LL5β Directs the Translocation of Filamin A and SHIP2 to Sites of Phosphatidylinositol 3,4,5-Triphosphate (PtdIns(3,4,5)P3) Accumulation, and PtdIns(3,4,5)P3 Localization Is Mutually Modified by Co-recruited SHIP2*

Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P3 regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P3 and the reciprocal regulation of PtdIns(3,4,5)P3 itself. LL5β, whose subcellular localization is directed by membrane PtdIns(3,4,5)P3, recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P3 accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5β by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5β or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5β without its PtdIns(3,4,5)P3-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P3 accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P3 at the same location.

Directed migration is crucial in many biological events, including morphogenesis during development, accumulation of immune cells to the site of infection, and metastasis of cancer cells. A lamellipodium (a sheet-like process composed of actin filaments) must be formed correctly at the leading edge of a cell for smooth and efficient migration. Following stimulation with a chemoattractant, phosphatidylinositol 3-kinase (PI3K) 6 is locally activated, resulting in the transient accumulation of PtdIns(3,4,5)P 3 on the leading edge of directed migrating amoebas and leukocytes (1). PtdIns(3,4,5)P 3 works, at least in part, as a cell compass that translates external signals into directed cell movement (2). However, how PtdIns(3,4,5)P 3 contributes to the appropriate formation of a lamellipodium, or conversely, how lamellipodium formation influences PtdIns(3,4,5)P 3 accumulation has not been fully elucidated. For proper lamellipodium formation, the intracellular network of actin filaments (F-actins) must be regulated both dynamically and precisely. Such networks are composed primarily of cross-linked and branched F-actins. Previous work has shown that Filamin A cross-links F-actins and is indispensable for lamellipodium formation, whereas the Wiskott-Aldrich syndrome protein family verprolin homologous (WAVE) proteins are involved in branch formation (3)(4)(5).
Proteins involved in signal transduction and cytoskeletal dynamics interact with membrane phosphoinositides through their pleckstrin homology (PH) domains; such interactions are critical for the subcellular localization of these proteins and the regulation of their activities (6,7). Although the core tertiary structure is conserved among PH domains, high sequence variability among their primary structures (ϳ120 amino acids) gives rise to different specificities of PH domains for different phosphoinositides (8,9). LL5␤ (PHLDB2; pleckstrin homology-like domain, family B, member 2) is an ϳ160-kDa protein that contains two predicted coiled-coil domains and a PH domain that is highly specific for PtdIns (3,4,5)P 3 (10,11). It has been reported that LL5␤ binds to exogenous Filamin A, as well as Filamin C (12), in vitro. However, the LL5␤ and Filamin A regions required for binding and the biological significance of LL5␤-Filamin A binding have not been experimentally explored.
It has been shown that SHIP2 is a critical component of a negative feedback loop that regulates PtdIns(3,4,5)P 3 levels in nerve growth factor-stimulated PC12 cells, together with the constitutively active phosphatidyl inositol-3 (PtdIns-3) phosphatase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (13). This suggests that SHIP2 contributes to establishing a narrow accumulation of PtdIns(3,4,5)P 3 in the membrane. Because PtdIns(3,4,5)P 3 works as an internal compass of polarity in directed migrating cells, SHIP2 is likely to help a cell respond accurately and dynamically to external signals that impart directionality by increasing PtdIns(3,4,5)P 3 levels in a leading edge of the cell. Previous studies have demonstrated that SHIP2 binds to Filamin A and that Filamin A is essential for the translocation of SHIP2 to the membrane ruffle. Moreover, recombinant SHIP2 regulates PtdIns(3,4,5)P 3 levels and the distribution of submembranous actin at membrane ruffles following growth factor stimulation (14). However, it remains elusive as to how and what molecules control SHIP2 and Filamin A translocation to the membrane, where in the cell periphery SHIP2 translocates, how membranous PtdIns(3,4,5)P 3 levels are regulated by translocation of SHIP2, and how submembranous actins are controlled.

EXPERIMENTAL PROCEDURES
Details of experimental procedures are provided in the supplemental Experimental Procedures.
Isolation of LL5␤ cDNA-The full-length human LL5␤ cDNA (GenBank TM accession number AJ496194) was cloned from a human placenta cDNA library (BD Biosciences).
Cell Culture and DNA Transfection-COS-7 cells were used. Vectors were transfected using PolyFect transfection reagent (Qiagen, Hilden, Germany) or FuGENE 6 transfection reagent (Roche Diagnostics). To increase PtdIns (3,4,5)P 3 in the plasma membrane, cells were cultured without serum for 16 -18 h and then cultured with epidermal growth factor (EGF) for 2 min or serum for 10 min. Human melanoma cell lines M2 and A7 were maintained as reported previously (4). FuGENE HD (Roche Diagnostics) was used to transfect these cells.
F-actin Staining, Immunocytochemistry, Analyses of Cell Motility, and Reverse Transcription-PCR-Conventional protocols were used.
Immunoprecipitation, Western Blotting (Immunoblotting) Analyses, and MALDI-TOF Mass Spectrometry-Immunoprecipitation was performed with Dynabeads (Dynal, Hamburg, Germany) coated with antibodies specific to rabbit or mouse IgG. Matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Billerica, MA) was used for protein identification.
Wound-healing Assay-Overconfluent COS-7 cells were cotransfected with pEGFP-C3 and mU6pro-LL5␤-RNAi vector or pEGFP-C3 and control vector. Empty shRNA vector was used as the control vector. After 36 -48 h, the confluent cell layer was disrupted followed by a 3-h incubation. After fixing and staining with rhodamine-phalloidin, cells at the defect edge expressing EGFP were observed, and the numbers of cells with or without lamellipodia were counted.
PI3K Inhibition and Time-lapse Observation-For PI3K inhibition experiments, cells were cultured for 18 -24 h followed by the addition of wortmannin (Sigma, final concentration 100 nM) or LY294002 (Sigma, final concentration 10 M). Timelapse observation was started (time 0) in a CO 2 incubation chamber (5% CO 2 at 37°C) fitted onto a confocal microscope. Images were obtained every 1-2 min for 15 min to 1 h. Confocal images or live cell images were captured. Images were obtained using an AxioCam (Carl Zeiss, Oberkochen, Germany).
Subcellular Fractionation-Subcellular fractionation was performed using the ProteoExtract subcellular proteome extraction kit (Calbiochem) in accordance with the manufacturer's instructions. Utilizing the different extraction buffers in the kit, four different fractions were separated. The Triton-insoluble membrane/organelle fractions are shown in Fig. 5E. The amounts of the pan-cadherin (as reference for membrane proteins) and glyceraldehyde-3-phosphate dehydrogenase were used as internal controls.
Statistical Analyses-Significant differences, as indicated by p Ͻ 0.01, were determined by the non-parametric Mann-Whitney U test or t test.
The interaction between LL5␤ and Filamin A was confirmed by immunoprecipitation. Cell lysates from COS-7 cells expressing EGFP-LL5␤, an anti-GFP antibody that is capable of recognizing EGFP, and an anti-Filamin A antibody were used. This anti-Filamin A antibody did not cross-react with Filamin C in lysates from Filamin C-overexpressing COS-7 cells (data not shown). Filamin A, which is ϳ280 kDa (3,20), was co-immu-  PtdIns(3,4,5 (Fig. 1D). We then searched for the region of Filamin A responsible for this interaction. Filamin A, which usually exists as a homodimer, contains an amino-terminal actin-binding domain that precedes 24 immunoglobulin-like ␤-sheet motifs (FR, filamin repeats) (Fig. 1E). The last FR (FR 24) is responsible for homodimerization (20). Using three deletion mutants of amino-terminally HA-tagged Filamin A that lack different regions of the immunoglobulin-like-␤sheet motifs (Filamin A⌬1-Filamin A⌬3), we identified the domain of Filamin A responsible for its interaction with LL5␤ (Fig. 1E). Even Filamin A⌬3, which includes hinge 1 but lacks FR1-23, was co-immunoprecipitated with EGFP-LL5␤ (Fig.  1E). We next assessed the ability of the two actin-binding motifs (CH1, CH2), FR24 and hinge 2 (H2) of Filamin A, to bind to EGFP-LL5␤. Two Filamin A fragments, CH1CH2 and FR24, but not FR23H2, were co-immunoprecipitated with EGFP-LL5␤ (Fig. 1, E and F). Furthermore, HA-tagged Filamin A without actin-binding domain (HA-Filamin A ⌬ABD) or without FR24 (HA-Filamin A ⌬FR24) was co-immunoprecipitated with EGFP-LL5␤ (Fig. 1, E, G, and H). These results suggest that both the actin-binding domain and the FR24 of Filamin A are responsible for an interaction with LL5␤.

LL5␤-Filamin A-SHIP2 Leads to Steeper
We next determined which domains of LL5␤ are required for this interaction (Fig. 2A). First, we generated a mutant of LL5␤ that lacks the PH domain (LL5␤⌬PH). HA-tagged Filamin A was co-expressed with EGFP-tagged LL5␤ (EGFP-LL5␤) or EGFP-tagged LL5␤⌬PH (EGFP-LL5␤⌬PH) in COS-7 cells followed by lysis and immunoprecipitation with an anti-GFP antibody. Co-immunoprecipitated HA-tagged Filamin A was observed with the similar intensity in lysates from cells co-expressing either EGFP-LL5␤ or EGFP-LL5␤⌬PH (Fig. 2B), indicating that the PH domain is not required for binding to Filamin A. We then used various truncated forms of LL5␤ with N-terminal EGFP tags to search for regions of LL5␤ responsible for Filamin A binding. A Filamin A interaction occurred with ⌬N1 and ⌬N2, but not with the ⌬N3, ⌬N4, or ⌬N5 mutants of LL5␤, indicating that a domain present in ⌬N2 but not ⌬N3 is responsible for this interaction. We then further truncated this domain and found that ⌬N2-a, but not ⌬N2-b or ⌬N2-c, could interact. Finally, we discovered that a 33-amino acid region (323-356, ⌬N2-e) was sufficient for binding (Fig. 2C). This binding region of LL5␤ was different from what has been previously documented (12). To confirm our observation, we expressed ⌬N2-e in COS-7 cells and identified co-immunoprecipitated proteins using MALDI-TOF mass spectrometry. Then, Filamin A was identified (Fig. 2D). We reconfirmed our results.
Because the PH domain of LL5␤ is specifically sensitive to PtdIns(3,4,5)P 3 (10), we next examined whether the recruitment of LL5␤ to the lamellipodia is driven by PtdIns(3,4,5)P 3 . Because the phosphorylation state of PtdIns(3,4,5)P 3 is regulated by the phosphorylation of phosphatidylinositol 4,5-biphosphate by PI3K, COS-7 cells with exogenous EGFP-LL5␤ were treated with the PI3K inhibitor, LY294002. After this FIGURE 3. Knockdown of LL5␤ decreases cell motility and lamellipodium formation. A, LL5␤-RNAi2 or LL5␤-RNAi3 were co-transfected with the EGFP-LL5␤ expression vector in COS-7 cells. The knockdown efficiency of each RNAi was evaluated by estimating the EGFP signal intensity. B, EGFP expression vector and empty mU6pro vector (control) (left) or EGFP expression vector and LL5␤-RNAi vector (LL5␤-RNAi 2) (right) were transfected into COS-7 cells that had been cultured under low cell density conditions. EGFP images (green) were acquired at an interval of 120 min and merged together after the color of the later images had been converted to red. Knockdown of LL5␤ resulted in a decrease in cell motility. Scale bar ϭ 50 m. C, to quantify cell motility, the migrated distance (mean Ϯ S.E.) of each nucleus during the 120min interval was measured for each group (n ϭ 50 each). * indicates statistical significance (p Ͻ 0.01). D, the EGFP expression vector was transfected into COS-7 cells together with an empty mU6pro vector (control) (upper panels) or LL5␤-RNAi vector (lower panels). EGFP-positive (transfected) cells were studied. F-actin localization was visualized simultaneously with rhodamine-phalloidin or Alexa Fluor 568 phalloidin. Knockdown of LL5␤ resulted in the frequent formation of small protrusions (arrowheads) and in a decrease in lamellipodium formation. Scale bar ϭ 10 m. E, the number of cells with lamellipodia (with an edge line equal to or longer than 10 m) in panel D was counted among randomly selected EGFP-positive cells (mean Ϯ S.E.). n ϭ 90 for both cases (three independent experiments, n ϭ 30 each). *, p Ͻ 0.01. F, the peripheral distribution of endogenous Filamin A was impaired by knocking down LL5␤. Only fiber-like Filamin A was observed in small protrusions (arrowheads). The EGFP expression vector was co-transfected, and EGFP-positive cells were studied (EGFP images were not shown). Scale bar ϭ 10 m. G, culture medium containing 100 ng/ml EGF was added (0 min) to COS-7 cells expressing EGFP-LL5␤ or EGFP-LL5␤⌬PH after being cultured without serum for 18 h. EGFP-LL5␤-expressing cells responded strongly and dramatically changed their morphology, whereas those with EGFP-LL5␤⌬PH did not. Scale bar ϭ 20 m. PtdIns(3,4,5)P 3 Accumulation treatment, EGFP fluorescence was distributed in a punctate manner and colocalized with endogenous Filamin A ( Fig. 2E; supplemental Fig. S3; supplemental Video 1). No accumulation of Filamin A was observed at the cell periphery. Our time-lapse study gave us more detailed information on how this redistribution took place. Puncta were formed in COS-7 cells about 14 min after treatment with the PI3K inhibitor, wortmannin (Fig. 2F). The EGFP-LL5␤ signal was not observed in lamellipodia after this treatment (Fig. 2F). This observation was further investigated by overexpressing EGFP-LL5␤⌬PH. EGFP-LL5␤⌬PH did not localize to the plasma membrane but colocalized well with endogenous Filamin A in a punctate distribution in the cytoplasm (Fig. 2G). This result is consistent with the demonstration of LL5␤⌬PH binding to HA-tagged Filamin A (Fig. 2B). We further examined the spatiotemporal regulation of LL5␤ in response to PtdIns(3,4,5)P 3 . We added EGF to the culture medium after serum depletion for 16 -18 h and continuously observed changes in EGFP-LL5␤ localization. Recruitment of EGFP-LL5␤ to the plasma membrane was first observed ϳ80 s after EGF application, and by 112 s, EGFP-LL5␤ accumulated along the plasma membrane ( Fig. 2H; supplemental Video 2). These data suggest that LL5␤ changes its subcellular localization quickly enough to chase changeable PtdIns(3,4,5)P 3 distribution (supplemental Fig. S4 and supplemental Video 3).

LL5␤-Filamin A-SHIP2 Leads to Steeper
The ability of LL5␤ to bind to Filamin A, a protein crucial for cell motility (20), suggests that LL5␤ is also involved in motility control. To investigate the function of LL5␤, we carried out LL5␤ knockdown experiments using RNA interference (RNAi). Of the three different shRNA constructs against LL5␤, we found that LL5␤-RNAi2 and LL5␤-RNAi3 suppressed the expression of EGFP-LL5␤ with similar efficiency ( Fig. 3A; supplemental Fig. S5). In the following experiments, we primarily used LL5␤-RNAi2 to suppress LL5␤ expression in COS-7 cells, but LL5␤-RNAi3 gave us similar results (supplemental Fig. S6). LL5␤-RNAi reduced cell motility when compared with control cells under low cell density conditions where cells could move freely (Fig. 3,  B and C). We then employed a wound-healing assay to determine whether there was any alteration to the cell-crawling machinery (Fig. 3,  D and E). In this assay, quiescent cells in an overconfluent state usually develop lamellipodia in response to the removal of neighboring cells. We found that "typical lamellipodia" (defined as edge lines equal to or more than 10 m long) were not properly formed in cells with LL5␤-RNAi. Instead, small protrusions that contained F-actin (with edge lines less than 10 m long) were observed (Fig. 3D). The frequency of cells with lamellipodia was estimated with or without LL5␤-RNAi. Knockdown of LL5␤ resulted in a decrease in lamellipodium formation (Fig. 3E), suggesting that LL5␤ is essential for lamellipodium formation in COS-7 cells. Moreover, when LL5␤ expression was down-regulated by the RNAi technique in COS-7 cells, the distribution of Filamin A near the plasma membrane was significantly altered (Fig. 3F). Reduced formation of lamellipodia and a fiber-like distribution of Filamin A were observed in the small protrusions seen in LL5␤ knockdown cells. In contrast, LL5␤ overexpression resulted in the formation of large lamellipodia (Figs. 3G, upper row, and 4A, left and middle panels). In response to EGF, COS-7 cells with exogenous LL5␤ became very active (supplemental Video 4), whereas those with exogenous LL5␤⌬PH did not (Fig. 3G, lower row), indicating that the . Very large lamellipodia were developed in the presence of EGFP-LL5␤ in COS-7 cells and A7 cells but not in the M2 cells. Scale bar ϭ 10 m. B, a whole-cell area was measured from 50 cells; cells were randomly selected in each case (mean Ϯ S.E.). * p Ͻ 0.01. C and D, M2 cells expressing EGFP-LL5␤ and Filamin A⌬ABD or EGFP and Filamin A⌬ABD were cultured without serum for 18 h. EGF (final concentration 100 ng/ml) was then added into the culture medium (time 0). Approximately 2 min after EGF addition, small pseudopods (arrowheads) were transiently generated. The images were merged with their differential interference contrast images. Scale bars ϭ 10 m.
Because LL5␤ overexpression in COS-7 cells resulted in the enlargement of the peripheral area (including lamellipodia) of cells, we next examined whether Filamin A is involved in this response. M2 cells, in which LL5␤ mRNA was not detected (data not shown), are derived from a human melanoma and do not express Filamin A, whereas A7 cells are generated by stably transfecting Filamin A cDNA into M2 cells (4). These cells express a molar ratio of Filamin A to actin comparable with that found in cells normally expressing Filamin A (4). The LL5␤induced enlargement was observed in A7 cells as well as COS-7 cells, but not in M2 cells (Fig. 4, A and B), suggesting that enlargement induced by LL5␤ overexpression requires Filamin A. The versatility of Filamin A led us to ask whether the F-actincross-linking activity of Filamin A is essential for this enlargement. Filamin A dimerized through their carboxyl termini, and then dimerized Filamin A cross-linked two F-actin molecules through their ABD at their amino termini (15,21). Thus, we transfected a Filamin A⌬ABD expression vector (15) into M2 cells and examined the Filamin A F-actin-cross-linking activity to that observed in A7 cells. Application of EGF resulted in the formation of small pseudopods, but not proper lamellipodia, in M2 cells transfected with EGFP-LL5␤ and Filamin A⌬ABD (Fig. 4C). Therefore, the actin binding activity, or probably the F-actin-cross-linking activity, of Filamin A is crucial for the LL5␤-mediated formation of lamellipodia. Because EGF induced small pseudopods in M2 cells transfected with Filamin A⌬ABD even in the absence of LL5␤ (Fig. 4D), it is unlikely that LL5␤ is involved in the formation of these small pseudopods.
We next sought to determine whether LL5␤ directs translocation of SHIP2-Filamin A complex. As reported (14), SHIP2 interacts with Filamin A (data not shown). By contrast, direct interaction between SHIP2 and LL5␤ was not observed (supplemental Fig. S7), suggesting that LL5␤ directs subcellular localization of SHIP2 via Filamin A. Two minutes after EGF stimulation, EGFP-tagged SHIP2 (EGFP-SHIP2) or mCherrytagged SHIP2 (SHIP2-mCherry) localized to the cell periphery with LL5␤ (Fig. 5, A, lower row, and B, upper row) but remained in the cytosol in the presence of LL5␤⌬PH (Fig. 5B, lower row). As expected, SHIP2 colocalized well with Filamin A (Fig. 5C, upper row), but translocation of SHIP2 to the cell membrane was observed less in LL5␤ knockdown COS-7 cells (Fig. 5, C,  lower row, and D). Supporting this observation, the amount of SHIP2 in the membrane fraction was reduced in LL5␤ knockdown COS-7 cells (Fig. 5E). These data indicate that Filamin A is an intermediate between LL5␤ and SHIP2. Indeed, translo- cation of EGFP-SHIP2 and LL5␤ to the cell periphery was observed in A7 cells (Fig. 5F). In M2 cells (Filamin A-deficient A7 cells), LL5␤ was translocated to the cell periphery, leaving SHIP2 in the cytosol, following EGF stimulation (Fig. 5G). It was of interest that the amount of ␣-tubulin was also decreased in LL5␤ knockdown cells (Fig. 5E). Because it has been reported that SHIP2 regulates PtdIns(3,4,5)P 3 at membrane ruffles (14) and because it has also been proposed that SHIP2 mediates negative feedback on the accumulation of PtdIns(3,4,5)P 3 (13), we examined whether SHIP2, which is directed by LL5␤ through Filamin A, regulates membrane PtdIns(3,4,5)P 3 . We found that ARNO-PH, one of the widely used PtdIns(3,4,5)P 3 detectors (16, 17) (supplemental Figs. S2 and S4 and supplemental Video 3), showed a greater accumulation at the plasma membrane in LL5␤ knockdown cells (Fig. 5, H and I). This suggests that SHIP2, which is directed by LL5␤, is very likely to regulate membrane PtdIns(3,4,5)P 3 .

DISCUSSION
We demonstrate here that LL5␤ is essential for quick Filamin A-dependent SHIP2 translocation in response to membrane PtdIns(3,4,5)P 3 , fast enough to chase PtdIns(3,4,5)P 3 changes, and the reciprocal control of membrane PtdIns(3,4,5)P 3 through SHIP2, as well as the enhanced lamellipodium formation induced by PtdIns(3,4,5)P 3 (Fig. 6). It has been reported that v-ral simian leukemia viral oncogene homolog A (RalA) targets Filamin A in filopodia (22). In addition, faint membrane localization of mutated SHIP2 that lacks Filamin A-interacting domain, is observed after 5 min of EGF stimulation (14). This is consistent with the fact that SHIP2 is capable of associating with p130 Cas and localizes to membrane ruffles and focal adhesions (23).
Because F-actin primarily grows near the plasma membrane by polymerizing globular actins at its plus end (24) and because cells do not form lamellipodia (large protrusions) in response to EGF application in the presence of the non-membrane-associating LL5␤⌬PH mutant, it is likely that increasing the crosslinking of newly polymerized F-actins by membrane-recruited Filamin A helps a cell to form lamellipodia. It is of interest that Filamin A can regulate the formation of small membrane protrusions without its actin-cross-linking activity. Indeed, small pseudopods were formed in M2 cells transfected with Filamin A⌬ABD in response to EGF.
LL5␤, as well as Filamin A, is apparently versatile because the amount of ␣-tubulin in the membrane fraction was decreased by LL5␤ knockdown. It has been reported that LL5␤ binds to cytoplasmic linker-associated proteins (CLASPs) (25), which attach to the plus ends of microtubules, thereby modulating microtubule stabilization (26). Therefore, it is possible that LL5␤ synchronizes actins and microtubules, thereby coordinating cytoskeletal components for efficient migration.
It has been shown that SHIP2 is a critical component of a negative feedback loop that regulates PtdIns(3,4,5)P 3 levels and is likely to help a cell respond accurately and dynamically to external signals that impart directionality (13). We show here that SHIP2 translocates directly to sites of PtdIns(3,4,5)P 3 accumulation with Filamin A, which is dependent on LL5␤. Consid-ering that LL5␤ accumulates to the membrane in response to EGF application within a few minutes, it is plausible that SHIP2 follows changes in PtdIns(3,4,5)P 3 without a substantial delay. Therefore, it is likely that SHIP2, Filamin A, and LL5␤ help a cell to move in one direction smoothly. Moreover, the ability of PtdIns(3,4,5)P 3 and SHIP2 to regulate various cellular responses, such as the insulin response (27), and the reciprocal regulation of PtdIns(3,4,5)P 3 with Filamin A-LL5␤ reported here suggest that these findings are significant for the general understanding of such events, and possibly, for the understanding of related diseases.