A Novel Interaction between Chemokine and Phosphoinositide Signaling in Metastatic Prostate Cancer.

Prostate cancer commonly metastasizes to bone due to its favorable microenvironment for cell growth and survival. Currently, the standard of care for metastatic prostate cancer is medical castration in conjunction with chemotherapeutic agents and newer anti-androgen/androgen receptor therapies. While these therapies aim to improve the quality of life in patients with advanced disease, resistance to these therapies is inevitable prompting the development of newer therapies to contain disease progression.

Adaptor proteins mediate CXCR4 and PI4KA crosstalk in prostate cancer cells and the significance of PI4KA in bone tumor growth.

The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4-PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells.

Adaptor proteins mediate CXCR4 and PI4KA crosstalk in prostate cancer cells and the significance of PI4KA in bone tumor growth.

The chemokine receptor, CXCR4 signaling regulates cell growth, invasion, and metastasis to the bone-marrow niche in prostate cancer (PCa). Previously, we established that CXCR4 interacts with phosphatidylinositol 4-kinase IIIα (PI4KIIIα encoded by PI4KA) through its adaptor proteins and PI4KA overexpressed in the PCa metastasis. To further characterize how the CXCR4-PI4KIIIα axis promotes PCa metastasis, here we identify CXCR4 binds to PI4KIIIα adaptor proteins TTC7 and this interaction induce plasma membrane PI4P production in prostate cancer cells.

Small molecule PIKfyve inhibitors as cancer therapeutics: Translational promises and limitations.

Through synthesis of two rare phosphoinositides, PtdIns(3,5)P and PtdIns5P, the ubiquitously expressed phosphoinositide kinase PIKfyve is implicated in pleiotropic cellular functions. Small molecules specifically inhibiting PIKfyve activity cause cytoplasmic vacuolation in all dividing cells in culture yet trigger non-apoptotic death through excessive vacuolation only in cancer cells. Intriguingly, cancer cell toxicity appears to be inhibitor-specific suggesting that additional targets beyond PIKfyve are affected.

Severe Consequences of SAC3/FIG4 Phosphatase Deficiency to Phosphoinositides in Patients with Charcot-Marie-Tooth Disease Type-4J.

Charcot-Marie-Tooth disease type-4J (CMT4J), an autosomal recessively inherited peripheral neuropathy characterized by neuronal degeneration, segmental demyelination, and limb muscle weakness, is caused by compound heterozygous mutations in the SAC3/FIG4 gene, resulting in SAC3/FIG4 protein deficiency. SAC3/FIG4 is a phosphatase that not only turns over PtdIns(3,5)P to PtdIns3P but also promotes PtdIns(3,5)P synthesis by activating the PIKFYVE kinase that also makes PtdIns5P. Whether CMT4J patients have alterations in PtdIns(3,5)P, PtdIns5P or in other phosphoinositides (PIs), and if yes, in what direction these changes might be, has never been examined.

Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation.

PIKfyve, an evolutionarily conserved kinase synthesizing PtdIns5P and PtdIns(3,5)P2, is crucial for mammalian cell proliferation and viability. Accordingly, PIKfyve inhibitors are now in clinical trials as anti-cancer drugs. Among those, apilimod is the most promising, yet its potency to inhibit PIKfyve and affect endomembrane homeostasis is only partially characterized.

A novel cross-talk between CXCR4 and PI4KIIIα in prostate cancer cells.

Chemokine signaling regulates cell migration and tumor metastasis. CXCL12, a member of the chemokine family, and its receptor, CXCR4, a G protein coupled receptor (GPCR), are key mediators of prostate-cancer (PC) bone metastasis. In PC cells androgens activate CXCR4 gene expression and receptor signaling on lipid rafts, which induces protease expression and cancer cell invasion.

PIKfyve inhibitor cytotoxicity requires AKT suppression and excessive cytoplasmic vacuolation.

PIKfyve phosphoinositide kinase produces PtdIns(3,5)P and PtdIns5P and governs a myriad of cellular processes including cytoskeleton rearrangements and cell proliferation. The latter entails rigorous investigation since the cytotoxicity of PIKfyve inhibition is a potential therapeutic modality for cancer. Here we report the effects of two PIKfyve-specific inhibitors on the attachment/spreading and viability of mouse embryonic fibroblasts (MEFs) and CC myoblasts.

Active vacuolar H+ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P2 loss under PIKfyve or Vps34 deficiency.

The two evolutionarily conserved mammalian lipid kinases Vps34 and PIKfyve are involved in an important physiological relationship, whereby the former produces phosphatidylinositol (PtdIns) 3P that is used as a substrate for PtdIns(3,5)P2 synthesis by the latter. Reduced production of PtdIns(3,5)P2 in proliferating mammalian cells is phenotypically manifested by the formation of multiple translucent cytoplasmic vacuoles, readily rescued upon exogenous delivery of PtdIns(3,5)P2 or overproduction of PIKfyve. Although the aberrant vacuolation phenomenon has been frequently used as a sensitive functional measure of localized PtdIns(3,5)P2 reduction, cellular factors governing the appearance of cytoplasmic vacuoles under PtdIns3P-PtdIns(3,5)P2 loss remain elusive.

Unexpected severe consequences of Pikfyve deletion by aP2- or Aq-promoter-driven Cre expression for glucose homeostasis and mammary gland development.

Systemic deficiency of PIKfyve, the evolutionarily conserved phosphoinositide kinase synthesizing cellular PtdIns5P and PtdIns(3,5)P2 and implicated in insulin signaling, causes early embryonic death in mice. In contrast, mice with muscle-specific Pikfyve disruption have normal lifespan but exhibit early-age whole-body glucose intolerance and muscle insulin resistance, thus establishing the key role of muscle PIKfyve in glucose homeostasis. Fat and muscle tissues control postprandial glucose clearance through different mechanisms, raising questions as to whether adipose Pikfyve disruption will also trigger whole-body metabolic abnormalities, and if so, what the mechanism might be.

The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation.

The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown.

Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.

The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media.

Plentiful PtdIns5P from scanty PtdIns(3,5)P2 or from ample PtdIns? PIKfyve-dependent models: Evidence and speculation (response to: DOI 10.1002/bies.201300012).

Recently, we have presented data supporting the notion that PIKfyve not only produces the majority of constitutive phosphatidylinositol 5-phosphate (PtdIns5P) in mammalian cells but that it does so through direct synthesis from PtdIns. Another group, albeit obtaining similar data, suggests an alternative pathway whereby the low-abundance PtdIns(3,5)P2 undergoes hydrolysis by unidentified 3-phosphatases, thereby serving as a precursor for most of PtdIns5P. Here, we review the experimental evidence supporting constitutive synthesis of PtdIns5P from PtdIns by PIKfyve.

cdc-like/dual-specificity tyrosine phosphorylation-regulated kinases inhibitor leucettine L41 induces mTOR-dependent autophagy: implication for Alzheimer's disease.

Leucettines, a family of pharmacological inhibitors of dual-specificity tyrosine phosphorylation regulated kinases and cdc-like kinases (CLKs), are currently under investigation for their potential therapeutic application to Down syndrome and Alzheimer's disease. We here report that leucettine L41 triggers bona fide autophagy in osteosarcoma U-2 OS cells and immortalized mouse hippocampal HT22 cells, characterized by microtubule-associated protein light chain 3 membrane translocation and foci formation. Leucettine L41-triggered autophagy requires the Unc-51-like kinase and is sensitive to the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and 3-methyladenine, suggesting that it acts through the mammalian target of rapamycin (mTOR)/PI3K-dependent pathway.

The PIKfyve-ArPIKfyve-Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines.

The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve-ArPIKfyve-Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P-PtdIns(3,5)P2 synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P-PtdIns(3,5)P2 conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy.

Muscle-specific Pikfyve gene disruption causes glucose intolerance, insulin resistance, adiposity, and hyperinsulinemia but not muscle fiber-type switching.

The evolutionarily conserved kinase PIKfyve that synthesizes PtdIns5P and PtdIns(3,5)P₂ has been implicated in insulin-regulated GLUT4 translocation/glucose entry in 3T3-L1 adipocytes. To decipher PIKfyve's role in muscle and systemic glucose metabolism, here we have developed a novel mouse model with Pikfyve gene disruption in striated muscle (MPIfKO). These mice exhibited systemic glucose intolerance and insulin resistance at an early age but had unaltered muscle mass or proportion of slow/fast-twitch muscle fibers.

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636.

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis.

The phosphoinositide kinase PIKfyve is vital in early embryonic development: preimplantation lethality of PIKfyve-/- embryos but normality of PIKfyve+/- mice.

Gene mutations in the phosphoinositide-metabolizing enzymes are linked to various human diseases. In mammals, PIKfyve synthesizes PtdIns(3,5)P(2) and PtdIns5P lipids that regulate endosomal trafficking and responses to extracellular stimuli. The consequence of pikfyve gene ablation in mammals is unknown.

ArPIKfyve regulates Sac3 protein abundance and turnover: disruption of the mechanism by Sac3I41T mutation causing Charcot-Marie-Tooth 4J disorder.

The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P(2)) phosphatase Sac3 and ArPIKfyve, the associated regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P(2) production. Whether the ArPIKfyve-Sac3 subcomplex functions outside the PIKfyve context is unknown. Here we show that stable or transient expression of ArPIKfyve(WT) in mammalian cells elevates steady-state protein levels and the PtdIns(3,5)P(2)-hydrolyzing activity of Sac3, whereas knockdown of ArPIKfyve has the opposite effect.

PIKfyve-ArPIKfyve-Sac3 core complex: contact sites and their consequence for Sac3 phosphatase activity and endocytic membrane homeostasis.

The phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) metabolizing enzymes, the kinase PIKfyve and the phosphatase Sac3, constitute a single multiprotein complex organized by the PIKfyve regulator ArPIKfyve and its ability to homodimerize. We previously established that PIKfyve is activated within the triple PIKfyve-ArPIKfyve-Sac3 (PAS) core. These data assign an atypical function for the phosphatase in PtdIns(3,5)P(2) biosynthesis, thus raising the question of whether Sac3 retains its PtdIns(3,5)P(2) hydrolyzing activity within the PAS complex.

Sac3 is an insulin-regulated phosphatidylinositol 3,5-bisphosphate phosphatase: gain in insulin responsiveness through Sac3 down-regulation in adipocytes.

Insulin-regulated stimulation of glucose entry and mobilization of fat/muscle-specific glucose transporter GLUT4 onto the cell surface require the phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) pathway for optimal performance. The reduced insulin responsiveness observed under ablation of the PtdIns(3,5)P(2)-synthesizing PIKfyve and its associated activator ArPIKfyve in 3T3L1 adipocytes suggests that dysfunction of the PtdIns(3,5)P(2)-specific phosphatase Sac3 may yield the opposite effect. Paradoxically, as uncovered recently, in addition to turnover Sac3 also supports PtdIns(3,5)P(2) biosynthesis by allowing optimal PIKfyve-ArPIKfyve association.

YM201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes.

Silencing of PIKfyve, the sole enzyme for PtdIns(3,5)P(2) biosynthesis that controls proper endosome dynamics, inhibits retroviral replication. A novel PIKfyve-specific inhibitor YM201636 disrupts retroviral budding at 800 nM, suggesting its potential use as an antiretroviral therapeutic. Because PIKfyve is also required for optimal insulin activation of GLUT4 surface translocation and glucose influx, we tested the outcome of YM201636 application on insulin responsiveness in 3T3L1 adipocytes.

Kinesin adapter JLP links PIKfyve to microtubule-based endosome-to-trans-Golgi network traffic of furin.

JIPs (c-Jun N-terminal kinase interacting proteins), which scaffold JNK/p38 MAP kinase signaling modules, also bind conventional kinesins and are implicated in microtubule-based membrane trafficking in neuronal cells. Here we have identified a novel splice variant of the Jip4 gene product JLP(L) (JNK-interacting leucine zipper protein) in yeast-two hybrid screens with the phosphoinositide kinase PIKfyve. The interaction was confirmed by pulldown and coimmunoprecipitation assays in native cells.

ArPIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3 in a complex to promote PIKfyve activity and functionality.

PtdIns(3,5)P(2) (with PtdIns indicating phosphatidylinositol) is vital in the differentiation and development of multicellular organisms because knockout of the PtdIns(3,5)P(2)-synthesizing enzyme PIKfyve (phosphoinositide kinase for position 5 containing a FYVE finger domain) or its associated regulator ArPIKfyve is lethal. In previous work with endogenous proteins, we identified that Sac3, a phosphatase that turns over PtdIns(3,5)P(2), associates with the PIKfyve-ArPIKfyve biosynthetic complex. However, whether the three proteins suffice for the organization/maintenance of this complex [referred to as the PAS (PIKfyve-ArPIKfyve-Sac3) complex], how they interact with one another, and what the functional relevance of this ternary association would be remained unresolved.

Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-PIKfyve complex.

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P2 in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P2-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P2-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P2, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown.