Skip to main content

Advertisement

SpringerLink
Log in

Palmitoylcarnitine Affects Localization of Growth Associated Protein GAP-43 in Plasma Membrane Subdomains and its Interaction with Gαo in Neuroblastoma NB-2a Cells

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

An Erratum to this article was published on 31 August 2013

Abstract

Palmitoylcarnitine was observed previously to promote differentiation of neuroblastoma NB-2a cells, and to affect protein kinase C (PKC). Palmitoylcarnitine was also observed to increase palmitoylation of several proteins, including a PKC substrate, whose expression augments during differentiation of neural cells—a growth associated protein GAP-43, known to bind phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Since palmitoylated proteins are preferentially localized in sphingolipid- and cholesterol-rich microdomains of plasma membrane, the present study has been focused on a possible effect of palmitoylcarnitine on GAP-43 localization in these microdomains. Palmitoylcarnitine treatment resulted in GAP-43 appearance in floating fractions (rafts) in sucrose gradient and increased co-localization with cholesterol and with PI(4,5)P2, although co-localization of both lipids decreased. GAP-43 disappeared from raft fraction upon treatment with 2-bromopalmitate (an inhibitor of palmitoylating enzymes) and after treatment with etomoxir (carnitine palmitoyltransferase I inhibitor). Raft localization of GAP-43 was completely abolished by treatment with methyl-β-cyclodextrin, a cholesterol binding agent, while there was no change upon sequestration of PI(4,5)P2 with neomycin. GAP-43 co-precipitated with a monomeric form of Gαo, a phenomenon diminished after palmitoylcarnitine treatment and paralleled by a decrease of Gαo in the raft fraction. These observations point to palmitoylation of GAP-43 as a mechanism leading to an increased localization of this protein in microdomains of plasma membrane rich in cholesterol, in majority different, however, from microdomains in which PI(4,5)P2 is present. This localization correlates with decreased interaction with Gαo and suppression of its activity—an important step regulating neural cell differentiation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

BSA:

Bovine serum albumin

CPT-I:

Carnitine palmitoyltransferase I

MβCD:

Methyl-β-cyclodextrin

OCTN2:

Organic cation/carnitine transporter

PBS:

Phosphate buffered saline

PI(4,5)P2 :

Phosphatidylinositol 4,5-bisphosphate

PKC:

Protein kinase C

References

  1. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572

    Article  PubMed  CAS  Google Scholar 

  2. Brown DA (2006) Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda) 21:430–439

    Article  CAS  Google Scholar 

  3. Resh MD (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta 1451:1–16

    Article  PubMed  CAS  Google Scholar 

  4. Resh MD (2004) Membrane targeting of lipid modified signal transduction proteins. Subcell Biochem 37:217–232

    Article  PubMed  CAS  Google Scholar 

  5. Aicart-Ramos C, Valero RA, Rodriguez-Crespo I (2011) Protein palmitoylation and subcellular trafficking. Biochim Biophys Acta 1808:2981–2994

    Article  PubMed  CAS  Google Scholar 

  6. Smotrys JE, Linder ME (2004) Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem 73:559–587

    Article  PubMed  CAS  Google Scholar 

  7. Skene JH (1989) Axonal growth-associated proteins. Annu Rev Neurosci 12:127–156

    Article  PubMed  CAS  Google Scholar 

  8. Skene JH, Virag I (1989) Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J Cell Biol 108:613–624

    Article  PubMed  CAS  Google Scholar 

  9. Strittmatter SM, Valenzuela D, Kennedy TE, Neer EJ, Fishman MC (1990) G0 is a major growth cone protein subject to regulation by GAP-43. Nature 344:836–841

    Article  PubMed  CAS  Google Scholar 

  10. Coggins PJ, Zwiers H (1989) Evidence for a single protein kinase C-mediated phosphorylation site in rat brain protein B-50. J Neurochem 53:1895–1901

    Article  PubMed  CAS  Google Scholar 

  11. Laux T, Fukami K, Thelen M, Golub T, Frey D, Caroni P (2000) GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism. J Cell Biol 149:1455–1472

    Article  PubMed  CAS  Google Scholar 

  12. Hope HR, Pike LJ (1996) Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains. Mol Biol Cell 7:843–851

    Article  PubMed  CAS  Google Scholar 

  13. Klopfenstein DR, Tomishige M, Stuurman N, Vale RD (2002) Role of phosphatidylinositol(4,5)bisphosphate organization in membrane transport by the Unc104 kinesin motor. Cell 109:347–358

    Article  PubMed  CAS  Google Scholar 

  14. van Rheenen J, Achame EM, Janssen H, Calafat J, Jalink K (2005) PIP2 signaling in lipid domains: a critical re-evaluation. EMBO J 24:1664–1673

    Article  PubMed  Google Scholar 

  15. Tong J, Nguyen L, Vidal A, Simon SA, Skene JH, McIntosh TJ (2008) Role of GAP-43 in sequestering phosphatidylinositol 4,5-bisphosphate to raft bilayers. Biophys J 94:125–133

    Article  PubMed  CAS  Google Scholar 

  16. Nałęcz KA, Mroczkowska JE, Berent U, Nałęcz MJ (1997) Effect of palmitoylcarnitine on the cellular differentiation, proliferation and protein kinase C activity in neuroblastoma nb-2a cells. Acta Neurobiol Exp (Wars) 57:263–274

    Google Scholar 

  17. Kerner J, Hoppel C (2000) Fatty acid import into mitochondria. Biochim Biophys Acta 1486:1–17

    Article  PubMed  CAS  Google Scholar 

  18. Lee K, Kerner J, Hoppel CL (2011) Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex. J Biol Chem 286:25655–25662

    Article  PubMed  CAS  Google Scholar 

  19. Ebert D, Haller RG, Walton ME (2003) Energy contribution of octanoate to intact rat brain metabolism measured by 13C nuclear magnetic resonance spectroscopy. J Neurosci 23:5928–5935

    PubMed  CAS  Google Scholar 

  20. Nałęcz KA, Szczepankowska D, Czeredys M, Kulikova N, Grześkiewicz S (2007) Palmitoylcarnitine regulates estrification of lipids and promotes palmitoylation of GAP-43. FEBS Lett 581:3950–3954

    Article  PubMed  Google Scholar 

  21. Tamai I, Ohashi R, Nezu J, Yabuuchi H, Oku A, Shimane M, Sai Y, Tsuji A (1998) Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2. J Biol Chem 273:20378–20382

    Article  PubMed  CAS  Google Scholar 

  22. Tamai I, Ohashi R, Nezu JI, Sai Y, Kobayashi D, Oku A, Shimane M, Tsuji A (2000) Molecular and functional characterization of organic cation/carnitine transporter family in mice. J Biol Chem 275:40064–40072

    Article  PubMed  CAS  Google Scholar 

  23. Januszewicz E, Bekisz M, Mozrzymas JW, Nałęcz KA (2010) High affinity carnitine transporters from OCTN family in neural cells. Neurochem Res 35:743–748

    Article  PubMed  CAS  Google Scholar 

  24. Shinawi M, Gruener N, Lerner A (1998) CSF levels of carnitine in children with meningitis, neurologic disorders, acute gastroenteritis, and seizure. Neurology 50:1869–1871

    Article  PubMed  CAS  Google Scholar 

  25. Szczepankowska D, Nałęcz KA (2003) Palmitoylcarnitine modulates palmitoylation of proteins: implication for differentiation of neural cells. Neurochem Res 28:645–651

    Article  PubMed  CAS  Google Scholar 

  26. Sobiesiak-Mirska J, Nałęcz MJ, Nałęcz KA (2003) Interaction of palmitoylcarnitine with protein kinase C in neuroblastoma NB-2a cells. Neurochem Int 42:45–55

    Article  PubMed  CAS  Google Scholar 

  27. Sobiesiak-Mirska J, Nałęcz KA (2006) Palmitoylcarnitine modulates interaction between protein kinase C betaII and its receptor RACK1. FEBS J 273:1300–1311

    Article  PubMed  CAS  Google Scholar 

  28. Mizgalska JA, Berent U, Mac M, Oestreicher B, De Graan PNE, Gispen WH, Nałęcz MJ, Nałęcz KA (1998) Accumulation of palmitoylcarnitine in neuroblastoma NB-2a cells affects the expression, phosphorylation and localization of B-50 protein. Neurosci Res Commun 22:73–82

    Article  CAS  Google Scholar 

  29. Milligan G, Parenti M, Magee AI (1995) The dynamic role of palmitoylation in signal transduction. Trends Biochem Sci 20:181–187

    Article  PubMed  CAS  Google Scholar 

  30. Igarashi M, Strittmatter SM, Vartanian T, Fishman MC (1993) Mediation by G proteins of signals that cause collapse of growth cones. Science 259:77–79

    Article  PubMed  CAS  Google Scholar 

  31. Sudo Y, Valenzuela D, Beck-Sickinger AG, Fishman MC, Strittmatter SM (1992) Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43. EMBO J 11:2095–2102

    PubMed  CAS  Google Scholar 

  32. Brockenbrough JS, Korc M (1987) Inhibition of epidermal growth factor binding in rat pancreatic acini by palmitoyl carnitine: evidence for Ca2+ and protein kinase C independent regulation. Cancer Res 47:1805–1810

    PubMed  CAS  Google Scholar 

  33. Lamprecht MR, Sabatini DM, Carpenter AE (2007) Cell Profiler: free, versatile software for automated biological image analysis. Biotechniques 42:71–75

    Article  PubMed  CAS  Google Scholar 

  34. Vokes MS, Carpenter AE (2008) Using CellProfiler for automatic identification and measurement of biological objects in images. Curr Protoc Mol Biol Chapter 14, Unit 14 17

  35. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  36. Miecz D, Januszewicz E, Czeredys M, Hinton BT, Berezowski V, Cecchelli R, Nałęcz KA (2008) Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood-brain barrier. J Neurochem 104:113–123

    PubMed  CAS  Google Scholar 

  37. Gray MC, Plant AL, Nicholson JM, May WE (1995) Microenzymatic fluorescence assay for serum cholesterol. Anal Biochem 224:286–292

    Article  PubMed  CAS  Google Scholar 

  38. Gorodinsky A, Harris DA (1995) Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. J Cell Biol 129:619–627

    Article  PubMed  CAS  Google Scholar 

  39. Sargiacomo M, Scherer PE, Tang Z, Kubler E, Song KS, Sanders MC, Lisanti MP (1995) Oligomeric structure of caveolin: implications for caveolae membrane organization. Proc Natl Acad Sci USA 92:9407–9411

    Article  PubMed  CAS  Google Scholar 

  40. Fukata M, Fukata Y, Adesnik H, Nicoll RA, Bredt DS (2004) Identification of PSD-95 palmitoylating enzymes. Neuron 44:987–996

    Article  PubMed  CAS  Google Scholar 

  41. Arduini A, Mancinelli G, Radatti GL, Dottori S, Molajoni F, Ramsay RR (1992) Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes. J Biol Chem 267:12673–12681

    PubMed  CAS  Google Scholar 

  42. Ramsay RR (2000) The carnitine acyltransferases: modulators of acyl-CoA-dependent reactions. Biochem Soc Trans 28:182–186

    PubMed  CAS  Google Scholar 

  43. Ramsay RR, Gandour RD, van der Leij FR (2001) Molecular enzymology of carnitine transfer and transport. Biochim Biophys Acta 1546:21–43

    Article  PubMed  CAS  Google Scholar 

  44. Kiorpes TC, Hoerr D, Ho W, Weaner LE, Inman MG, Tutwiler GF (1984) Identification of 2-tetradecylglycidyl coenzyme A as the active form of methyl 2-tetradecylglycidate (methyl palmoxirate) and its characterization as an irreversible, active site-directed inhibitor of carnitine palmitoyltransferase A in isolated rat liver mitochondria. J Biol Chem 259:9750–9755

    PubMed  CAS  Google Scholar 

  45. Kerner J, Zaluzec E, Gage D, Bieber LL (1994) Characterization of the malonyl-CoA-sensitive carnitine palmitoyltransferase (CPTo) of a rat heart mitochondrial particle. Evidence that the catalytic unit is CPTi. J Biol Chem 269:8209–8219

    PubMed  CAS  Google Scholar 

  46. Hao M, Mukherjee S, Maxfield FR (2001) Cholesterol depletion induces large scale domain segregation in living cell membranes. Proc Natl Acad Sci USA 98:13072–13077

    Article  PubMed  CAS  Google Scholar 

  47. Adkins EM, Samuvel DJ, Fog JU, Eriksen J, Jayanthi LD, Vaegter CB, Ramamoorthy S, Gether U (2007) Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Biochemistry 46:10484–10497

    Article  PubMed  CAS  Google Scholar 

  48. Wu J, McNicholas CM, Bevensee MO (2009) Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates the electrogenic Na/HCO3 cotransporter NBCe1-A expressed in Xenopus oocytes. Proc Natl Acad Sci USA 106:14150–14155

    Article  PubMed  CAS  Google Scholar 

  49. Strittmatter SM, Valenzuela D, Sudo Y, Linder ME, Fishman MC (1991) An intracellular guanine nucleotide release protein for G0. GAP-43 stimulates isolated alpha subunits by a novel mechanism. J Biol Chem 266:22465–22471

    PubMed  CAS  Google Scholar 

  50. Strittmatter SM, Valenzuela D, Vartanian T, Sudo Y, Zuber MX, Fishman MC (1991) Growth cone transduction: Go and GAP-43. J Cell Sci Suppl 15:27–33

    Article  PubMed  CAS  Google Scholar 

  51. Yang H, Qu L, Ni J, Wang M, Huang Y (2008) Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization. Mol Membr Biol 25:58–71

    Article  PubMed  Google Scholar 

  52. Carter BD, Medzihradsky F (1993) Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain. Proc Natl Acad Sci USA 90:4062–4066

    Article  PubMed  CAS  Google Scholar 

  53. Brown DA, Sihra TS (2008) Presynaptic signaling by heterotrimeric G-proteins. Handb Exp Pharmacol 184:207–260

    Article  PubMed  CAS  Google Scholar 

  54. Koenig JA, Edwardson JM, Humphrey PP (1997) Somatostatin receptors in Neuro2A neuroblastoma cells: operational characteristics. Br J Pharmacol 120:45–51

    Article  PubMed  CAS  Google Scholar 

  55. Arduini A, Denisova N, Virmani A, Avrova N, Federici G, Arrigoni-Martelli E (1994) Evidence for the involvement of carnitine-dependent long-chain acyltransferases in neuronal triglyceride and phospholipid fatty acid turnover. J Neurochem 62:1530–1538

    Article  PubMed  CAS  Google Scholar 

  56. Yang H, Wan L, Song F, Wang M, Huang Y (2009) Palmitoylation modification of Galpha o depresses its susceptibility to GAP-43 activation. Int J Biochem Cell Biol 41:1495–1501

    Article  PubMed  CAS  Google Scholar 

  57. Bates CA, Meyer RL (1996) Heterotrimeric G protein activation rapidly inhibits outgrowth of optic axons from adult and embryonic mouse, and goldfish retinal explants. Brain Res 714:65–75

    Article  PubMed  CAS  Google Scholar 

  58. Li S, Okamoto T, Chun M, Sargiacomo M, Casanova JE, Hansen SH, Nishimoto I, Lisanti MP (1995) Evidence for a regulated interaction between heterotrimeric G proteins and caveolin. J Biol Chem 270:15693–15701

    Article  PubMed  CAS  Google Scholar 

  59. Chisari M, Saini DK, Kalyanaraman V, Gautam N (2007) Shuttling of G protein subunits between the plasma membrane and intracellular membranes. J Biol Chem 282:24092–24098

    Article  PubMed  CAS  Google Scholar 

  60. Jia L, Linder ME, Blumer KJ (2011) Gi/o signaling and the palmitoyltransferase DHHC2 regulate palmitate cycling and shuttling of RGS7 family-binding protein. J Biol Chem 286:13695–13703

    Article  PubMed  CAS  Google Scholar 

  61. Tsutsumi R, Fukata Y, Noritake J, Iwanaga T, Perez F, Fukata M (2009) Identification of G protein alpha subunit-palmitoylating enzyme. Mol Cell Biol 29:435–447

    Article  PubMed  CAS  Google Scholar 

  62. Duncan JA, Gilman AG (1998) A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J Biol Chem 273:15830–15837

    Article  PubMed  CAS  Google Scholar 

  63. Tomatis VM, Trenchi A, Gomez GA, Daniotti JL (2010) Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43. PLoS ONE 5:e15045

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was financed by the statutory funds of the Nencki Institute. We would like to thank Dr. Katarzyna Piwocka from the Laboratory of Cytometry of the Nencki Institute of Experimental Biology for advice and the help in flow cytometry experiments, as well as Dr. Tytus Bernaś for assistance with confocal microscopy and help in co-localization analysis.

Author information

Authors and Affiliations

  1. Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093, Warsaw, Poland

    Karolina Tułodziecka, Magdalena Czeredys & Katarzyna A. Nałęcz

Authors
  1. Karolina Tułodziecka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Magdalena Czeredys
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katarzyna A. Nałęcz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Katarzyna A. Nałęcz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tułodziecka, K., Czeredys, M. & Nałęcz, K.A. Palmitoylcarnitine Affects Localization of Growth Associated Protein GAP-43 in Plasma Membrane Subdomains and its Interaction with Gαo in Neuroblastoma NB-2a Cells. Neurochem Res 38, 519–529 (2013). https://doi.org/10.1007/s11064-012-0944-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-012-0944-5

Keywords

Search

Navigation