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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 9, 2006 as doi:10.1096/fj.05-4683fje. |
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,
,,,1
* Department of Nutrition and Food Science,
Department of Veterinary Integrative Biosciences,
Center for Environmental and Rural Health, and
Departments of Medical Biochemistry and Genetics, Chemistry, and Biochemistry and Biophysics, Texas A&M University, College Station, Texas USA
1Correspondence: Faculty of Nutrition, Texas A&M University, TAMU 2253, College Station, TX 77843-2253, USA. E-mail: r-chapkin{at}tamu.edu
SPECIFIC AIMS
Membrane localization of lipidated cytosolic signaling proteins is mediated by interactions between specific lipid anchors and nonpolar membrane components. The aim of this study was to investigate the effect of docosahexaenoic acid (DHA: 22:6, n-3), a membrane lipid-modifying dietary fatty acid, on the plasma membrane (PM) targeting and subcellular localization of lipidated cytosolic proteins.
PRINCIPAL FINDINGS
1. DHA inhibits PM targeting of N- and H-Ras but not K-Ras4B
Ras isoforms, N-Ras, H-Ras, and K-Ras4B (hereafter designated K-Ras), use different membrane anchors and trafficking routes for PM targeting. Upon completion of the common processing in the endoplasmic reticulum (ER), farnesylated and palmitoylated N- and H-Ras traffic to the PM via the Golgi-mediated exocytic pathway as cytoplasmic cargo in vesicular transport. K-Ras bypasses the Golgi, taking an uncharacterized route to the PM. To determine how DHA affects the subcellular localization of each Ras isoform, especially at the PM, we conducted a quantitative localization study using z-serial confocal microscopy in living cells and digital image analysis. Normal mouse colonocyte YAMC cells were incubated with a physiological dose (50 µM) of oleic acid (OA: 18:1, n-9), linoleic acid (LA: 18:2, n-6), or DHA, or left untreated for 24 h prior to and 36–48 h after transfection with green fluorescent protein (GFP) fusion constructs of wild-type Ras isoforms (GFP-ras-wt). Live cells were stained with a vital fluorescence dye, FM 4-64, to visualize the PM, and relative localization of GFP-ras-wt at the PM and Golgi was quantified as % of total cellular GFP-ras-wt using digital processing of z-serial confocal images. Subcellular distribution of GFP-Nras-wt was similar in untreated and LA-treated cells, indicating that fatty acid supplementation per se does not cause significant alterations (Fig. 1
). However, in DHA-treated cells GFP-Nras-wt fluorescence was substantially reduced at the PM and increased at endomembranes, with extensive staining at the Golgi. Quantitatively, DHA decreased PM localization of GFP-Nras-wt by 
56% and increased Golgi association by 55%. DHA inhibited PM targeting of GFP-Hras-wt in a similar manner compared with LA and OA, resulting in increased endomembrane association (Fig. 1)
. However, subcellular distribution of GFP-Kras-wt, which localized mainly at the PM and the ER, was unaffected by fatty acid treatment (Fig. 1)
.
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2. DHA differentially alters PM targeting of Ras isoforms regardless of their mutation status
In view of the critical role of constitutively active oncogenic Ras in human malignancies, we determined the ability of DHA to alter the PM localization of GFP-labeled oncogenic (G12V) H- and K-Ras. Similar to the effects on GFP-Hras-wt, DHA treatment dramatically altered the subcellular localization of GFP-Hras-G12V relative to LA and OA by inhibiting PM localization by 32% (P<0.0001) and increasing endomembrane association as reflected by an 85% increase in the Golgi fraction (P<0.02). There was no difference among fatty acid-treated cells with regard to the overall distribution pattern or PM localization of GFP-Kras-G12V, similar to GFP-Kras-wt.
3. DHA-induced alterations in Ras localization are independent of Ras signaling
To investigate the possibility that the DHA-induced selective inhibition of PM targeting of N- and H-Ras but not K-Ras may result from interference with isoform-specific functions of ectopically overexpressed Ras, we determined whether DHA affects the ability of isolated PM targeting sequences of H- and K-Ras to direct GFP to the PM. GFP-tH and GFP-tK contain PM targeting sequences of H- and K-Ras, respectively, at the C terminus of GFP, and are biologically inert, although they are lipidated and traffic like their full-length cognates. DHA significantly inhibited PM localization and increased the endomembrane pool of GFP-tH but had no effect on GFP-tK. Quantitatively, of the three H-Ras-derived GFP chimeras, the relative PM localization in DHA-treated cells (36–42%), as well as the extent of DHA-induced inhibition relative to OA and LA (by 32–40%), was similar despite differences in the functional state. We conclude that the differential effect of DHA on Ras isoform localization is independent of Ras signaling.
4. The protein trafficking route dictates DHA selectivity
We determined whether 1) DHA specifically affects Ras isoforms or instead broadly affects other lipidated cytosolic proteins as well and 2) DHA selectivity is dictated by a specific membrane anchor(s) or intracellular trafficking route. Lck and Fyn are Src-related nonreceptor tyrosine kinases that play a key role in T cell-mediated immune responses and inflammatory diseases. Unlike Ras isoforms using C-terminal membrane anchors (farnesyl+palmitoyl for N- and H-Ras; farnesyl+polybasic region for K-Ras), Lck and Fyn are N-terminally lipidated with myristate and palmitate for PM targeting. However, while Lck traffics through the exocytic pathway as do N- and H-Ras, Fyn is targeted directly and rapidly to the PM after biosynthesis. PM targeting of Fyn is insensitive to inhibitors of vesicular transport, somewhat analogous to K-Ras. DHA inhibited the PM localization of Lck-GFP by 28% (P<0.0001), increasing the intracellular pool markedly at perinuclear vesicular structures as well as the cytoplasm. However, the subcellular distribution of Fyn-GFP was identical in all fatty acid-treated cells, with the majority of fluorescence emanating from the PM. These results indicate that DHA selectively affects lipidated proteins that traffic as cytoplasmic cargo in the exocytic pathway regardless of the location (C-terminal vs. N-terminal) and composition of membrane anchors.
5. DHA does not compromise the integrity of the exocytic pathway
Because DHA inhibits PM targeting of cytoplasmic cargo of the exocytic pathway, we investigated whether DHA affects the secretory vesicular transport process by examining PM delivery of conventional exocytic cargo. Since the subcellular distribution and PM localization of GFP chimeras of vesicular stomatitis virus glycoprotein (VSVG), a transmembrane protein cargo in the exocytic pathway, were unaffected by fatty acid treatment, DHA does not appear to affect the bulk flow of secretory vesicular traffic.
6. DHA enrichment in cell membranes is essential to the inhibition of lipidated protein PM targeting
To determine whether the DHA-induced decrease in PM targeting of lipidated proteins is reversible as is the DHA level in membranes, GFP-tH-expressing cells were initially treated with DHA, then washed and subsequently incubated with OA or LA. After a 24 h washout incubation with OA or LA, the DHA-induced unique distribution of GFP-tH, characterized by a predominant endomembrane association, was dramatically changed to a more prominent PM-staining pattern similar to that observed with OA- and LA-treated cells.
To further examine the relationship between membrane lipid composition and lipidated protein PM targeting, we determined the fatty acyl composition of membrane phospholipids. DHA treatment dramatically increased phospholipid DHA content, concomitantly decreasing PM localization of GFP-tH relative to OA and LA. DHA-treated cell membranes also exhibited unique characteristics compared with both OA- and LA-enriched cell membranes, including increases in the unsaturation index and saturated fatty acid content and a decrease in arachidonic acid (20:4, n-6) content. Further incubation of DHA-treated cells with OA or LA effectively decreased phospholipid DHA content and abolished all other changes induced by DHA, and this coincided with a loss of the inhibitory effect on GFP-tH PM localization. However, neither the n-6/n-3 nor the polyunsaturated/saturated fatty acid ratios, although affected by fatty acid treatment, were related to the DHA-induced inhibition of GFP-tH PM targeting. Taken together, these results strongly indicate that DHA enrichment in cell membranes is essential to the inhibitory effect of DHA on protein PM targeting, and that some, but not all of the related changes in membrane lipid composition, are correlated with this DHA-dependent effect.
CONCLUSIONS AND SIGNIFICANCE
Lipidation-mediated protein targeting mechanisms are increasingly recognized to be vital to the regulation of biological functions as well as correct subcellular localization of signaling proteins. While the major driving force for membrane anchoring of lipidated cytosolic proteins is hydrophobic interactions between specific lipid anchors and nonpolar membrane components, little is known about the regulatory role of membrane lipid composition in lipidated protein membrane targeting.
Diet greatly influences cellular membrane composition. DHA, a dietary polyunsaturated fatty acid found in fatty fish, alters membrane structure and function via its incorporation into membrane phospholipids, and these changes have been linked to protection from heart disease, cancer, and immune disorders. We had reported that DHA and dietary fish oil decrease membrane association of Ras, a lipidated proto-oncogene product implicated in colon cancer, and concomitantly reduce Ras-dependent signaling, cell proliferation, and tumor incidence in transformed mouse colonocytes and colonic epithelium from carcinogen-injected rats. As Ras lipidation was unaffected by DHA, we hypothesized that DHA may influence Ras membrane association by modifying membrane lipid composition. Yet, what remains unknown is the effect of DHA on the subcellular distribution of Ras isoforms. Ras is present not only in the PM, the major Ras signaling platform, but also in endomembranes, including the ER and Golgi. Ras isoforms exhibit differential subcellular distribution, and not all Ras isoforms appear to be inhibited by DHA.
We show that DHA selectively alters the subcellular distribution of lipidated cytosolic proteins, including Ras isoforms, by modifying membrane lipid composition (Fig. 2
). Our present data not only corroborate the findings of the earlier in vivo diet studies but also provide a novel molecular mechanism whereby DHA influences cellular signaling. What is intriguing is the implication that the subcellular distribution of signaling proteins and hence their signaling activity can dynamically change in response to a variable diet.
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Lipidated cytosolic signaling proteins localized at the PM, such as Ras and Src-related tyrosine kinases, play critical roles in various cellular functions by relaying extracellular signals from surface receptors to downstream signaling networks. However, since overactivation of these proteins has been linked to deleterious cellular events, including oncogenesis and immune disorders, therapeutic strategies have been developed to inhibit lipidated cytosolic signaling proteins. Our unprecedented findings that membrane lipid composition can directly influence the intracellular trafficking and subcellular localization of lipidated proteins reveal an unanticipated value of dietary consumption of DHA. The dramatic influence of DHA on PM targeting of N-, H-Ras, and Lck underscores the pharmacological potential of DHA.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4683fje;
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