Therefore, phosphorylation of Thr induces significant conformational changes in the cytoplasmic region of APP Figure 3 that affect its interaction with FE65, a neuronal adaptor protein [ 10 ].
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- Lipid rafts.
The usual procedure to explore the function of a protein phosphorylation site is to mimic the phosphorylation state by amino acid substitutions of Asp or Glu for the appropriate Thr and Ser residues. However, this strategy may not be suitable in the case of APP phosphorylation, as the substitution of Asp for Thr did not alter the carboxyl terminal helix state as remarkably as phosphorylation of Thr Figure 3A.
By contrast, substitution of Thr with Ala in APP has been found to mimic effectively the nonphosphorylated state in the helix structure of the APP cytoplasmic domain.
Figure 3B presents a schematic illustration of the Thrdependent conformational changes. Therefore, to reveal the role of APP phosphorylation at Thr, careful analysis for the phosphorylation state of both APP and the APP metabolic fragments in the brain are described here. The substitution of Asp for Thr did not alter the carboxyl terminal helix state as remarkably as phosphorylation of Thr Dynamic and highly ordered membrane microdomains, termed lipid rafts, are rich in cholesterol and sphingolipids such as seramide, gangliosides, glycerophospholipids, and sterols.
The average diameter of lipid rafts has been estimated at 50 nm. However, several classes of lipid rafts that vary in size and duration can exist in a cell [ 11 ]. Lipid rafts are formed in the Golgi and transported to the plasma membrane [ 12 ], where they serve as platforms for cell signaling, pathogen entry, cell adhesion, and protein sorting.
Lipid rafts are biochemically defined as the detergent-resistant membrane DRM fraction [ 12 ]. Taken together, lipid raft localization of secretases involved in amyloidogenic APP cleavage is regulated by their post-translational modification. However, the factors that determine lipid raft localization of APP remain unclear. The molecular mechanisms underlying the suppression of APP amyloidogenic metabolism by X11 and X11L have been addressed in a recent analysis. The arrows indicate translocation direction of APP.
The Dysfunction of X11s in aged neurons may thus contribute to sporadic AD etiology. Alteration in the lipid composition of membranes may enlarge lipid raft areas or increase the number of lipid rafts, which could also enhance APP translocation to DRMs. They also describe techniques for the isolation of lipid rafts, the analysis of the lipid and protein components of lipid rafts, the imaging of lipid rafts in living cells, and the analysis of signal transduction in lipid rafts.
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Life Sciences Cell Biology. Free Preview. Buy eBook. Buy Hardcover. We found that FRS2 was expressed at each of the stages of development examined and that in all stages FRS2 was specifically localized to the lipid raft fractions Fig. Therefore, we have used the mobility shift as an indicator of FRS2 serine-threonine levels. FRS2 was only detected in lipid raft fractions and not in the Triton Xsoluble fraction of the cells Fig. Characterization of FRS2 serine-threonine and tyrosine phosphorylation-dependent signaling in lipid rafts.
Analysis of Grb2 levels showed that Grb2 was present in the soluble fractions but not in the raft fractions and was recruited to lipid rafts upon FGF2 stimulation. The recruitment of Grb2 to lipid rafts was enhanced by treatment with PP2 or U Caveolin levels confirm equal loading of lipid raft fractions. An analysis of Western blots showing phosphotyrosine levels in lipid rafts revealed that FGF2 treatment induced tyrosine phosphorylation of FRS2, which was not blocked by pretreatment of the cells with PP2, Bis I, or U inhibitors that blocked serine-threonine phosphorylation of FRS2.
Rather, under these conditions, tyrosine phosphorylation of FRS2 appeared to be enhanced Fig. These results suggest that serine-threonine phosphorylation of FRS2 is not required for tyrosine phosphorylation of FRS2 to occur and that blocking the serine-threonine phosphorylation of FRS2 can lead to enhanced tyrosine phosphorylation. Because FRS2 was associated exclusively with lipid rafts, we examined the level of Grb2 recruitment to lipid rafts after cell stimulation. Grb2 was present in the Triton Xsoluble cell fraction under all of the conditions examined Fig.
Under control conditions, Grb2 was either not present in lipid rafts or at extremely low levels. As was observed for tyrosine phosphorylation of FRS2, it appeared that Grb2 recruitment to lipid rafts was actually enhanced under conditions that inhibit serine-threonine phosphorylation of FRS2 Fig. These data suggest that serine-threonine phosphorylation of FRS2 is neither sufficient nor necessary for the recruitment of Grb2 to lipid rafts; however, enhancing tyrosine phosphorylation of FRS2 recruits more Grb2 to lipid rafts.
Equal loading of the lipid raft fractions was confirmed by Western blotting for caveolin protein levels Fig. We have already shown that Grb2 is recruited to tyrosine-phosphorylated FRS2 in lipid rafts in the absence of FRS2 serine-threonine phosphorylation. As observed previously, neither PP2 nor Bis I inhibited the tyrosine phosphorylation of FRS2; however, there was an increase in the tyrosine phosphorylated FRS2 seen at 75 kDa that corresponds to the accumulation of FRS2 in the non-serine-threonine-phosphorylated form Fig.
B , whole cell lysates treated the same as above were examined for MAPK activation. Whole cell lysates from cells treated with the same conditions were used to determine the phosphorylation levels of MAPK. When the cells were pretreated with the MEK inhibitor U, the FGF2-induced cell rounding was completely blocked and the cells remained in a flattened adherent morphology Fig.
Plasma membrane microdomains: organisation, function and trafficking
A , FGF2 treatment induced the rounding of the cells, whereas TPA and thymeleatoxin only had a slight effect on cell rounding. In summary, we have shown that FRS2 is a lipid raft-associated protein, both in the cell culture model used here and also in vivo. We have also shown that FGF2 signaling through FRS2 involves multiple pathways that appear to regulate distinct phenotypic responses in the cell. FRS2 was initially described as an adaptor protein involved in FGF signaling that was modified at the N terminus with the addition of a myristoylation moiety 7.
The acyl modification functions to target FRS2 to the membrane, and several reports 7 , 40 have shown that FRS2 is indeed localized to the membrane portion of fractionated cells and that membrane association is required for efficient signaling to occur through FRS2. Here, we have shown that FRS2 is not only targeted to the plasma membrane but that it is localized specifically to lipid rafts within the membrane, both in cultured LAN-1 cells and also in lipid rafts isolated from various stages of developing mouse brain.
Many signaling molecules have been reported to be present within lipid rafts, and membrane microdomains are thought to act as sights for regulated formation of signal transduction complexes that influence downstream signaling from a variety of membrane receptors The localization of FRS2 to lipid rafts is probably important in regulating its interaction with other molecules such as bringing FRS2 into close proximity with FGFR to enable the association of the two proteins that has been demonstrated to occur between the juxtamembrane region of FGFR and the phosphotyrosine binding domain of FRS2 FRS2 is known to be expressed in the developing nervous system Our observation that FRS2 is also localized to lipid rafts in developing mouse brain provides evidence that this localization may be functionally important for efficient signaling through FRS2 in vivo during neural development.
We have been unable to determine whether any of the FGFRs are localized to lipid rafts 35 , possibly because of the fact that the receptors are extracted from the lipid raft fraction under the conditions used as has been noted for the epidermal growth factor receptor FRS2 contains several tyrosine residues that have been shown to be phosphorylated in response to growth factor signaling 7 , 8.
The tyrosine phosphorylation of FRS2 is important in signal propagation and has been shown to be required for association with Grb2 and downstream activation of the MAPK pathway 40 , 42 as well as in the recruitment of several other signaling molecules such as Cbl 45 and phosphatidylinositol 3-kinase However, it has also been shown that the majority of phosphorylation on FRS2 is on serine and threonine rather than tyrosine 7 , Previous reports of the molecular weight of FRS2 have varied somewhat, possibly because of the change in electrophoretic mobility that is associated with serine-threonine kinase activity toward FRS2 In this report, we have examined the tyrosine and serine-threonine levels of FRS2 under various conditions to determine the effect they have on FRS2 signaling within lipid rafts.
PKC can associate with Src family kinases through adaptor proteins, and tyrosine phosphorylation of PKC has been implicated in the modulation of downstream signaling 48 , During the preparation of this paper, it has been demonstrated that the activation of MAPK results in serine phosphorylation of FRS2, which acts to negatively regulate the tyrosine phosphorylation-dependent signaling through FRS2 We also observed that the inhibition of serine-threonine phosphorylation of FRS2 leads to increased tyrosine phosphorylation levels of FRS2 as well as a correlating increase in the level of Grb2 recruited to lipid rafts that suggests a negative regulation of FRS2 phosphotyrosine by serine-threonine phosphorylation.
Although Lax et al. A model showing FRS2 signaling pathways is shown in Fig. Tyrosine phosphorylation of FRS2 Y , possibly through the action of the tyrosine kinase receptor itself, leads to activation of MAPK through multiple pathways. In addition to FGF, a diverse array of signaling molecules has been shown to utilize lipid rafts for various aspects of their signaling mechanisms We have previously shown that the glycosylphosphatidylinositol-anchored ephrins are also able to induce a compartmentalized signaling response within the lipid rafts of cells including neural cells.
As is the case for FGF, ephrin signaling induces the phosphorylation of a p75—80 phosphoprotein 35 ; however, this protein is not FRS2. In summary, we have shown that the FGF-responsive adaptor protein FRS2 is localized exclusively to lipid rafts within the cell membrane and that this localization is probably an important factor in the regulation of signaling via FRS2 through the regulation of interacting signaling molecules that either regulate the phosphorylation of FRS2 or are responsive to FRS2 phosphorylation levels.
We thank the members of the laboratory for their constructive comments and discussions during the course of this work.
Membrane Microdomain Signaling: Lipid Rafts in Biology and Medicine - Google Libros
We also thank Dr. Alice Davy for valuable input during the initial stages of this work. The costs of publication of this article were defrayed in part by the payment of page charges. Section solely to indicate this fact. To whom correspondence should be addressed: Depts. Davy, M. Ridyard, and S.
Robbins, unpublished data. You'll be in good company. Journal of Lipid Research. Previous Section Next Section. Immunoprecipitation and Western Blotting Cells were rinsed once with phosphate-buffered saline, pH 7.