Phosphatidylinositol 4-kinase Beta Research Articles

Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

With the escalating prevalence of global heat waves, heat stroke has become a prominent health concern, leading to substantial liver damage. Unlike other forms of liver injury, heat stroke-induced damage is characterized by heat cytotoxicity and heightened inflammation, directly contributing to elevated mortality rates. While clinical assessments have identified elevated bilirubin levels as indicative of Kupffer cell dysfunction, their specific correlation with heat stroke liver injury remains unclear. Our hypothesis proposes the involvement of Kupffer cell ferroptosis during heat stroke, initiating IL-1β-mediated inflammation. Using single-cell RNA sequencing of murine macrophages, a distinct and highly susceptible Kupffer cell subtype, Clec4F+/CD206+, emerged, with heme oxygenase 1 (HMOX-1) playing a pivotal role. Mechanistically, heat-induced HMOX-1, regulated by early growth response factor 1, mediated ferroptosis in Kupffer cells, specifically in the Clec4F+/CD206+ subtype (KC2), activating phosphatidylinositol 4-kinase beta and promoting PI4P production. This cascade triggered NLRP3 inflammasome activation and maturation of IL-1β. These findings underscore the critical role of targeted therapy against HMOX-1 in ferroptosis within Kupffer cells, particularly in Clec4F+/CD206+ KCs. Such an approach has the potential to mitigate inflammation and alleviate acute liver injury in the context of heat stroke, offering a promising avenue for future therapeutic interventions.

Locational and functional characterization of PI4KB in the mouse embryo.

Phosphatidylinositol 4-kinase beta (PI4KB) is a member of the PI4K family, which is mainly enriched and functions in the Golgi apparatus. The kinase domain of PI4KB catalyzes the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a process that regulates various sub-cellular events, such as non-vesicular cholesterol and ceramide transport, protein glycosylation, and vesicle transport, as well as cytoplasmic division. In this study, a strain of PI4KB knockout mouse, immunofluorescence, reverse transcription polymerase chain reaction and microinjection were used to characterize the cytological location and biological function of PI4KB in the mouse embryos. we found that knocking down Pi4kb in mouse embryos resulted in embryonic lethality at around embryonic day (E) 7.5. Additionally, we observed dramatic fluctuations in PI4KB expression during the development of preimplantation embryos, with high expression in the 4-cell and morula stages. PI4KB colocalized with the Golgi marker protein TGN46 in the perinuclear and cytoplasmic regions in early blastomeres. Postimplantation, PI4KB was highly expressed in the epiblast of E7.5 embryos. Treatment of embryos with PI4KB inhibitors was found to inhibit the development of the morula into a blastocyst and the normal progression of cytoplasmic division during the formation of a 4-cell embryo. These findings suggest that PI4KB plays an important role in mouse embryogenesis by regulating various intracellular vital functions of embryonic cells.

ARMH3-mediated recruitment of PI4KB directs Golgi-to-endosome trafficking and activation of the antiviral effector STING

The cGAS-STING pathway mediates cytoplasmic DNA-triggered innate immunity. STING activation is initiated by cyclic-GMP-AMP (cGAMP)-induced translocation from the endoplasmic reticulum and sulfated glycosaminoglycans-induced polymerization at the Golgi. Here, we examine the mechanisms underlying STING transport and activation beyond the Golgi. A genome-wide CRISPR-Cas9 screen identified Armadillo-like helical domain-containing protein 3 (ARMH3) as critical for STING activation. Upon cGAMP-triggered translocation, ARMH3 interacted with STING at the Golgi and recruited phosphatidylinositol 4-kinase beta (PI4KB) to synthesize PI4P, which directed STING Golgi-to-endosome trafficking via PI4P-binding proteins AP-1 and GGA2. Disrupting PI4P-dependent lipid transport through RNAi of other PI4P-binding proteins impaired STING activation. Consistently, disturbed lipid composition inhibited STING activation, whereas aberrantly elevated cellular PI4P led to cGAS-independent STING activation. Armh3fl/fllLyzCre/Cre mice were susceptible to DNA virus challenge invivo. Thus, ARMH3 bridges STING and PIK4B to generate PI4P for STING transportation and activation, an interaction conserved in all eukaryotes.

Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

134. KRP-A218, an Orally Active and Selective PI4KB Inhibitor with Broad-Spectrum Anti-Rhinovirus Activity, Has Potent Therapeutic Antiviral Activity In vivo

BackgroundRhinovirus (RV) is a major respiratory virus that poses a threat to immunocompromised people and those with underlying disease. However, there are no approved therapies. Moreover, RV infection cannot be prevented by a vaccine because there are over 100 serotypes. Here we report the pharmacological profile of a novel small-molecule host-targeted antiviral (HTA), KRP-A218 (A218). A highly potent and selective inhibitor of phosphatidylinositol 4 kinase beta (PI4KB), a key host factor of RV replication, A218 is undergoing clinical study.Methods in vitro antiviral activities of A218 and Vapendavir (Vap), a virus-targeted antiviral, were examined by inhibition of CPE, viral load, or replication. in vivo antiviral activity and pathological analysis of A218 were examined in coxsackievirus B3 (CVB3; belong to the genus enterovirus as with RV)-infected mice as a surrogate model of RV infection as CVB3, unlike RV, replicates very well in both mouse and human tissue. Daily oral dosing of A218 (1-10 mg/kg) was started 2 days post intraperitoneal infection with CVB3. Tissue viral load, pancreas pathological change at 4 days post infection, and survival rate up to 14 days were evaluated. PI4KB heterozygous kinase-dead mice (PI4KB KD) were established by a CRISPR-Cas9 system. Viral load and survival rate following viral infection were evaluated in these mice.ResultsA218 showed broad antiviral activity for RV and enteroviruses (Table) and has a higher barrier to drug resistance than Vap. These results are consistent with expectations for HTAs. Repeated dosing of A218 starting 2 days post infection decreased viral load and improved acute pancreatitis, accompanied by decrease of inflammatory and pancreatitis markers in plasma. Moreover, therapeutic dosing of A218 improved survival rate in a CVB3-infected lethal mouse model (Figure). These results show the first evidence that a PI4KB inhibitor has potent therapeutic efficacy in a severe viral infection model. Similar effects were observed in PI4KB KD, supporting the on-target effect of A218.Table. Antiviral activity of A218 and Vap against RV/EV infection Figure. Therapeutic effect of A218 on survival rate in CVB3-infected miceConclusionA218 is a promising therapeutic agent for improving the exacerbation of pathological conditions caused by RV infection. Nonclinical package including GLP-Tox also supports the ongoing first-in-human study of A218.Disclosures Toshiyuki Matsui, MPharm, Kyorin pharmaceutical Co., LTD (Employee) Motomichi Fujita, PhD, Kyorin Pharmaceutical Co., Ltd. (Employee) Yuji Ishibashi, PhD, Kyorin Pharmaceutical co., ltd. (Employee) Tyzoon Nomanbhoy, PhD, ActivX Biosciences (Other Financial or Material Support, Full time employee of ActivX, a wholly owned subsidiary of Kyorin Pharmaceuticals) Jonathan S. Rosenblum, PhD, ActivX Biosciences (Employee) Michiaki Nagasawa, PhD, Kyorin Pharmaceutical Co., Ltd (Employee)

Myelination of peripheral nerves is controlled by PI4KB through regulation of Schwann cell Golgi function

Better understanding myelination of peripheral nerves would benefit patients affected by peripheral neuropathies, including Charcot-Marie-Tooth disease. Little is known about the role the Golgi compartment plays in Schwann cell (SC) functions. Here, we studied the role of Golgi in myelination of peripheral nerves in mice through SC-specific genetic inactivation of phosphatidylinositol 4-kinase beta (PI4KB), a Golgi-associated lipid kinase. Sciatic nerves of such mice showed thinner myelin of large diameter axons and gross aberrations in myelin organization affecting the nodes of Ranvier, the Schmidt-Lanterman incisures, and Cajal bands. Nonmyelinating SCs showed a striking inability to engulf small diameter nerve fibers. SCs of mutant mice showed a distorted Golgi morphology and disappearance of OSBP at the cis-Golgi compartment, together with a complete loss of GOLPH3 from the entire Golgi. Accordingly, the cholesterol and sphingomyelin contents of sciatic nerves were greatly reduced and so was the number of caveolae observed in SCs. Although the conduction velocity of sciatic nerves of mutant mice showed an 80% decrease, the mice displayed only subtle impairment in their motor functions. Our analysis revealed that Golgi functions supported by PI4KB are critically important for proper myelination through control of lipid metabolism, protein glycosylation, and organization of microvilli in the nodes of Ranvier of peripheral nerves.

Defining how viruses manipulate lipid phosphoinositides through activation of PI4P kinases to mediate viral replication

Phosphoinositide kinases are central regulators of myriad cellular processes, and their misregulation is causative of multiple human diseases. We are currently focusing on the structural basis for the regulation of phosphoinositide kinases, specifically the Golgi localised phosphatidylinositol 4‐kinase beta (PI4KB). We have identified a previously uncharacterised protein binding partner of PI4KB, the GTPase regulatory protein c10orf76 1, and revealed novel insight into an unexpected PI4K positive feedback loop. Using a combined hydrogen deuterium exchange mass spectrometry (HDX‐MS) and structural biology approach we have defined the molecular basis of regulation by the protein c10orf76, revealing novel insight into the role of these PI4KB in viral replication. This has revealed key insight into unexpected feedback loops with Ras superfamily GTPases. Overall, this approach has provided a novel toolbox of engineered mutations to tease out the molecular basis of regulation of phosphoinositide kinases in both homeostasis and disease, and has revealed novel opportunities for the development of small molecule therapeutics for multiple pathogenic viral infections.Support or Funding InformationResearch was supported by the Canadian Institute of Health Research (CRN‐142393), and salary award for the CIHR new investigator and MSFHR scholar programs.

PI4KB on Inclusion Bodies Formed by ER Membrane Remodeling Facilitates Replication of Human Parainfluenza Virus Type 3

Many positive-strand RNA viruses remodel the endomembrane to form specialized replication organelles. However, knowledge regarding whether negative-strand RNA viruses take advantage of intracellular membranes for replication is limited. Here we show that a negative-strand RNA virus, human parainfluenza virus type 3 (HPIV3), remodels the endoplasmic reticulum (ER) membrane to form inclusion bodies (IBs), whereby the phosphoprotein (P) of HPIV3 recruits phosphatidylinositol 4-kinase beta (PI4KB) to IBs to generate PI4P, creating a PI4P-enriched microenvironment to promote HPIV3 replication. In addition, we find that human respiratory syncytial virus (HRSV) also takes advantage of the ER to form IBs and that these IBs are also enriched with PI4P. The nucleoprotein of HRSV recruits PI4KB to IBs. These results suggest that paramyxoviruses also exploit the host endomembrane to form IBs and that PI4KB is recruited by viral proteins to enrich IBs with PI4P to facilitate viral replication.

Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant.

We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kβ1β2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kβ1β2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kβ1 and PI4Kβ2. To achieve this we used the sid2pi4kβ1β2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kβ1β2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kβ1β2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kβ1β2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kβ1β2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kβ1β2 triple mutant revealed the role of PI4Kβ1/β2 per se, thus showing the importance of these enzymes in plant defence responses.

RAB30 regulates PI4KB (phosphatidylinositol 4-kinase beta)-dependent autophagy against group A Streptococcus

ABSTRACTMacroautophagy/autophagy plays an important role in the immune response to invasion by intracellular pathogens such as group A Streptococcus (GAS; Streptococcus pyogenes). We previously identified RAB30, a Golgi-resident GTPase, as a novel anti-bacterial autophagic regulator in the formation of GAS-containing autophagosome-like vacuoles (GcAVs); however, the precise mechanism underlying this process remains elusive. Here, we elucidate a novel property of RAB30: the ability to recruit PI4KB (phosphatidylinositol 4-kinase beta) to the Golgi apparatus and GcAVs. We found that trans-Golgi network (TGN) vesicles were incorporated into GcAVs via RAB30 to promote GcAV formation. Moreover, depletion of phosphatidylinositol-4-phosphate (PtdIns4P), a phosphatidylinositol enriched in the TGN, by wortmannin and phenylarsine oxide, followed by subsequent repletion with exogenous PtdIns4P revealed that PtdIns4P is crucial for GcAV formation. Furthermore, we identify an interaction between RAB30 and PI4KB, in which the knockdown of RAB30 decreased the localization of PI4KB to the TGN and GcAVs. Finally, PI4KB knockout suppressed autophagy by inhibiting GcAV formation, resulting in the increased survival of GAS. Our results demonstrate a novel autophagosomal formation mechanism involving coordinative functions of RAB30 and PI4KB distinct from those utilized in canonical autophagy.Abbreviations: GAS: group A Streptococcus; GcAVs: GAS-containing autophagosome-like vacuoles; PI4KB: phosphatidylinositol 4-kinase beta; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; PtdIns5P: phosphatidylinositol-5-phosphate; SLO: streptolysin O; TGN: trans-Golgi network; TGOLN2: trans-golgi network protein 2; PH: plekstrin homology; OSBP: oxysterol binding protein

Deep Proteomic Quantification of Inner Medullary Collecting Duct Protein Phosphorylation: Response to Vasopressin

Progress in protein mass spectrometry instrumentation and labeling methods have led to dramatic increases in the depth of phospho‐peptide identification and quantification. Here we used tandem mass tag (TMT) labeling and LC‐MS/MS for deep identification of phospho‐proteomic responses to the vasopressin analog dDAVP (1 nM for 30 minutes) in inner medullary collecting duct (IMCD) suspensions from rats. We analyzed three pairs of samples (dDAVP vs. vehicle) on different days. The analysis yielded 16 GB of spectral data mapping to 11,658 phospho‐peptides in 1758 distinct proteins. Vehicle vs. vehicle comparisons allowed assessment of the variability of the method and permitted dual criteria to be applied for stringent identification of abundance changes (based on t‐statistic and z‐score). Among all phospho‐peptides quantified, only 93 were increased in abundance, and only 49 were decreased, indicating very selective regulation within the broad range of phospho‐peptides quantified. A substantial number of the increases were in aquaporin‐2 and the UT‐A urea channel, including a previously unidentified phosphorylation site at Ser918 of UT‐A1 (sequence RAS*MITK) that was more than doubled in abundance. Abundance changes in aquaporin‐2 peptides confirmed prior findings. Of interest, phosphorylation changes in Raf1 (increased phosphorylation at Ser259, a kinase inhibitory site) and A‐Raf (decreased phosphorylation of Ser255, a kinase activating site) provide an explanation for findings that vasopressin decreases ERK activation and AQP2 phosphorylation at Ser261 in the collecting duct. Finally, vasopressin significantly increased phosphorylation of protein kinase D1 (Prkd1) at Ser255 and of phosphatidylinositol 4‐kinase beta at Ser551. Both proteins play central roles in regulated exocytosis of ENaC, and we speculate that they could play similar roles in vasopressin‐stimulated exocytotic apical insertion of aquaporin‐2 and UT‐A1.Support or Funding InformationV.D. was a member of the 2017 Biomedical Engineering Student Internship Program at the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract PR12: A genome-wide shRNA screen reveals that inhibiting kinases potentiates the anti-breast cancer activity of fluvastatin

Abstract Background: Statins are widely used to manage hypercholesterolemia, and have also been shown to possess anti-tumor effects. Breast cancer clinical trials have demonstrated that statins are effective in some but not all patients. We aim to identify combination treatments that can expand the anti-tumor benefit of statins to a larger subset of breast cancer patients. We hypothesize that a genome-wide shRNA screen will identify novel genomic targets to inhibit in co-therapies with fluvastatin. Methods: We used a pooled shRNA dropout screen to determine if knocking down specific genomic targets increases the anti-proliferative effects of fluvastatin. Cells transduced with the 80K TRC1 shRNA library were treated with either sublethal doses of fluvastatin or vehicle control over 12 days. Genomic DNA was collected from these cells every three days for hybridization to custom Affymetrix Gene Modulation Array Platform (GMAP) arrays. Candidate shRNA dropout hits were validated using shRNAs, siRNAs, and pharmacological inhibitors. Results: Our shRNA screen identified several kinase targets as dropouts, suggesting that knocking down specific kinases can potentiate the anti-proliferative effects of fluvastatin. We validated two candidate hits, PI4KB (phosphatidylinositol 4-kinase beta) and CSNK2B (casein kinase 2, beta polypeptide) in two breast cancer cell lines: MDA-MB-231 cells, which are highly sensitive to fluvastatin, and MCF-7 cells, which are less sensitive to fluvastatin. We used shRNAs and siRNAs targeting PI4KB or CSNK2B to confirm that knockdown of these kinases potentiates the anti-proliferative effects of fluvastatin. We also used pharmacological inhibitors of PI4KB and CSNK2B, which also increased the anti-proliferative activity of fluvastatin. Conclusions: Statins show promising anti-tumor effects, but co-treatments will be required to increase both their efficacy and the number of patients who will respond. We show here that kinases are a class of targets that can potentiate fluvastatin efficacy in breast cancer cell lines. We are now performing a small-molecule kinase inhibitor library screen that is designed to identify FDA-approved kinase inhibitors to combine with fluvastatin. The screen readout involves high-content confocal imaging and is currently underway. This work may lead to the discovery of effective and novel co-treatments for breast cancer that will better impact patient care. This abstract is also presented as Poster A16. Citation Format: Janice Pong, Aleksandra Pandyra, Carolyn Goard, Elke Ericson, Kevin Brown, Jarkko Ylanko, David Andrews, Corey Nislow, Jason Moffat, Linda Penn. A genome-wide shRNA screen reveals that inhibiting kinases potentiates the anti-breast cancer activity of fluvastatin. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR12.

The mast cell: where endocytosis and regulated exocytosis meet

We have investigated whether Ca(2+)-binding proteins, which have been implicated in the control of neurons and neuroendocrine secretion, play a role in controlling mast cell function. These studies have identified synaptotagmins (Syts) II, III, and IX as well as neuronal Ca(2+) sensor 1 (NCS-1) as important regulators of mast cell function. Strikingly, we find that these Ca(2+)-binding proteins contribute to mast cell function by regulating specific endocytic pathways. Syt II, the most abundant Syt homologue in mast cells, resides in an amine-free lysosomal compartment. Studying the function of Syt II-knocked down rat basophilic leukemia cells has shown a dual function of this homologue. Syt II is required for the downregulation of protein kinase Calpha, but it negatively regulates lysosomal exocytosis. Syt III, the next most abundant homologue, localizes to early endosomes and is required for the formation of the endocytic recycling compartment (ERC). Syt IX and NCS-1 localize to the ERC and regulate ERC export, NCS-1 by activating phosphatidylinositol 4-kinase beta. Finally, we show that recycling through the ERC is needed for secretory granule protein sorting as well as for the activation of the mitogen-activated protein kinases, extracellular signal-regulated kinase 1 and 2. Accordingly, NCS-1 stimulates Fc epsilon RI-triggered exocytosis and release of arachidonic acid metabolites.

Phosphatidylinositol 4-Kinase IIIβ Regulates the Transport of Ceramide between the Endoplasmic Reticulum and Golgi

The recently identified ceramide transfer protein, CERT, is responsible for the bulk of ceramide transport from the endoplasmic reticulum (ER) to the Golgi. CERT has a C-terminal START domain for ceramide binding and an N-terminal pleck-strin homology domain that binds phosphatidylinositol 4-phosphate suggesting that phosphatidylinositol (PI) 4-kinases are involved in the regulation of CERT-mediated ceramide transport. In the present study fluorescent analogues were used to follow the ER to Golgi transport of ceramide to determine which of the four mammalian PI 4-kinases are involved in this process. Overexpression of pleckstrin homology domains that bind phosphatidylinositol 4-phosphate strongly inhibited the transport of C5-BODIPY-ceramide to the Golgi. A newly identified PI 3-kinase inhibitor, PIK93 that selectively inhibits the type III PI 4-kinase beta enzyme, and small interfering RNA-mediated down-regulation of the individual PI 4-kinase enzymes, revealed that PI 4-kinase beta has a dominant role in ceramide transport between the ER and Golgi. Accordingly, inhibition of PI 4-kinase III beta either by wortmannin or PIK93 inhibited the conversion of [3H]serine-labeled endogenous ceramide to sphingomyelin. Therefore, PI 4-kinase beta is a key enzyme in the control of spingomyelin synthesis by controlling the flow of ceramide from the ER to the Golgi compartment.

335 Type II phosphatidylinositol 4-kinase beta expression reverts epidermal growth factors-stimulated invasiveness of hepatocarcinoma cells

335 Type II phosphatidylinositol 4-kinase beta expression reverts epidermal growth factors-stimulated invasiveness of hepatocarcinoma cells

Parallel Activation of Phosphatidylinositol 4-Kinase and Phospholipase C by the Extracellular Calcium-sensing Receptor

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that regulates physiological processes including Ca(2+) metabolism, Na(+), Cl(-), K(+), and H(2)0 balance, and the growth of some epithelial cells through diverse signaling pathways. Although many effects of CaR are mediated by the heterotrimeric G proteins Galpha(q) and Galpha(i), not all signaling pathways regulated by CaR have been identified. We used human embryonic kidney (HEK)-293 cells that stably express human CaR to study the regulation of inositol lipid metabolism by CaR. The nonfunctional mutant CaR(R796W) was used as a negative control. We found that CaR regulates phosphatidylinositol (PI) 4-kinase, the first step in inositol lipid biosynthesis. In cells pretreated with to inhibit phospholipase C activation and to block the degradation of PI 4,5-bisphosphate to form [(3)H]inositol trisphosphate (IP(3)), CaR stimulated the accumulation of [(3)H]PI monophosphate (PIP). Additionally, wortmannin, an inhibitor of both PI 3-kinase and type III PI 4-kinase, blocked CaR-stimulated accumulation of [(3)H]PIP and inhibited [(3)H]IP(3) production. CaR-stimulated inositol lipid synthesis was attributable to PI 4-kinase and not PI 3-kinase because CaR did not activate Akt, a downstream target of PI 3-kinase. CaR associates with PI 4-kinase based on the findings that CaR and the 110-kDa PI 4-kinase beta can be co-immunoprecipitated with antibodies against either CaR or PI 4-kinase. The PI-4 kinase in co-immunoprecipitates with anti-CaR antibody was activated in Ca(2+)-stimulated HEK-293 cells, which stably express the wild type CaR. Pertussis toxin did not affect the formation of [(3)H]IP(3) or the rise in intracellular Ca(2+) (Handlogten, M. E., Huang, C. F., Shiraishi, N., Awata, H., and Miller, R. T. (2001) J. Biol. Chem. 276, 13941-13948). RGS4, an accelerator of GTPase activity of members of the Galpha(i) and Galpha(q) families, attenuated the CaR-stimulated PLC activation and IP(3) accumulation, which is mediated by Galpha(q), but did not inhibit CaR-stimulated [(3)H]PIP formation. In HEK-293 cells, which express wild type CaR, Rho was enriched in immune complexes co-immunoprecipitated with the anti-CaR antibody. C(3) toxin, an inhibitor of Rho, also inhibited the CaR-stimulated [(3)H]IP(3) production but did not lead to CaR-stimulated [(3)H]PIP formation, reflecting inhibition of PI 4-kinase. Taken together, our data demonstrate that CaR stimulates PI 4-kinase, the first step in inositol lipid biosynthesis conversion of PI to PI 4-P by Rho-dependent and Galpha(q)- and Galpha(i)-independent pathways.

Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells.

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue, frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.

Alterations in exocytosis induced by neuronal Ca2+ sensor-1 in bovine chromaffin cells.

A variety of Ca2+ binding proteins are known to play an integral role in catecholamine release from synapses as well as secretory cells, such as chromaffin cells. The Drosophila protein frequenin and its mammalian homolog neuronal Ca2+ sensor-1 (NCS-1) belong to a family of Ca2+ sensors with EF hands that bind Ca2+ and then interact with other proteins. Frequenin/NCS-1 has been shown to enhance exocytotic activity in addition to altering Ca2+ channel regulation. To better understand how NCS-1 regulates stimulus-secretion coupling, bovine chromaffin cells were infected with Semliki Forest virus (SFV) vectors containing the rat NCS-1 gene. Cells were studied in the perforated whole-cell patch-clamp configuration. Membrane capacitance was monitored as an indicator of exocytosis-endocytosis. Exocytosis elicited by membrane depolarization was not significantly different between cells infected with SFV expressing green fluorescent protein (GFP) or GFP plus NCS-1, except that the overexpression of NCS-1 resulted in a faster rundown in exocytosis. When cells were stimulated with histamine, NCS-1 overexpression led to higher exocytosis, as well as [Ca2+]i elevation. Immunocytochemistry showed a similar distribution of NCS-1 and phosphatidylinositol 4-kinase beta (PI4Kbeta). NCS-1 and PI4Kbeta coimmunoprecipitate, opening up the possibility that the two proteins directly interact. These results suggest that NCS-1 may regulate cellular activity through the modulation of the phosphatidylinositol signaling pathway.

Axonal transport of neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta in the adult rat sciatic nerve.

Neuronal calcium sensor-1 (NCS-1) and its putative substrate phosphatidylinositol 4-kinase beta (PtdIns 4-kinase beta) both indirectly regulate synaptic vesicle exocytosis and are located in DRG neurites. In this study we have tested whether NCS-1 and PtdIns 4-kinase beta are transported in axons using the analysis of double ligation approach in the adult rat sciatic nerve. We show that NCS-1 accumulates on both the distal and proximal side of the nerve ligation indicating that this protein undergoes bidirectional transport in axons. In contrast, PtdIns 4-kinase beta accumulated on the distal side which suggests that it undergoes retrograde axonal transport and unlike NCS-1 was also present in non-neuronal cells.