Here we show that expression of Edg-1 in Sf9 and COS-7 cells results in inhibition of adenylate cyclase and activation of MAP kinase (Gi-mediated), but not Ca2+ mobilization, in response to S1P. These responses are specific in that (i) S1P action is not mimicked by LPA, and (ii) Vzg-1/Edg-2 cannot substitute for Edg-1. Thus the Edg-1 receptor is capable of mediating a subset of the cellular responses to S1P.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Malignant effusions contain lysophosphatidic acid (LPA)-like activity.

BACKGROUND: Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids with mitogenic and growth factor-like activities that act via specific cell-surface receptors present in many normal and transformed cell types. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. The presence of LPA-like activity and the hypothesis that levels of this bioactivity in effusions of ovarian cancer patients are higher than those in effusions of other cancer patients was studied. MATERIALS AND METHODS: A neurite retraction bioassay in a neuroblastoma cell line previously developed for in vitro detection of LPA activity on cell lines was employed and bioactivity was expressed in virtual LPA-equivalent levels. LPA-equivalent levels were tested in effusions of 62 patients with a range of malignancies, including 13 ovarian cancer patients. Biochemical and clinical parameters were evaluated for correlations with LPA-equivalent levels. RESULTS: Average LPA-equivalent levels were 50.2 microns (range 5.4-200) for all patients, and 94.5 microns (range 15-200) for ovarian cancer patients (P = 0.004). There were no additional independent significant correlations between LPA-equivalent levels in effusions and a range of other biochemical and clinical characteristics. CONCLUSION: These data suggest a role for LPA-like lipids in the peritoneal spread of ovarian cancer and possibly that of other predominantly intraperitoneal malignancies.

Adenocarcinoma↗
PubMed1997

Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology.

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein-coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116(Rip), that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116(Rip) stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116(Rip) fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein-coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116(Rip) inhibits RhoA-stimulated contractility and promotes neurite outgrowth.

Amino Acid Sequence↗
PubMed1997

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-zeta.

The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (<45 min). Down-regulation of protein kinase C (PKC) -alpha, -delta and -epsilon isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-zeta but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-zeta is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated epsilon-peptide as a substrate. Furthermore, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-zeta.

Animals↗
PubMed1997

Receptor protein tyrosine phosphatases: involvement in cell-cell interaction and signaling.

Receptor protein tyrosine phosphatases (RPTPs) represent a relatively new family of cell-surface receptors consisting of a variable, putative ligand-binding ectodomain followed by a single transmembrane segment and one or two intracellular catalytic domains. The RPTPs are thought to transduce extracellular signals by dephosphorylating tyrosine-phosphorylated intracellular substrates. As such, they are the enzymatic counterparts of the well studied receptor tyrosine kinases. However, little is known about the signaling mechanisms and biological functions of the RPTPs. Recent studies show that the extracellular domain of certain RPTPs can mediate either homophilic or heterophilic interactions and suggest a role in cadherin-mediated cell-cell adhesion, possibly via an action on catenins. This review will focus on the role of RPTPs in cell-cell interaction and the possible biological implications.

Animals↗
PubMed1997

Lysophosphatidic acid: G-protein signalling and cellular responses.

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates a specific G protein coupled receptor to evoke multiple cellular responses. Recent work has identified two cDNAs encoding putative LPA receptors, various LPA-like agonists that act on distinct receptors, and new pathways that link the receptor(s) to such diverse events as Ras signalling, cytoskeletal remodelling and membrane depolarization.

Animals↗
PubMed1997

Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA.

Addition of lysophosphatidic acid (LPA) to serum-deprived N1E-115 neuronal cells results in rapid f-actin assembly accompanied by neurite retraction and rounding of the cell body due to contraction of the cortical actin cytoskeleton. LPA action is mimicked by activated RhoA, while it is blocked by dominant-negative RhoA (N19RhoA) and the Rho-inactivating C3 toxin. Using immunofluorescence analysis and high speed centrifugation we show that activated RhoA is localized to the plasma membrane. Wild-type RhoA and N19RhoA, however, are mainly cytosolic. We find that LPA-induced shape changes are preceded by translocation of RhoA from the cytosol to the cell periphery. LPA also stimulates translocation of inactive N19RhoA in the absence of ensuing shape changes. When membrane localization of RhoA is prevented by lovastatin, an inhibitor of protein isoprenylation, or by CAAX motif mutation, cytoskeletal contraction is blocked. However, the assembly of f-actin into stress fibers is not affected under these conditions. The effects of both LPA and activated RhoA are blocked by tyrosine kinase inhibitors (herbimycin, genistein, tyrphostin), but not by dominant-negative Src. We conclude that: (1) LPA-induced cytoskeletal contraction, but not stress fiber formation, requires translocation of RhoA from the cytosol to the plasma membrane; (2) translocation of RhoA occurs independently of its activation; and (3), a non-Src tyrosine kinase is involved in RhoA-stimulated contractility.

Actins↗
PubMed1996

Truncated, desensitization-defective neurokinin receptors mediate sustained MAP kinase activation, cell growth and transformation by a Ras-independent mechanism.

We have used the neurokinin NK-2 receptor as a model to examine how receptor desensitization affects cellular responses. The liganded receptor transiently activates phospholipase C (PLC) and is rapidly phosphorylated on Ser/Thr residues in its C-terminal domain. Mutant receptors lacking this domain mediate persistent activation of PLC. We now show that, in transfected Rat-1 cells, mutant receptor mediates ligand-induced DNA synthesis, morphological transformation and growth in soft agar, whereas wild-type (wt) receptor does not. Wt receptor causes only transient MAP kinase activation. In contrast, MAP kinase activation by mutant receptor is sustained for >4 h. Neither wt nor mutant receptor couples to Ras activation. Downregulation of protein kinase C (PKC) has little effect on MAP kinase activation, DNA synthesis and transformation. Mutant receptors also promote stronger protein tyrosine phosphorylation and stress fibre formation than does wt receptor. Thus, C-terminal truncation allows the NK-2 receptor to signal sustained MAP kinase activation, cell growth and transformation by a Ras- and PKC-independent mechanism. Our results reveal the importance of the C-terminal 'desensitization domain' in suppressing the oncogenic potential of a prototypic PLC-coupled receptor.

ADP Ribose Transferases↗
PubMed1996

Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein-coupled receptors to mediate similar actions.

Cell Line↗