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W2069847632 abstract "Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca2+-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kβ with functional consequences affecting the kinase. Recombinant PI4Kβ, but not its glutathioneS-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kβ in PI4Kβ-immunoprecipitates. When expressed in COS-7 cells, PI4Kβ and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kβ primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kβ inhibited the development of this vesicular phenotype. Transfection of PI4Kβ and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [32P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca2+-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kβ also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking. Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca2+-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kβ with functional consequences affecting the kinase. Recombinant PI4Kβ, but not its glutathioneS-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kβ in PI4Kβ-immunoprecipitates. When expressed in COS-7 cells, PI4Kβ and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kβ primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kβ inhibited the development of this vesicular phenotype. Transfection of PI4Kβ and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [32P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca2+-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kβ also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking. phosphatidylinositol phosphatidylinositol 4-monophosphate 5)P2, phosphatidylinositol 4,5-bisphosphate phosphatidylinositol 4-kinase neuronal calcium sensor-1 gluthathione S-transferase polyacrylamide gel electrophoresis phosphate-buffered saline green fluorescent protein Inositol lipid kinases are increasingly recognized as regulators of membrane remodeling events whether in Golgi-related transport, endocytosis, or exocytosis (1Martin T.F. Curr. Opin. Neurobiol. 1997; 7: 331-338Crossref PubMed Scopus (84) Google Scholar). These enzymes catalyze the formation of specific inositol phospholipids, which, in turn, contributes to the membrane recruitment and stabilization of molecular complexes via interaction of inositides with protein motifs present in several regulatory proteins. Phosphatidylinositol (PI)1 4-kinases (PI4Ks) are the enzymes that catalyze the formation of PI(4)P, the main precursor of several other polyphosphoinositides with important regulatory functions. PI 4-kinase activities have been characterized some 15–20 years ago and have been classified as type II and type III enzymes, based on their catalytic properties (2Carpenter C.L. Cantley L.C. Biochemistry. 1990; 29: 11147-11156Crossref PubMed Scopus (296) Google Scholar). Molecular identification of these proteins has been relatively slow, but two forms of type III PI4Ks have been cloned from various species. These enzymes, a larger (∼200 kDa) α, and a smaller (∼100 kDa) β form, are mammalian homologues of the yeast STT4 and PIK1 gene products, respectively, and are greatly conserved in all eukaryotes, including plants (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1305) Google Scholar, 4Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Crossref PubMed Scopus (85) Google Scholar, 5Gehrmann T. Heilmayer L.G. Eur. J. Biochem. 1998; 253: 357-370Crossref PubMed Scopus (109) Google Scholar). Type II PI4K(s) have been purified from several tissues, but their molecular identity has only recently been elucidated (6Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 7Minogue S. Anderson J.S. Waugh M.G. dos Santos M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Although both Stt4 and Pik1 are bona fide PI4Ks, they appear to serve nonredundant functions in yeast. Stt4 has been shown to participate in cell wall synthesis, while Pik1 is involved in Golgi-related trafficking (8Trotter P.J. Wu W.-I. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1998; 273: 13189-13196Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Walch-Solimena C. Novick P. Nat. Cell Biol. 1999; 1: 523-525Crossref PubMed Scopus (268) Google Scholar, 10Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar). In mammalian cells, PI4Kβ is primarily localized to the Golgi (11Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), where it has been reported to be regulated by Arf proteins (12Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (449) Google Scholar). The function(s) and exact site(s) of PI4Kα action(s) still await clarification. Both of these enzymes can be inhibited in mammalian cells by micromolar concentrations of the PI 3-kinase inhibitor, wortmannin, and at these higher concentrations, wortmannin completely inhibits the resynthesis of PI(4)P and PI(4,5)P2 in agonist-stimulated cells. This observation led to the assumption that hormone-sensitive inositide pools are also synthesized by type III PI 4-kinase(s) (13Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar). Both the α and β forms are present in high concentrations in the brain and may participate in the membrane recycling events that are associated with synaptic transmission, since phosphoinositides have also been implicated in this process (14Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar).Recently, it has been reported that the yeast homologue of the Ca2+-dependent regulatory protein, NCS-1, is able to stimulate PI 4-kinase activity of yeast homogenates apparently through interaction with the Pik1 protein (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). NCS-1 was first identified in Drososphila (where it was named frequenin) as an important determinant of synaptic plasticity and a regulator of synaptic development (16Pongs O. Lindemeier J. Zhu X.R. Theil T. Engelkamp D. Krah-Jentgens I. Lambrecht H.-G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (279) Google Scholar). Homologues of NCS-1 have been found inXenopus (17Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 2000; 92: 8001-8005Crossref Scopus (86) Google Scholar) as well as in avian (18Nef S. Fiumelli H. de Castro E. Raes M.B. Nef P. J. Recept. Signal Transduct. Res. 1995; 15: 365-378Crossref PubMed Scopus (59) Google Scholar) and mammalian tissues (19Martone M.E. Edelmann V.M. Ellisman M.H. Nef P. Cell Tissue Res. 2000; 295: 395-407Crossref Scopus (71) Google Scholar), and together with recoverin/neurocalcin they form a group of small Ca2+-binding proteins distinct from calmodulin (20Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar). The present study was undertaken to investigate whether mammalian NCS-1 is able to interact and regulate PI4Kβ in mammalian cells. Our results indicate that the two proteins can directly interact in vitro and that NCS-1 exerts a moderate stimulatory effect on the lipid kinase activity of PI4Kβ. The present data also show that myristoylation of NCS-1 is critical for its ability to interact with PI4Kβ and that expressed NCS-1-YFP co-localizes with PI4Kβ in the Golgi and induces the appearance of multiple perinuclear vacuoles. Analysis of the synthesis of endogenous PI(4)P in permeabilized COS-7 cells indicate that although overexpression of PI4Kβ and NCS-1 has detectable effects on PI(4)P synthesis during Ca2+-activated PI(4,5)P2 hydrolysis, these effects are relatively minor compared with the amounts of the expressed proteins. These data suggest that PI4Kβ interacts with NCS-1, but this complex probably requires additional factor(s) to access the endogenous substrate PI and, hence, regulate PI(4)P synthesis in a specific cellular compartment.DISCUSSIONIn the present study we provide evidence for the association and possible regulation of PI4Kβ activity by the Ca2+-binding regulatory protein, NCS-1, in mammalian cells. This regulation has been shown previously in Saccharomyces cerevisiae based on both genetic and biochemical evidence (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). Our data using recombinant proteins indicate that PI4Kβ and NCS-1 can interact without any additional binding partner, although it cannot be ruled out that, in the intact cell, additional proteins or lipids may participate and modify the interaction between these two proteins. Our studies indicate that myristoylation of NCS-1 is critical for efficient interaction and stimulation of PI4Kβ, although in the yeast study a similar difference was not observed (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). However, in the same yeast study the myristoylation-defective mutant of yeast frequenin was found much less effective than wild type in suppressing a temperature-sensitivepik1 allele (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). In the yeast, the N-terminal lipid kinase unique domain was found to be the site of NCS-1 binding to PIK1. The activity of GST-fused PI4Kβ was not affected by NCS-1 in our studies, also indicating the involvement of N-terminal sequences on PI4Kβ in the association. Although we found that Ca2+ can further stimulate the activity of the NCS-1-PI4Kβ complex, Ca2+was not essential for the association of the two proteins, and the presence or absence of Ca2+ did not make a noticeable difference in our immunoprecipitation experiments. Similarly, Ca2+ was found not to be required for the association of the two proteins in yeast (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar).Overexpression of PI4Kβ with or without NCS-1 had little if any impact on the [32P]phosphate labeling of endogenous PI(4)P in permeabilized COS-7 cells under basal condition. However, the two proteins still exerted a small but significant effect on [32P]phosphate labeling of PI(4)P during Ca2+-induced phospholipase C activation, confirming the functional interaction between the two proteins. These results are consistent with our observation that recombinant PI4Kβ is not able to phosphorylate the endogenous PI of red blood cell membranes, 3T. Balla, unpublished results. indicating that a putative adapter molecule assists the kinase in its access to the membrane-bound substrate. These data, as well as those on the cellular localization of the two proteins (see below), also suggest that NCS-1 alone is not the adapter that determines the localization of the kinase and that NCS-1 stimulates the kinase only in a membrane subdomain, most likely related to the Golgi.Our studies on the localization of the two proteins expressed in COS-7 cells are consistent with the biochemical data showing interaction of the two proteins. Both proteins are localized to the Golgi, as are their endogenous forms as shown in a recent study (29Bourne Y. Dannenberg J. Pollmann V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). However, NCS-1-YFP is also found in certain membranes (plasma membrane, nuclear membrane, and vacuolar membranes) where the localization of PI4Kβ is less prominent or completely lacking. We found no indication that NCS-1 would change its localization in response to the Ca2+ionophore, ionomycin. This is in agreement with recent findings on the Ca2+ insensitivity of myristoylated NCS-1 binding to rat brain membranes (30McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2000; 274: 30258-30265Abstract Full Text Full Text PDF Scopus (97) Google Scholar). The most prominent effect of the overexpression of NCS-1-YFP was the formation of large perinuclear vacuoles with NCS-1 present in their membranes in the majority of the cells expressing this protein. Although these structures did not show particular enrichment in PI4Kβ, the co-expression of a catalytically inactive mutant PI4Kβ, or its catalytically inactive GFP fusion form, was able to prevent the development of this characteristic vacuolar phenotype. These data strongly argue that PI4Kβ mediates the effects of NCS-1, leading to the development of the morphological changes. Intriguingly, in a recent report Weisz et al. (21Weisz O.A. Gibson G.A. Leung S.M. Roder J. Jeromin A. J. Biol. Chem. 2000; 275: 24341-24347Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) have shown that overexpression of NCS-1 caused a defect in the apical transport of influenza hemagglutinin from the trans-Golgi network, without affecting early transport steps from the Golgi in MDCK cells. All these data together are consistent with an important role of Arf-1, NCS-1, and PI4Kβ in the Golgi (12Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (449) Google Scholar) and subsequent vesicular trafficking steps, similarly to the role of PIK1 in yeast (9Walch-Solimena C. Novick P. Nat. Cell Biol. 1999; 1: 523-525Crossref PubMed Scopus (268) Google Scholar).The tissue distribution and reported effects of NCS-1 together suggest that the physiological function(s) of this protein is to regulate neuroendocrine secretion and transmitter release (31McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). While overexpression of NCS-1 significantly enhanced purinergic stimulation of secretion in adrenal chromaffin cells, it failed to affect Ca2+-induced secretion in the same permeabilized cell preparation (30McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2000; 274: 30258-30265Abstract Full Text Full Text PDF Scopus (97) Google Scholar, 31McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). This finding indicates that despite being a Ca2+-binding protein, NCS-1 may not serve at the Ca2+-dependent final step of exocytosis. A connection between the function(s) of NCS-1 and PI4Kβ in the secretory process and/or presynaptic events is quite feasible in light of several studies, indicating the importance of inositides in the exocytic fusion event (32Martin T.F.J. Loyet K.M. Barry V.A. Kowalchik J.A. Biochem. Soc. Trans. 1997; 25: 1137-1141Crossref PubMed Scopus (33) Google Scholar) as well as in the process of neurotransmitter release (14Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Phosphoinositides, and the kinases and phosphatases that regulate their levels, are clearly emerging as critical players at many cellular processes involving membrane budding or fusion events. Therefore, while NCS-1 and PI4Kβ might act in concert in regulating Golgi-related vesicular transport steps in COS-7 cells, the association between the two proteins could affect additional membrane events in neurons or secretory cells. While the present study demonstrates that the two proteins can physically associate with functional consequences on PI4Kβ activity, it still remains to be determined in which membrane compartment they function together to control exocytosis and neurotransmitter release. Inositol lipid kinases are increasingly recognized as regulators of membrane remodeling events whether in Golgi-related transport, endocytosis, or exocytosis (1Martin T.F. Curr. Opin. Neurobiol. 1997; 7: 331-338Crossref PubMed Scopus (84) Google Scholar). These enzymes catalyze the formation of specific inositol phospholipids, which, in turn, contributes to the membrane recruitment and stabilization of molecular complexes via interaction of inositides with protein motifs present in several regulatory proteins. Phosphatidylinositol (PI)1 4-kinases (PI4Ks) are the enzymes that catalyze the formation of PI(4)P, the main precursor of several other polyphosphoinositides with important regulatory functions. PI 4-kinase activities have been characterized some 15–20 years ago and have been classified as type II and type III enzymes, based on their catalytic properties (2Carpenter C.L. Cantley L.C. Biochemistry. 1990; 29: 11147-11156Crossref PubMed Scopus (296) Google Scholar). Molecular identification of these proteins has been relatively slow, but two forms of type III PI4Ks have been cloned from various species. These enzymes, a larger (∼200 kDa) α, and a smaller (∼100 kDa) β form, are mammalian homologues of the yeast STT4 and PIK1 gene products, respectively, and are greatly conserved in all eukaryotes, including plants (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1305) Google Scholar, 4Balla T. Biochim. Biophys. Acta. 1998; 1436: 69-85Crossref PubMed Scopus (85) Google Scholar, 5Gehrmann T. Heilmayer L.G. Eur. J. Biochem. 1998; 253: 357-370Crossref PubMed Scopus (109) Google Scholar). Type II PI4K(s) have been purified from several tissues, but their molecular identity has only recently been elucidated (6Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 7Minogue S. Anderson J.S. Waugh M.G. dos Santos M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Although both Stt4 and Pik1 are bona fide PI4Ks, they appear to serve nonredundant functions in yeast. Stt4 has been shown to participate in cell wall synthesis, while Pik1 is involved in Golgi-related trafficking (8Trotter P.J. Wu W.-I. Pedretti J. Yates R. Voelker D.R. J. Biol. Chem. 1998; 273: 13189-13196Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Walch-Solimena C. Novick P. Nat. Cell Biol. 1999; 1: 523-525Crossref PubMed Scopus (268) Google Scholar, 10Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar). In mammalian cells, PI4Kβ is primarily localized to the Golgi (11Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), where it has been reported to be regulated by Arf proteins (12Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (449) Google Scholar). The function(s) and exact site(s) of PI4Kα action(s) still await clarification. Both of these enzymes can be inhibited in mammalian cells by micromolar concentrations of the PI 3-kinase inhibitor, wortmannin, and at these higher concentrations, wortmannin completely inhibits the resynthesis of PI(4)P and PI(4,5)P2 in agonist-stimulated cells. This observation led to the assumption that hormone-sensitive inositide pools are also synthesized by type III PI 4-kinase(s) (13Nakanishi S. Catt K.J. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar). Both the α and β forms are present in high concentrations in the brain and may participate in the membrane recycling events that are associated with synaptic transmission, since phosphoinositides have also been implicated in this process (14Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Recently, it has been reported that the yeast homologue of the Ca2+-dependent regulatory protein, NCS-1, is able to stimulate PI 4-kinase activity of yeast homogenates apparently through interaction with the Pik1 protein (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). NCS-1 was first identified in Drososphila (where it was named frequenin) as an important determinant of synaptic plasticity and a regulator of synaptic development (16Pongs O. Lindemeier J. Zhu X.R. Theil T. Engelkamp D. Krah-Jentgens I. Lambrecht H.-G. Koch K.W. Schwemer J. Rivosecchi R. Mallart A. Galceran J. Canal I. Barbas J.A. Ferrus A. Neuron. 1993; 11: 15-28Abstract Full Text PDF PubMed Scopus (279) Google Scholar). Homologues of NCS-1 have been found inXenopus (17Olafsson P. Wang T. Lu B. Proc. Natl. Acad. Sci. U. S. A. 2000; 92: 8001-8005Crossref Scopus (86) Google Scholar) as well as in avian (18Nef S. Fiumelli H. de Castro E. Raes M.B. Nef P. J. Recept. Signal Transduct. Res. 1995; 15: 365-378Crossref PubMed Scopus (59) Google Scholar) and mammalian tissues (19Martone M.E. Edelmann V.M. Ellisman M.H. Nef P. Cell Tissue Res. 2000; 295: 395-407Crossref Scopus (71) Google Scholar), and together with recoverin/neurocalcin they form a group of small Ca2+-binding proteins distinct from calmodulin (20Burgoyne R.D. Weiss J.L. Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar). The present study was undertaken to investigate whether mammalian NCS-1 is able to interact and regulate PI4Kβ in mammalian cells. Our results indicate that the two proteins can directly interact in vitro and that NCS-1 exerts a moderate stimulatory effect on the lipid kinase activity of PI4Kβ. The present data also show that myristoylation of NCS-1 is critical for its ability to interact with PI4Kβ and that expressed NCS-1-YFP co-localizes with PI4Kβ in the Golgi and induces the appearance of multiple perinuclear vacuoles. Analysis of the synthesis of endogenous PI(4)P in permeabilized COS-7 cells indicate that although overexpression of PI4Kβ and NCS-1 has detectable effects on PI(4)P synthesis during Ca2+-activated PI(4,5)P2 hydrolysis, these effects are relatively minor compared with the amounts of the expressed proteins. These data suggest that PI4Kβ interacts with NCS-1, but this complex probably requires additional factor(s) to access the endogenous substrate PI and, hence, regulate PI(4)P synthesis in a specific cellular compartment. DISCUSSIONIn the present study we provide evidence for the association and possible regulation of PI4Kβ activity by the Ca2+-binding regulatory protein, NCS-1, in mammalian cells. This regulation has been shown previously in Saccharomyces cerevisiae based on both genetic and biochemical evidence (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). Our data using recombinant proteins indicate that PI4Kβ and NCS-1 can interact without any additional binding partner, although it cannot be ruled out that, in the intact cell, additional proteins or lipids may participate and modify the interaction between these two proteins. Our studies indicate that myristoylation of NCS-1 is critical for efficient interaction and stimulation of PI4Kβ, although in the yeast study a similar difference was not observed (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). However, in the same yeast study the myristoylation-defective mutant of yeast frequenin was found much less effective than wild type in suppressing a temperature-sensitivepik1 allele (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). In the yeast, the N-terminal lipid kinase unique domain was found to be the site of NCS-1 binding to PIK1. The activity of GST-fused PI4Kβ was not affected by NCS-1 in our studies, also indicating the involvement of N-terminal sequences on PI4Kβ in the association. Although we found that Ca2+ can further stimulate the activity of the NCS-1-PI4Kβ complex, Ca2+was not essential for the association of the two proteins, and the presence or absence of Ca2+ did not make a noticeable difference in our immunoprecipitation experiments. Similarly, Ca2+ was found not to be required for the association of the two proteins in yeast (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar).Overexpression of PI4Kβ with or without NCS-1 had little if any impact on the [32P]phosphate labeling of endogenous PI(4)P in permeabilized COS-7 cells under basal condition. However, the two proteins still exerted a small but significant effect on [32P]phosphate labeling of PI(4)P during Ca2+-induced phospholipase C activation, confirming the functional interaction between the two proteins. These results are consistent with our observation that recombinant PI4Kβ is not able to phosphorylate the endogenous PI of red blood cell membranes, 3T. Balla, unpublished results. indicating that a putative adapter molecule assists the kinase in its access to the membrane-bound substrate. These data, as well as those on the cellular localization of the two proteins (see below), also suggest that NCS-1 alone is not the adapter that determines the localization of the kinase and that NCS-1 stimulates the kinase only in a membrane subdomain, most likely related to the Golgi.Our studies on the localization of the two proteins expressed in COS-7 cells are consistent with the biochemical data showing interaction of the two proteins. Both proteins are localized to the Golgi, as are their endogenous forms as shown in a recent study (29Bourne Y. Dannenberg J. Pollmann V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). However, NCS-1-YFP is also found in certain membranes (plasma membrane, nuclear membrane, and vacuolar membranes) where the localization of PI4Kβ is less prominent or completely lacking. We found no indication that NCS-1 would change its localization in response to the Ca2+ionophore, ionomycin. This is in agreement with recent findings on the Ca2+ insensitivity of myristoylated NCS-1 binding to rat brain membranes (30McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2000; 274: 30258-30265Abstract Full Text Full Text PDF Scopus (97) Google Scholar). The most prominent effect of the overexpression of NCS-1-YFP was the formation of large perinuclear vacuoles with NCS-1 present in their membranes in the majority of the cells expressing this protein. Although these structures did not show particular enrichment in PI4Kβ, the co-expression of a catalytically inactive mutant PI4Kβ, or its catalytically inactive GFP fusion form, was able to prevent the development of this characteristic vacuolar phenotype. These data strongly argue that PI4Kβ mediates the effects of NCS-1, leading to the development of the morphological changes. Intriguingly, in a recent report Weisz et al. (21Weisz O.A. Gibson G.A. Leung S.M. Roder J. Jeromin A. J. Biol. Chem. 2000; 275: 24341-24347Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) have shown that overexpression of NCS-1 caused a defect in the apical transport of influenza hemagglutinin from the trans-Golgi network, without affecting early transport steps from the Golgi in MDCK cells. All these data together are consistent with an important role of Arf-1, NCS-1, and PI4Kβ in the Golgi (12Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (449) Google Scholar) and subsequent vesicular trafficking steps, similarly to the role of PIK1 in yeast (9Walch-Solimena C. Novick P. Nat. Cell Biol. 1999; 1: 523-525Crossref PubMed Scopus (268) Google Scholar).The tissue distribution and reported effects of NCS-1 together suggest that the physiological function(s) of this protein is to regulate neuroendocrine secretion and transmitter release (31McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). While overexpression of NCS-1 significantly enhanced purinergic stimulation of secretion in adrenal chromaffin cells, it failed to affect Ca2+-induced secretion in the same permeabilized cell preparation (30McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2000; 274: 30258-30265Abstract Full Text Full Text PDF Scopus (97) Google Scholar, 31McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). This finding indicates that despite being a Ca2+-binding protein, NCS-1 may not serve at the Ca2+-dependent final step of exocytosis. A connection between the function(s) of NCS-1 and PI4Kβ in the secretory process and/or presynaptic events is quite feasible in light of several studies, indicating the importance of inositides in the exocytic fusion event (32Martin T.F.J. Loyet K.M. Barry V.A. Kowalchik J.A. Biochem. Soc. Trans. 1997; 25: 1137-1141Crossref PubMed Scopus (33) Google Scholar) as well as in the process of neurotransmitter release (14Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Phosphoinositides, and the kinases and phosphatases that regulate their levels, are clearly emerging as critical players at many cellular processes involving membrane budding or fusion events. Therefore, while NCS-1 and PI4Kβ might act in concert in regulating Golgi-related vesicular transport steps in COS-7 cells, the association between the two proteins could affect additional membrane events in neurons or secretory cells. While the present study demonstrates that the two proteins can physically associate with functional consequences on PI4Kβ activity, it still remains to be determined in which membrane compartment they function together to control exocytosis and neurotransmitter release. In the present study we provide evidence for the association and possible regulation of PI4Kβ activity by the Ca2+-binding regulatory protein, NCS-1, in mammalian cells. This regulation has been shown previously in Saccharomyces cerevisiae based on both genetic and biochemical evidence (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). Our data using recombinant proteins indicate that PI4Kβ and NCS-1 can interact without any additional binding partner, although it cannot be ruled out that, in the intact cell, additional proteins or lipids may participate and modify the interaction between these two proteins. Our studies indicate that myristoylation of NCS-1 is critical for efficient interaction and stimulation of PI4Kβ, although in the yeast study a similar difference was not observed (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). However, in the same yeast study the myristoylation-defective mutant of yeast frequenin was found much less effective than wild type in suppressing a temperature-sensitivepik1 allele (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). In the yeast, the N-terminal lipid kinase unique domain was found to be the site of NCS-1 binding to PIK1. The activity of GST-fused PI4Kβ was not affected by NCS-1 in our studies, also indicating the involvement of N-terminal sequences on PI4Kβ in the association. Although we found that Ca2+ can further stimulate the activity of the NCS-1-PI4Kβ complex, Ca2+was not essential for the association of the two proteins, and the presence or absence of Ca2+ did not make a noticeable difference in our immunoprecipitation experiments. Similarly, Ca2+ was found not to be required for the association of the two proteins in yeast (15Hendricks K.B. Wang B.Q. Schnieders E.A. Thorner J. Nat. Cell Biol. 1999; 1: 234-241Crossref PubMed Scopus (216) Google Scholar). Overexpression of PI4Kβ with or without NCS-1 had little if any impact on the [32P]phosphate labeling of endogenous PI(4)P in permeabilized COS-7 cells under basal condition. However, the two proteins still exerted a small but significant effect on [32P]phosphate labeling of PI(4)P during Ca2+-induced phospholipase C activation, confirming the functional interaction between the two proteins. These results are consistent with our observation that recombinant PI4Kβ is not able to phosphorylate the endogenous PI of red blood cell membranes, 3T. Balla, unpublished results. indicating that a putative adapter molecule assists the kinase in its access to the membrane-bound substrate. These data, as well as those on the cellular localization of the two proteins (see below), also suggest that NCS-1 alone is not the adapter that determines the localization of the kinase and that NCS-1 stimulates the kinase only in a membrane subdomain, most likely related to the Golgi. Our studies on the localization of the two proteins expressed in COS-7 cells are consistent with the biochemical data showing interaction of the two proteins. Both proteins are localized to the Golgi, as are their endogenous forms as shown in a recent study (29Bourne Y. Dannenberg J. Pollmann V. Marchot P. Pongs O. J. Biol. Chem. 2001; 276: 11949-11955Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). However, NCS-1-YFP is also found in certain membranes (plasma membrane, nuclear membrane, and vacuolar membranes) where the localization of PI4Kβ is less prominent or completely lacking. We found no indication that NCS-1 would change its localization in response to the Ca2+ionophore, ionomycin. This is in agreement with recent findings on the Ca2+ insensitivity of myristoylated NCS-1 binding to rat brain membranes (30McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2000; 274: 30258-30265Abstract Full Text Full Text PDF Scopus (97) Google Scholar). The most prominent effect of the overexpression of NCS-1-YFP was the formation of large perinuclear vacuoles with NCS-1 present in their membranes in the majority of the cells expressing this protein. Although these structures did not show particular enrichment in PI4Kβ, the co-expression of a catalytically inactive mutant PI4Kβ, or its catalytically inactive GFP fusion form, was able to prevent the development of this characteristic vacuolar phenotype. These data strongly argue that PI4Kβ mediates the effects of NCS-1, leading to the development of the morphological changes. Intriguingly, in a recent report Weisz et al. (21Weisz O.A. Gibson G.A. Leung S.M. Roder J. Jeromin A. J. Biol. Chem. 2000; 275: 24341-24347Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) have shown that overexpression of NCS-1 caused a defect in the apical transport of influenza hemagglutinin from the trans-Golgi network, without affecting early transport steps from the Golgi in MDCK cells. All these data together are consistent with an important role of Arf-1, NCS-1, and PI4Kβ in the Golgi (12Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (449) Google Scholar) and subsequent vesicular trafficking steps, similarly to the role of PIK1 in yeast (9Walch-Solimena C. Novick P. Nat. Cell Biol. 1999; 1: 523-525Crossref PubMed Scopus (268) Google Scholar). The tissue distribution and reported effects of NCS-1 together suggest that the physiological function(s) of this protein is to regulate neuroendocrine secretion and transmitter release (31McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). While overexpression of NCS-1 significantly enhanced purinergic stimulation of secretion in adrenal chromaffin cells, it failed to affect Ca2+-induced secretion in the same permeabilized cell preparation (30McFerran B.W. Weiss J.L. Burgoyne R.D. J. Biol. Chem. 2000; 274: 30258-30265Abstract Full Text Full Text PDF Scopus (97) Google Scholar, 31McFerran B.W. Graham M.E. Burgoyne R.D. J. Biol. Chem. 1998; 273: 22768-22772Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). This finding indicates that despite being a Ca2+-binding protein, NCS-1 may not serve at the Ca2+-dependent final step of exocytosis. A connection between the function(s) of NCS-1 and PI4Kβ in the secretory process and/or presynaptic events is quite feasible in light of several studies, indicating the importance of inositides in the exocytic fusion event (32Martin T.F.J. Loyet K.M. Barry V.A. Kowalchik J.A. Biochem. Soc. Trans. 1997; 25: 1137-1141Crossref PubMed Scopus (33) Google Scholar) as well as in the process of neurotransmitter release (14Cremona O. Di Paolo G. Wenk M.R. Luthi A. Kim W.T. Takei K. Daniell L. Nemoto Y. Shears S.B. Flavell R.A. McCormick D.A. De Camilli P. Cell. 1999; 99: 179-188Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar). Phosphoinositides, and the kinases and phosphatases that regulate their levels, are clearly emerging as critical players at many cellular processes involving membrane budding or fusion events. Therefore, while NCS-1 and PI4Kβ might act in concert in regulating Golgi-related vesicular transport steps in COS-7 cells, the association between the two proteins could affect additional membrane events in neurons or secretory cells. While the present study demonstrates that the two proteins can physically associate with functional consequences on PI4Kβ activity, it still remains to be determined in which membrane compartment they function together to control exocytosis and neurotransmitter release. We express our thanks to Dr. Margarida Barroso (Department of Biology, University of Virginia, Charlottesville, VA) for the myristoylated p22 and the polyclonal antibody against this protein. We are grateful to Tuula Torkkeli for help during preparation of the recombinant virus for NCS-1 production in insect cells, as well as to Pirjo Kapyla for excellent technical assistance." @default.
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W2069847632 title "Interaction of Neuronal Calcium Sensor-1 (NCS-1) with Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Affects Membrane Trafficking in COS-7 Cells" @default.
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