The membrane phospholipid phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] is a critical signal transducer in eukaryotic cells. However, the physiological roles of the type I phosphatidylinositol phosphate kinases (PIPKIs) that synthesize PI(4,5)P2 are largely unknown. Here, we show that the α isozyme of PIPKI (PIPKIα) negatively regulates mast cell functions and anaphylactic responses. In vitro, PIPKIα-deficient mast cells exhibited increased degranulation and cytokine production after Fcɛ receptor-I cross-linking. In vivo, PIPKIα−/− mice displayed enhanced passive cutaneous and systemic anaphylaxis. Filamentous actin was diminished in PIPKIα−/− mast cells, and enhanced degranulation observed in the absence of PIPKIα was also seen in wild-type mast cells treated with latrunculin, a pharmacological inhibitor of actin polymerization. Moreover, the association of FcɛRI with lipid rafts and FcɛRI-mediated activation of signaling proteins was augmented in PIPKIα−/− mast cells. Thus, PIPKIα is a negative regulator of FcɛRI-mediated cellular responses and anaphylaxis, which functions by controlling the actin cytoskeleton and dynamics of FcɛRI signaling. Our results indicate that the different PIPKI isoforms might be functionally specialized.
Phosphatidylinositol 4-phosphate 5-kinase (PIP-5kin) regulates actin cytoskeletal reorganization through its product phosphatidylinositol 4,5-bisphosphate. In the present study we demonstrate that PIP-5kin is essential for neurite remodeling, which is regulated by actin cytoskeletal reorganization in neuroblastoma N1E-115 cells. Overexpression of wild-type mouse PIP-5kin-␣ inhibits the neurite formation that is normally stimulated by serum depletion, whereas a lipid kinase-defective mutant of PIP-5kin-␣, D266A, triggers neurite extension even in the presence of serum and blocks lysophosphatidic acid-induced neurite retraction. These results phenocopy those previously reported for the small GTPase RhoA and its effector p160 Rho-associated coiled coilforming protein kinase (ROCK). However, the ROCKspecific inhibitor Y-27632 failed to block the inhibition by PIP-5kin-␣ of neurite extension, whereas D266A did block the neurite retraction induced by overexpression of ROCK. These results, taken together, suggest that PIP-5kin-␣ functions as a downstream effector for RhoA/ ROCK to couple lysophosphatidic acid signaling to neurite retraction presumably through its product phosphatidylinositol 4,5-bisphosphate.Axon guidance is a critical event in the establishment of neuronal networks during embryogenesis and is regulated by extracellular cues such as chemoattractants and chemorepellents, which are recognized by growth cones at the tips of axons (1, 2). Axons extend toward chemoattractants and away from chemorepellents through mechanisms involving actin cytoskeletal reorganization in the growth cone (3, 4).Recently accumulated evidence suggests that the Rho family GTPases (Rho, Rac, and Cdc42) play crucial roles in reorganizing the actin cytoskeleton in neurons, thereby regulating the morphology of neurites and growth cones (5-8). In particular, Rho appears to be implicated in the repulsive signaling pathway. In mouse neuroblastoma N1E-115 cells, overexpression of a constitutively active mutant of Rho prevents neurite formation (9, 10), and inactivation of endogenous Rho by Clostridium botulinum C3 exoenzyme inhibits the growth cone collapse and neurite retraction induced by lysophosphatidic acid (LPA) 1 (9, 10). Inhibition of growth cone collapse by inactivation of Rho has also been shown for myelin-stimulated central nervous system neurons (11) and Ephrin-A5-stimulated retinal neurons (12).Rho-associated kinases termed ROCK, Rho-kinase, and ROK mediate Rho-induced actin cytoskeletal reorganization (13-15) and subsequent repulsive responses of neurites including growth cone collapse and neurite retraction. Evidence for this includes the findings that overexpression of ROCK as well as the active mutant of RhoA, RhoAV14, inhibits neurite formation in N1E-115 cells (16,17). In addition, pharmacological inactivation of endogenous ROCK, similar to inhibition of Rho activity, interferes with LPA-induced neurite retraction in N1E-115 cells (16,17) and Ephrin-A5-induced growth cone collapse in retinal neurons (12).Although the ...
Stimulation of the neuronal cell adhesion molecule L1 in cerebellar granule neurons (CGNs) enhances neurite outgrowth and this response is inhibited by the primary alcohol ethanol. Because primary alcohols suppress the formation of the signaling lipid phosphatidic acid (PA) by phospholipase D (PLD), this observation prompted us to investigate whether PLD plays a role in the L1-mediated neurite outgrowth in CGNs. In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 expression was not detected. The L1-stimulated neurite outgrowth was inhibited by primary alcohols and by overexpression of lipasedeficient PLD2. Increases in cellular PA levels by direct PA application or overexpression of wild-type PLD2 mimicked the L1-dependent stimulation of neurite outgrowth. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signalregulated kinase (ERK), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcoholrelated neurodevelopmental disorders.
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