Phosphatidylinositol 4-phosphate 5-kinase (PIPK) catalyzes a final step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP 2 ), a lipid signaling molecule. Strict regulation of PIPK activity is thought to be essential in intact cells. Here we show that type I enzymes of PIPK (PIPKI) are phosphorylated by cyclic AMPdependent protein kinase (PKA), and phosphorylation of PIPKI suppresses its activity. Serine 214 was found to be a major phosphorylation site of PIPK type I␣ (PIPKI␣) that is catalyzed by PKA. In contrast, lysophosphatidic acid-induced protein kinase C activation increased PIPKI␣ activity. Activation of PIPKI␣ was induced by dephosphorylation, which was catalyzed by an okadaic acid-sensitive phosphatase, protein phosphatase 1 (PP1). In vitro dephosphorylation of PIPKI␣ with PP1 increased PIPK activity, indicating that PP1 plays a role in lysophosphatidic acid-induced dephosphorylation of PIPKI␣. These results strongly suggest that activity of PIPKI␣ in NIH 3T3 cells is regulated by the reversible balance between PKA-dependent phosphorylation and PP1-dependent dephosphorylation.Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 is a signalgenerating phospholipid with crucial roles in various cellular processes. PIP 2 is the best substrate for phosphoinositide-specific phospholipase C, and PIP 2 hydrolysis generates two second messengers, 1,2-diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Inositol 1,4,5-trisphosphate binds to specific receptors and induces release of calcium from intracellular stores (1), whereas DG activates protein kinase C (PKC) (2). In mammalian cells, PIP 2 can be further phosphorylated by phosphoinositide 3-kinase for generation of phosphatidylinositol 3,4,5-trisphosphate, a mediator of cell growth and survival (3, 4). In addition, PIP 2 directly modulates the activity of numerous enzymes and proteins involved in diverse cellular processes including exocytosis (5), cytoskeletal re-organization (6 -8), and membrane trafficking (9).Consistent with this important role played by PIP 2 in cellular signaling, intracellular PIP 2 levels are strictly regulated (10, 11). In response to various extracellular stimuli, a hydrolysis of PIP 2 has taken place, and its levels are rapidly decreased, resulting in the shortage of PIP 2 . However, since compensatory synthesis of PIP 2 is rapidly induced, PIP 2 levels are constantly maintained, and the subsequent generation of second messengers inositol 1,4,5-trisphosphate and DG is renewed (12) .PIP 2 is synthesized from phosphatidylinositol (PI) by two lipid kinases, phosphatidylinositol kinase (PIK) and PIPK. At least two immunologically distinct PIPK subtypes, PIPK type I and PIPK type II (13), exist in mammalian cells and are composed of three isoforms ␣, ␤, and ␥ (13-18). Most PIP 2 synthesis is catalyzed by type I enzymes, which phosphorylate the D-5 position of the inositol ring of PI 4-phosphate (PI4P). Recently, type II enzymes have been reported to be a PI 5-phosphate 4-kinase (19), which suggests the presence of an alternat...

Reactive oxygen species (ROS) generation is linked to dynamic actin cytoskeleton reorganization, which is involved in tumor cell motility and metastasis. Thus, inhibition of ROS generation and actin polymerization in tumor cells may represent an effective anticancer strategy. However, the molecular basis of this signaling pathway is currently unknown. Here, we show that the Ecklonia cava-derived antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Steady-state intracellular ROS levels were higher in malignant B16F10 cells than in parental, nonmetastatic B16F0 cells. Elevation of ROS by H(2)O(2) treatment increased migration and invasion ability of B16F0 cells to level similar to that of B16F10 cells, suggesting that intracellular ROS signaling mediates the prometastatic properties of B16 mouse melanoma cells. ROS levels and the cell migration and invasion ability of B16 melanoma cells correlated with Rac1 activation and WAVE2 expression. Overexpression of dominant negative Rac1 and depletion of WAVE2 by siRNA suppressed H(2)O(2)-induced cell invasion of B16F0 and B16F10 cells. Similarly, dieckol attenuates the ROS-mediated Rac1 activation and WAVE2 expression, resulting in decreased migration and invasion of B16 melanoma cells. In addition, we found that dieckol decreases association between WAVE2 and NADPH oxidase subunit p47(phox). Therefore, this finding suggests that WAVE2 acts to couple intracellular Rac1/ROS signaling to the invasive migration of B16 melanoma cells, which is inhibited by dieckol.

We have previously isolated dieckol, a nutrient polyphenol compound, from the brown alga, Ecklonia cava (Lee et al.,2010a). Dieckol shows both antitumor and antioxidant activity and thus is of special interest for the development of chemopreventive and chemotherapeutic agents against cancer. However, the mechanism by which dieckol exerts its antitumor activity is poorly understood. Here, we show that dieckol, derived from E. cava, inhibits migration and invasion of HT1080 cells by scavenging intracellular reactive oxygen species (ROS). H2O2 or integrin signal-mediated ROS generation increases migration and invasion of HT1080 cells, which correlates with Rac1 activation and increased expression and phosphorylation of focal adhesion kinase (FAK). Rac1 activation is required for ROS generation. Depletion of FAK by siRNA suppresses Rac1-ROS-induced cell migration and invasion. Dieckol treatment attenuated intracellular ROS levels and activation of Rac1 as well as expression and phosphorylation of FAK. Dieckol treatment also decreases complex formation of FAK-Src-p130C as and expression of MMP2, 9, and 13. These results suggest that the Rac1-ROS-linked cascade enhances migration and invasion of HT1080 cells by inducing expression of MMPs through activation of the FAK signaling pathway, whereas dieckol downregulates FAK signaling through scavenging intracellular ROS. This finding provides new insights into the mechanisms by which dieckol is able to suppress human cancer progresssion and metastasis. Therefore, we suggest that dieckol is a potential therapeutic agent for cancer treatment.

Neural Wiskott–Aldrich syndrome protein (N-WASP) regulates reorganization of the actin cytoskeleton through activation of the Arp2/3 complex. Here, we show that heat shock protein 90 (HSP90) regulates N-WASP-induced actin polymerization in cooperation with phosphorylation of N-WASP. HSP90 binds directly to N-WASP, but binding alone does not affect the rate of N-WASP/Arp2/3 complex-induced in vitro actin polymerization. An Src family tyrosine kinase, v-Src, phosphorylates and activates N-WASP. HSP90 increases the phosphorylation of N-WASP by v-Src, leading to enhanced N-WASP-dependent actin polymerization. In addition, HSP90 protects phosphorylated and activated N-WASP from proteasome-dependent degradation, resulting in amplification of N-WASP-dependent actin polymerization. Association between HSP90 and N-WASP is increased in proportion to activation of N-WASP by phosphorylation. HSP90 is colocalized and associated with active N-WASP at podosomes in 3Y1/v-Src cells and at growing neurites in PC12 cells, whose actin structures are clearly inhibited by blocking the binding of HSP90 to N-WASP. These findings suggest that HSP90 induces efficient activation of N-WASP downstream of phosphorylation signal by Src family kinases and is critical for N-WASP-dependent podosome formation and neurite extension

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP-and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are requiredforthisstimulation.Acidicliposome-triggered,N-WASPdependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 colocalizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.The dynamic nature of the actin cytoskeleton is crucial for a variety of cellular events, including cell morphogenesis, cell migration, and intracellular membrane traffic (1). Actin polymerization is stimulated by a variety of actin regulatory proteins, prominent among which are WASP family proteins that function by triggering Arp2/3-mediated actin nucleation (2). Activation of WASP family proteins, in turn, is controlled by factors that bind these proteins and release an autoinhibitory intramolecular interaction that prevents their VCA domain from interacting with the Arp2/3 complex. As extensively shown for N-WASP, many such factors are proteins that bind to the N-WASP proline-rich region via the SH3 4 domain. Recently, we have found that the SH3 domain containing protein amphiphysin 1 stimulates actin polymerization during phagocytosis in testicular Sertoli cells, and this effect requires interactions of its C-terminal SH3 domain (3). Amphiphysin 1 is an endocytic adaptor present at high levels in brain at neuronal synapses but is also expressed at significant levels in Sertoli cells (4,5). In addition to a C-terminal SH3 domain, known to bind the GTPase dynamin and the phosphoinositide phosphatase synaptojanin (6), amphiphysin 1 contains an N-terminal BAR domain, a curved protein module that binds lipid bilayers and generates and senses curvature (7,8). It also contains binding motifs for clathrin and for the clathrin adaptor AP-2 (9). Hence, amphiphysin 1 was primarily studied as an endocytic protein capable of assembling at the neck of endocytic pits and of coupling clathrin-mediated budding to dynamin-mediated fission (10, 11). However, regulatory roles of amphiphysin 1 in actin cytoskeleton have also been suggested by studies of neuronal growth cones (12) and by the function of the amphiphysin homologue in yeast, . 81-86-235-7126; E-mail: kohji@md.okayama-u.ac.jp. 4 The abbrevi...

BackgroundAMP-activated protein kinase (AMPK) not only functions as an intracellular energy sensor and regulator, but is also a general sensor of oxidative stress. Furthermore, there is recent evidence that it participates in limiting acute inflammatory reactions, apoptosis and cellular senescence. Thus, it may oppose the development of chronic obstructive pulmonary disease.MethodsTo investigate the role of AMPK in cigarette smoke-induced lung inflammation and emphysema we first compared cigarette smoking and polyinosinic-polycytidylic acid [poly(I:C)]-induced lung inflammation and emphysema in AMPKα1-deficient (AMPKα1-HT) mice and wild-type mice of the same genetic background. We then investigated the role of AMPK in the induction of interleukin-8 (IL-8) by cigarette smoke extract (CSE) in A549 cells.ResultsCigarette smoking and poly(I:C)-induced lung inflammation and emphysema were elevated in AMPKα1-HT compared to wild-type mice. CSE increased AMPK activation in a CSE concentration- and time-dependent manner. 5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an AMPK activator, decreased CSE-induced IL-8 production while Compound C, an AMPK inhibitor, increased it, as did pretreatment with an AMPKα1-specific small interfering RNA.ConclusionAMPKα1-deficient mice have increased susceptibility to lung inflammation and emphysema when exposed to cigarette smoke, and AMPK appears to reduce lung inflammation and emphysema by lowering IL-8 production.

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