PI(3,4)P2 plays critical roles in the regulation of focal adhesion dynamics of MDA-MB-231 breast cancer cells.

Phosphoinositides play pivotal roles in the regulation of cancer cell phenotypes. Among them, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2 ) localizes to the invadopodia, and positively regulates tumor cell invasion. In this study, we examined the effect of PI(3,4)P2 on focal adhesion dynamics in MDA-MB-231 basal breast cancer cells. Knockdown of SHIP2, a phosphatidylinositol 3,4,5-trisphosphatase (PIP3 ) 5-phosphatase that generates PI(3,4)P2 , in MDA-MB-231 breast cancer cells, induced the development of focal adhesions and cell spreading, leading to the suppression of invasion. In contrast, knockdown of PTEN, a 3-phosphatase that de-phosphorylates PIP3 and PI(3,4)P2 , induced cell shrinkage and increased cell invasion. Interestingly, additional knockdown of SHIP2 rescued these phenotypes. Overexpression of the TAPP1 PH domain, which binds to PI(3,4)P2 , and knockdown of Lpd, a downstream effector of PI(3,4)P2 , resulted in similar phenotypes to those induced by SHIP2 knockdown. Taken together, our results suggest that inhibition of PI(3,4)P2 generation and/or downstream signaling could be useful for inhibiting breast cancer metastasis.

Regulation of CD44 expression and focal adhesion by Golgi phosphatidylinositol 4-phosphate in breast cancer.

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol-4-phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK-BR-3 cells. In contrast, knockdown of PI 4-kinase IIIß in highly invasive MDA-MB-231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.

Glucose-regulated protein 78 (GRP78) binds directly to PIP3 phosphatase SKIP and determines its localization.

Skeletal muscle and kidney-enriched inositol polyphosphate phosphatase (SKIP), a PIP3 phosphatase, has been implicated in the regulation of insulin signaling in skeletal muscle. SKIP interacts with Pak1 and glucose-regulated protein 78 (GRP78), both of which are necessary for the regulation of insulin signaling. In this study, we showed that GRP78 directly binds to the SKIP C-terminal homology (SKICH) domain of SKIP and that this binding is necessary for the localization of SKIP at the ER. In addition, in vitro binding analysis showed that GRP78 and Pak1 competitively bind to SKIP. Taken together, these findings suggest a model by which GRP78 regulates intracellular localization of SKIP and how SKIP binds to Pak1 on insulin stimulation.

Phosphatidylinositol 3,4,5-Trisphosphate Phosphatase SKIP Links Endoplasmic Reticulum Stress in Skeletal Muscle to Insulin Resistance.

Insulin resistance is critical in the pathogenesis of type 2 diabetes. Endoplasmic reticulum (ER) stress in liver and adipose tissues plays an important role in the development of insulin resistance. Although skeletal muscle is a primary site for insulin-dependent glucose disposal, it is unclear if ER stress in those tissues contributes to insulin resistance. In this study, we show that skeletal muscle kidney-enriched inositol polyphosphate phosphatase (SKIP), a PIP3 (phosphatidylinositol-3,4,5-trisphosphate) phosphatase, links ER stress to insulin resistance in skeletal muscle. SKIP expression was increased due to ER stress and was higher in the skeletal muscle isolated from high-fat-diet-fed mice and db/db mice than in that from wild-type mice. Mechanistically, ER stress promotes activating transcription factor 6 (ATF6) and X-box binding protein 1 (XBP1)-dependent expression of SKIP. These findings underscore the specific and prominent role of SKIP in the development of insulin resistance in skeletal muscle.

IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues.

Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3- cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2.

Regulation of insulin signaling in skeletal muscle by PIP3 phosphatase, SKIP, and endoplasmic reticulum molecular chaperone glucose-regulated protein 78.

Insulin resistance is characterized as a pathogenic factor in type 2 diabetes. Despite skeletal muscle being primarily responsible for systemic glucose disposal, the mechanisms underlying the induction of insulin resistance in skeletal muscle have not been fully elucidated. A number of studies have shown that it is characterized by the inhibition of the phosphatidylinositol (PI) 3-kinase signaling pathway. Here, we show that skeletal muscle- and kidney-enriched inositol polyphosphate phosphatase (SKIP), a phosphatidylinositol-3,4,5-trisphosphate (PIP3) phosphatase, and glucose-regulated protein 78 (GRP78) are implicated in the inhibition of insulin-dependent PI 3-kinase signaling in skeletal muscle. Mechanistically, under resting conditions, SKIP forms a complex with GRP78 at the endoplasmic reticulum (ER). Insulin stimulation facilitates the dissociation of SKIP from GRP78 and its binding to the activated form of Pak1. GRP78 is necessary for membrane localization and Pak1-binding of SKIP, which facilitates inactivation of the insulin signaling pathway. These findings underscore the specific and prominent role of SKIP and GRP78 in the regulation of insulin-dependent PI 3-kinase signaling in skeletal muscle.

Feedback regulation between plasma membrane tension and membrane-bending proteins organizes cell polarity during leading edge formation.

Tension applied to the plasma membrane (PM) is a global mechanical parameter involved in cell migration. However, how membrane tension regulates actin assembly is unknown. Here, we demonstrate that FBP17, a membrane-bending protein and an activator of WASP/N-WASP-dependent actin nucleation, is a PM tension sensor involved in leading edge formation. In migrating cells, FBP17 localizes to short membrane invaginations at the leading edge, while diminishing from the cell rear in response to PM tension increase. Conversely, following reduced PM tension, FBP17 dots randomly distribute throughout the cell, correlating with loss of polarized actin assembly on PM tension reduction. Actin protrusive force is required for the polarized accumulation, indicating a role for FBP17-mediated activation of WASP/N-WASP in PM tension generation. In vitro experiments show that FBP17 membrane-bending activity depends on liposomal membrane tension. Thus, FBP17 is the local activator of actin polymerization that is inhibited by PM tension in the feedback loop that regulates cell migration.

α-Actinin-4 enhances colorectal cancer cell invasion by suppressing focal adhesion maturation.

α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion.

Pancreatic fistulae secondary to trypsinogen activation by Pseudomonas aeruginosa infection after pancreatoduodenectomy.

BACKGROUND: Pancreatic fistula after pancreatoduodenectomy (PD) is associated with high mortality and morbidity. Trypsinogen activation and bacteria, although hypothesized to be interrelated etiopathogenetically, have not had their relationship and pathogenic mechanisms elucidated. This study investigated bacterial involvement in pancreatic juice activation perioperatively after PD at sites of pancreatic fistula formation. METHODS: Fifty patients underwent PD; postoperative pancreatic fistulae were graded based on the International Study Group for Pancreatic Fistula grading criteria. Bacteria were isolated from cultures of drainage fluid. Digested peptides from trypsinogen and bacterial culture supernatants underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and mass spectrometric analysis. Zymography was used to detect the trypsinogen activator. RESULTS: Pseudomonas aeruginosa and Enterobacter cloacae isolated from drainage fluid in patients with grades B and C pancreatic fistulae could cause trypsinogen activation. Trypsinogen activation by P. aeruginosa and E. cloacae were preventable by the use of a serine protease inhibitor in vitro. A protease in the supernatant from P. aeruginosa-positive cultures acted as the trypsinogen activator. CONCLUSIONS: Infection with P. aeruginosa perioperatively to PD entails secretion of a protease activator of trypsinogen to trypsin. Bacterial infection control in the perioperative PD period could be crucial to prevent development of pancreatic fistula.

Improvement of insulin signaling in myoblast cells by an addition of SKIP-binding peptide within Pak1 kinase domain.

Abnormalities in insulin-induced glucose incorporation in skeletal muscle were observed in Type 2 diabetes. Our previous studies revealed that the binding between skeletal muscle and kidney-enriched inositol polyphosphate phosphatase (SKIP) and p21-activated protein kinase (Pak1) at the plasma membrane is induced insulin-dependently and that this binding mediated a rapid and efficient termination of insulin signaling and a subsequent glucose uptake into skeletal muscle cells. Here, we identified 11-amino-acids peptide within kinase domain of Pak1, necessary and sufficient for SKIP binding. Expression of this region in C2C12 cells resulted in an increase in insulin signaling. Supplementation of a synthetic peptide of this sequence increased insulin signaling and insulin-induced glucose uptake into skeletal muscle cell lines. These findings suggest the physiological role of Pak1-SKIP binding in the regulation of insulin signaling in skeletal muscle.

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes.

Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhesion and invasive cell migration in human breast cancer.

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 IIIß, 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.

Nucleoredoxin regulates glucose metabolism via phosphofructokinase 1.

Phosphofructokinase (PFK) 1 is a glycolytic enzyme, and its abnormality contributes to the development of multiple human diseases, such as cancer. Here, we report that nucleoredoxin (NRX), a thioredoxin-related oxidoreductase, is a novel interacting partner of PFK1. NRX binds directly to PFK1, and endogenous NRX and PFK1 interact in vivo. In NRX(-/-) mouse embryonic fibroblasts (MEFs), the oligomerization status of PFK1 is altered and the catalytic activity of PFK1 is decreased. NRX deficiency augmented levels of NADPH and reduced glutathione, two major cellular antioxidants generated through the pentose phosphate pathway. Indeed, NRX(-/-) MEFs are significantly more resistant to oxidative stress than NRX(+/+) MEFs. These results reveal a novel role of NRX in the regulation of PFK1 activity and in the balance between glycolysis and the pentose phosphate pathway.

srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity.

The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.

Actin dynamics regulated by the balance of neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin activities determines the biphasic response of glucose-induced insulin secretion.

Actin dynamics in pancreatic ß-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic ß-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic ß-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic ß-cells (MIN6-K8 ß-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 ß-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 ß-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.

A novel serum metabolomics-based diagnostic approach to pancreatic cancer.

BACKGROUND: To improve the prognosis of patients with pancreatic cancer, more accurate serum diagnostic methods are required. We used serum metabolomics as a diagnostic method for pancreatic cancer. METHODS: Sera from patients with pancreatic cancer, healthy volunteers, and chronic pancreatitis were collected at multiple institutions. The pancreatic cancer and healthy volunteers were randomly allocated to the training or the validation set. All of the chronic pancreatitis cases were included in the validation set. In each study, the subjects' serum metabolites were analyzed by gas chromatography mass spectrometry (GC/MS) and a data processing system using an in-house library. The diagnostic model constructed via multiple logistic regression analysis in the training set study was evaluated on the basis of its sensitivity and specificity, and the results were confirmed by the validation set study. RESULTS: In the training set study, which included 43 patients with pancreatic cancer and 42 healthy volunteers, the model possessed high sensitivity (86.0%) and specificity (88.1%) for pancreatic cancer. The use of the model was confirmed in the validation set study, which included 42 pancreatic cancer, 41 healthy volunteers, and 23 chronic pancreatitis; that is, it displayed high sensitivity (71.4%) and specificity (78.1%); and furthermore, it displayed higher sensitivity (77.8%) in resectable pancreatic cancer and lower false-positive rate (17.4%) in chronic pancreatitis than conventional markers. CONCLUSIONS: Our model possessed higher accuracy than conventional tumor markers at detecting the resectable patients with pancreatic cancer in cohort including patients with chronic pancreatitis. IMPACT: It is a promising method for improving the prognosis of pancreatic cancer via its early detection and accurate discrimination from chronic pancreatitis.

IRSp53 mediates podosome formation via VASP in NIH-Src cells.

Podosomes are cellular "feet," characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation.

Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation.

FBP17, an F-BAR domain protein, has emerged as a crucial factor linking the plasma membrane to WASP-mediated actin polymerization. Although it is well established that FBP17 has a powerful self-polymerizing ability that promotes actin nucleation on membranes in vitro, knowledge of inhibitory factors that counteract this activity in vivo is limited. Here, we demonstrate that the assembly of FBP17 on the plasma membranes is antagonized by PSTPIP2, another F-BAR protein implicated in auto-inflammatory disorder. Knockdown of PSTPIP2 in macrophage promotes the assembly of FBP17 as well as subsequent actin nucleation at podosomes, resulting in an enhancement of matrix degradation. This phenotype is rescued by expression of PSTPIP2 in a manner dependent on its F-BAR domain. Time-lapse total internal reflection fluorescence (TIRF) microscopy observations reveal that the self-assembly of FBP17 at the podosomal membrane initiates actin polymerization, whereas the clustering of PSTPIP2 has an opposite effect. Biochemical analysis and live-cell imaging show that PSTPIP2 inhibits actin polymerization by competing with FBP17 for assembly at artificial as well as the plasma membrane. Interestingly, the assembly of FBP17 is dependent on WASP, and its dissociation by WASP inhibition strongly induces a self-organization of PSTPIP2 at podosomes. Thus, our data uncover a previously unappreciated antagonism between different F-BAR domain assemblies that determines the threshold of actin polymerization for the formation of functional podosomes and may explain how the absence of PSTPIP2 causes auto-inflammatory disorder.