These data demonstrate a coupling between the machinery for PtdIns(3,5)P 2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P 2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.

Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P(3) gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P(3) through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P(3)-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet-derived growth factor (PDGF). Production of PtdIns(3,4,5)P(3) at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P(3) alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P(3), recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.

Myotubularin and related proteins constitute a large and highly conserved family possessing phosphoinositide 3-phosphatase activity, although not all members possess this activity. This family contains a conserved region called the GRAM domain that is found in a variety of proteins associated with membrane-coupled processes and signal transduction. Mutations of myotubularin are found in X-linked myotubular myopathy, a severe muscle disease. Mutations in the GRAM domain are responsible for this condition, suggesting crucial roles for this region. Here, we show that the GRAM domain of myotubularin binds to phosphoinositide with the highest affinity to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ). In patients with myotubular myopathy, mutations in the myotubularin GRAM domain eliminate this binding, indicating that the PtdIns(3,5)P 2 binding ability of the GRAM (glucosyltransferases, Rablike GTPase activators and myotubularin) domain is crucial for the functions of myotubularin in vivo. Stimulation of epidermal growth factor recruits myotubularin to the late endosomal compartment in a manner dependent on the phosphoinositide binding. Overexpression of myotubularin inhibits epidermal growth factor receptor trafficking from late endosome to lysosome and induces the large endosomal vacuoles. Thus, our data suggest that myotubularin phosphatase physiologically functions in late endosomal trafficking and vacuolar morphology through interaction with PtdIns(3,5)P 2 .In eukaryotic cells, D3-phosphorylated phosphoinositides such as phosphatidylinositol 3-phosphate (PtdIns3P) 1 play key roles in the vesicular trafficking through direct interaction with phosphoinositide-binding domains such as the PH domain, FYVE finger domain, and PX (Phox) domain found in effector proteins that control vesicular trafficking (1-3). Previous studies have revealed that PtdIns3P binding is essential for the recruitment/activation of these effector proteins at unique membrane sites (4, 5). PtdIns(3,5)P 2 was one of the phosphoinositide species identified recently in both yeast and mammalian cells (6). PtdIns(3,5)P 2 is thought to be involved in osmotic stress responsiveness and essential for the maintenance of vacuole size and homeostasis in yeast (7). Recently, it was reported that PtdIns(3,5)P 2 is necessary for late endosomal trafficking in yeast (8). However, the mechanisms for cellular PtdIns(3,5)P 2 regulation are unknown. Intracellular levels of these phosphoinositide species are strictly regulated by enzymes that dephosphorylate at the D3-position of the inositol ring. Myotubularin and its related proteins (myotubularinrelated proteins; MTMRs) constitute a large and highly conserved subfamily of dual specific phosphatases that were recently revealed to be phosphoinositide 3-phosphatases (9 -11). Among those proteins, myotubularin is encoded by the MTM1 gene, which is mutated in X-linked myotubular myopathy (12), whereas MTMR2 is associated with neurodegenerative disorder Charcot-Marie-tooth disease type 4B (13). Myopathy patie...

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4,5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K m ‫؍‬ 180 M) than for inositol 1,4,5-trisphosphate (K m ‫؍‬ 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 is known to play a role in the generation of two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) and diacylglycerol (DG), by phospholipase C. Ins(1,4,5)P 3 mobilizes Ca 2ϩ stores, and DG activates protein kinase C (1, 2).In addition to its role as a lipid second messenger, PtdIns(4,5)P 2 binds to several actin-regulating proteins such as vinculin, profilin, cofilin, and gelsolin (3-6) to suppress their functions. In contrast, PtdIns(4,5)P 2 binds also to ␣-actinin to activate the actin-gelating activity (7). An inositol polyphosphate 5-phosphatase (IP5Pase), synaptojanin 1, previously demonstrated to hydrolyze PtdIns(4,5)P 2 , bound to vinculin, ␣-actinin, and profilin in vitro (8). However, the molecular mechanism by which IP5Pase regulates the actin cytoskeleton remains to be elucidated.IP5Pases hydrolyze the D-5 position of inositol phosphates and corresponding phospholipids. Eight IP5Pases and several splicing variants were isolated and classified into three groups according to their substrate specificity (9 -24). Type I 5-phosphatases hydrolyze only water-soluble substrates, Ins(1,4,5)P 3 and Ins(1,3,4,5)P 4 , indicating roles in regulating intracellular calcium levels (11). Type II enzymes hydrolyze PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 as well as soluble inositol phosphates. OCRL encodes the X chromosome, and its mutation causes Lowe's oculocerebrorenal syndrome (10). Synaptojanin 1 and 2 are related enzymes (14,19,20). They contain C-terminal proline-rich regions that bind SH3 homology domains of Ash/Grb2 or amphiphysin and an N-terminal domain homologous to yeast protein SacI, recently identified as inositol polyphosphate phosphatase (24). PIPP (Proline-rich Inositol Polyphosphate 5-Phosphatase) is a recently identified Type II IP5Pase that localizes at membrane ruffles (25). Type III...

Skeletal muscle and kidney enriched inositol phosphatase (SKIP) is an inositol polyphosphate 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] to downregulate intracellular levels. In this study, we show that SKIP inhibits phosphoinositide 3-kinase signaling in insulin-stimulated CHO cells. Ectopic expression of SKIP did not inhibit insulin-induced PI(3,4,5)P 3 generation but did rapidly decrease insulin-induced intracellular PI(3,4,5)P 3 levels compared with those in control cells. Further, insulininduced phosphorylation of some downstream targets such as Akt and p70 S6 kinase was markedly inhibited by the ectopic expression of SKIP, whereas phosphorylation of mitogen-activated protein kinase was not. In contrast, downregulation of intracellular SKIP levels by antisense oligonucleotides dramatically enhanced Akt (protein kinase B) phosphorylation in response to insulin, suggesting that endogenous SKIP downregulates insulin signaling. SKIP also markedly inhibited GLUT4 translocation and membrane ruffle formation. We conclude that SKIP preferentially regulates glucose transport and actin cytoskeletal rearrangement among a variety of PI(3,4,5)P 3 downstream events.The binding of insulin to its receptor stimulates tyrosine autophosphorylation of the receptor, leading to the activation of its intrinsic tyrosine kinase. This kinase phosphorylates insulin receptor substrates (IRSs), Shc, and other proteins (21,22,28,41,49). Tyrosine phosphorylation of IRS-1 and Shc promotes their interaction with the regulatory subunit of phosphoinositide (PI) 3-kinase (52). PI 3-kinase is a heterodimer composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Activated PI 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] to generate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] (15), which plays a crucial role in the biological responses induced by insulin (43).One of the major roles of insulin is the activation of glucose uptake in muscle and adipose tissue through the translocation of the glucose transporter GLUT4 (8). PI 3-kinase is necessary for this process (9, 25). Akt (protein kinase B [PKB]) is a serine/threonine kinase located downstream of PI 3-kinase, and in its constitutively active form, Akt causes increased glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes (18). For the activation of Akt, phosphorylation by Akt kinase phosphoinositide-dependent kinase (PDK) is also important (39,40). In this case, binding of PI(3,4,5)P 3 and PI(3,4)P 2 to the pleckstrin homology domain of Akt, in addition to phosphorylation by PDK, is required for its full activation (6, 40). However, recent data suggest that PI(3,4,5)P 3 is a more potent activator of Akt than is PI(3,4)P 2 (17). Growth factors such as insulin and platelet-derived growth factor induce actin cytoskeletal rearrangement, leading to stress fiber breakdown and membrane ruffling (25,26). Such membrane ruffling requires PI(3,4,5)P 3 formation, which presumably causes the activat...

Here, we identify a novel rat phosphatidylinositol-5-phosphate 4-kinase, phosphatidylinositol-phosphate kinase II␥ (PIPKII␥ Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) 1 is a phospholipid with a variety of functions in vivo including not only the production of second messengers such as diacylglycerol and inositol 1,4,5-trisphosphate, but also the regulation of actin regulatory proteins and the activation of phospholipase D and ADP-ribosylation factor. It has also been reported that PI(4,5)P 2 synthesis is potentiated by various stimuli including GTP␥S (1-3), phorbol esters (4), tyrosine kinases (5), and integrins (6). The variations in its function and the regulation of its synthesis indicate that enzymes responsible for the production of PIP 2 , such as PI kinase and PIPK, also show large diversities. Among PIPKs, two major subtypes (types I and II), each comprising two isoforms (I␣, I␤, II␣, and II␤), have been identified to date (13, 16 -18), and it is thought that the role for each subtype in vivo is different. The type

Downregulation of cell-cell adhesion and upregulation of cell migration play critical roles in the conversion of benign tumors to aggressive invasive cancers. In this study, we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity depend on the level of phosphatidylinositol 4-phosphate [PI(4)P] in the Golgi apparatus in breast cancer cells. Attenuating SAC1, a PI(4)P phosphatase localized in the Golgi apparatus, resulted in decreased cell-cell adhesion and increased cell migration in weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase IIIb, which generates PI(4)P in the Golgi apparatus, increased cell-cell adhesion and decreased invasion in highly invasive cells. Furthermore, a PI(4)P effector, Golgi phosphoprotein 3, was found to be involved in the generation of these phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a new model for breast cancer cell progression in which progression is controlled by PI(4)P levels in the Golgi apparatus. Cancer Res; 74(11); 3054-66. Ó2014 AACR.

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