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. 2009 Apr 1;122(Pt 7):912-8.
doi: 10.1242/jcs.039024. Epub 2009 Mar 3.

Endocytosis of flotillin-1 and flotillin-2 is regulated by Fyn kinase

Kirsi Riento  1 , Manfred Frick, Ingmar Schafer, Benjamin J Nichols
Affiliations

Endocytosis of flotillin-1 and flotillin-2 is regulated by Fyn kinase

Kirsi Riento et al. J Cell Sci. .

Abstract

Flotillin-1 and flotillin-2 co-assemble into plasma membrane microdomains that are involved in the endocytosis of molecules such as glycosyl phosphatidylinositol (GPI)-linked proteins. Previous studies suggest that budding of flotillin microdomains from the plasma membrane is a tightly regulated process. Here, we demonstrate that endocytosis of flotillins is regulated by the Src family kinase Fyn. The Src kinase inhibitor PP2 prevents EGF-induced flotillin internalisation, and EGF-induced internalisation does not occur in SYF cells lacking Src, Yes and Fyn. Expression of Fyn, but not Src or Yes, restores EGF-induced internalisation in SYF cells. Expression of an active form of Fyn but not other Src kinases is sufficient to induce redistribution of flotillins from the plasma membrane to late endosomes and lysosomes. Using two partial Fyn constructs that form a functional kinase upon addition of rapamycin to cells, we show that flotillin internalisation from the plasma membrane occurs shortly after Fyn activation. Tyr160 in flotillin-1 and Tyr163 in flotillin-2 are directly phosphorylated by Fyn, and mutation of these residues to phenylalanine prevents Fyn-induced flotillin internalisation. Uptake of the GPI-linked protein CD59 is reduced by expression of the phenylalanine-mutated flotillins. These data establish uptake of flotillin microdomains as a tyrosine-kinase-regulated endocytic process.

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Figures

Fig. 1.
Fig. 1.
Flotillin microdomains redistribute in response to stimulation of cells with EGF. (A) Colocalisation of endogenous flotillin-1 and flotillin-2 revealed by indirect immunofluorescence. Flotillin-1 was detected with an affinity-purified anti-flotillin-1 polyclonal antibody (Frick et al., 2007), flotillin-2 with a specific monoclonal antibody. Insets show magnified view of the boxed areas. Scale bars: 5 μm. (B) Single confocal sections of NIH3T3 cells either starved, or stimulated for 30 minutes with EGF. Flotillin-1 and flotillin-2 was detected as in A. Scale bars: 20 μm. (C) Flotillin-2 detected with a monoclonal antibody, in HeLa cells treated as shown. TIR images showing the bottom ∼100 nm of cells. EGF was added for 30 minutes. Scale bars: 20 μm. (D) Quantification of the mean fluorescence intensity of flotillin-2 in TIR images of HeLa and NIH3T3 cells treated as shown. Error bars represent s.e.m.; n>10.
Fig. 2.
Fig. 2.
Flotillin microdomains redistribute from the plasma membrane to intracellular organelles upon expression of active Fyn. (A) Indirect immunofluorescence indicating that when FynY531F-GFP is expressed in HeLa cells endogenous flotillin-1 and flotillin-2 redistribute from peripheral, membrane-associated puncta to larger perinuclear organelles. A basal confocal section is shown. Merged image is shown on right. Scale bar: 20 μm. (B) Indirect immunofluorescence indicating that when FynY531F-GFP is expressed in HeLa cells, flotillin-1 redistributes from peripheral, membrane-associated puncta to larger perinuclear organelles. The FynY531F-GFP transfected cells are indicated with a red asterisk. The image is a projection of 24 confocal sections, so that all fluorescence in the cells is represented. Scale bar: 20 μm. (C) Total internal reflection (TIR) imaging of flotillin-2 or caveolin-1 labelled by indirect immunofluorescence at the bottom ∼100 nm of HeLa cells transfected with FynY531-mCh, and adjacent untransfected cells. Scale bars: 20 μm. (D) Quantification of the amount of flotillin-2 or caveolin-1 labelled by indirect immunofluorescence in TIR images of the bottom ∼100 nm of HeLa cells transfected with the kinases indicated. All kinase constructs had mCherry fused to the C-terminus, and control cells expressed mCh alone. n=10; error bars represent s.e.m. (E) Quantification of the amount of flotillin-2 labelled by indirect immunofluorescence in TIR images of the bottom ∼100 nm of SYF cells transfected with the constructs indicated, and treated as shown. All kinase constructs had mCherry fused to the C-terminus. n=15, error bars represent s.e.m.
Fig. 3.
Fig. 3.
Use of heterobivalent crosslinking to recruit active Fyn to the plasma membrane. (A) Scheme of experimental design and composition of constructs. FKBP, FK506-binding protein; FRB, FKBP-rapamycin-binding domain. (B) Addition of rapamycin causes rapid recruitment of FKBP-FynY531F31-537-GFP from the cytoplasm to the plasma membrane in cells also expressing Fyn1-30-FRB. Confocal sections approximately 2 μm from the base of the cells are shown. Scale bars: 20 μm. (C) Rapamycin-induced recruitment of FKBP-FynY531F31-537-GFP to the plasma membrane causes visible redistribution of flotillin microdomains from the plasma membrane to intracellular organelles, in COS-7 cells pretreated with 20 μg ml–1 cycloheximide. Basal confocal sections are shown, so the recruitment of FKBP-FynY531F31-537-GFP to the plasma membrane is less readily visualised than in B. Merged images are shown on the right. Scale bars: 20 μm. (D) Quantification of the amount of flotillin-2 labelled by indirect immunofluorescence in TIR images of the bottom ∼100 nm of HeLa cells treated with rapamycin. Open circles are untransfected cells, closed circles are cells transfected with FKBP-FynY531F31-537-GFP and Fyn1-30-FRB. n>12; error bars represent s.e.m.
Fig. 4.
Fig. 4.
Total internal reflection (TIR) imaging showing apparent co-internalisation of flotillin-1-GFP and Fyn-mRFP. (A) Distribution of flotillin-1-GFP and Fyn-mRFP by TIR. Arrows highlight flotillin-1-positive puncta where Fyn is also concentrated. Time-lapse images of this cell are shown in supplementary material Movie 1. Scale bar: 10 μm. (B) Cointernalisation of flotillin-1-GFP and Fyn-mRFP. Yellow arrows highlight a punctum where flotillin-1-GFP and Fyn-mRFP colocalise (merged images in bottom row) before disappearing, presumably as a result of budding into the cell. White arrows highlight the same area of the cell after apparent budding. Times shown on individual panels refer to the budding event. Note that similar apparent budding events were also detected without visible concentration of Fyn-mRFP. See also supplementary material Movie 2.
Fig. 5.
Fig. 5.
Fyn directly phosphorylates critical tyrosine residues Y160 of flotillin-1 and Y163 of flotillin-2. (A) In vitro phosphorylation of flotillin-1 and flotillin-2 by Fyn and Src. Top panel shows Coomassie stain of different combinations of recombinant protein as shown. Note that Src and Fyn are present in the indicated lanes but in insufficient amounts to be detected with the Coomassie stain. Central panel shows an autoradiograph of the same samples after in vitro phosphorylation with 32P. Note that TRX-His-flotillin-1 has approximately the same molecular mass as Fyn, so the band at this size in the Fyn+, flotillin1&2+ lane could be due either to autophosphorylated Fyn or to phosphorylation of TRX-His-flotillin-1. In the lower panel, the same in vitro kinase reactions were subjected to immunoblotting with the phospho-specific flotillin antibodies, confirming that Fyn phosphorylates both flotillin-1 and flotillin-2. (B) Immunoprecipitation of phosphorylated flotillin-1 using phospho-specific antibody to Y160-P (PY160), at the times indicated after EGF stimulation of NIH3T3 cells. Precipitated flotillin-1 was detected on the western blot with monoclonal anti-flotillin-1.
Fig. 6.
Fig. 6.
Flotillin-1 Y160F and flotillin-2 Y163F are not internalised in response to FynY531F and reduce uptake of CD59. (A) Flotillin-1 and flotillin-2 with Y160 and Y163, respectively mutated to phenylalanines still bind normally to the opposite flotillin. Wild-type and mutant flotillin-GFP constructs were immunoprecipitated from HeLa cells using anti-GFP antibodies; control has same antibodies on lysates from untransfected cells. (B) Active Fyn is unable to translocate flotillin-1 Y160F and flotillin-2 Y163F from the plasma membrane. HeLa cells expressing GFP-tagged wild type or the phenylalanine mutant forms of both flotillins, together with FynY531F-mRFP. Scale bars: 15 μm. (C) Coexpression of flotillin-1 Y160F-GFP and flotillin-2 Y163F-GFP has a dominant negative effect on internalisation of antibodies against the GPI-linked protein CD59. Noninternalised antibody was removed by low-pH wash after 40 minutes of continuous uptake at 37°C. Scale bar: 20 μm. (D) Quantification of CD59 uptake in untransfected cells, cells expressing GFP-tagged wild-type and mutant flotillins, as shown. Mean anti-CD59 fluorescence per cell is shown. Error bars represent s.e.m.; n>40; P values are results of unpaired t-test.
Fig. 7.
Fig. 7.
Coassembly of flotillin-1 Y160F and flotillin-2 Y163F produces microdomains that are not competent for internalisation. (A) Use of cell fusion to study assembly of flotillins into microdomains and consequent endocytosis. A flotillin-1-GFP-expressing cell has fused with a cell expressing flotillin-2-YFP. Assembly of the two proteins into microdomains at the area of plasma-membrane fusion has occurred. Merged image is shown on the right. Image acquired 30 minutes after initiation of fusion reaction. Scale bar: 20 μm. (B) Endocytic structures containing flotillins and CD59 are generated in the region of cell fusion when cells expressing wild-type flotillins fuse, but not when the tyrosine mutants are expressed instead. Basal confocal sections (z=0 μm) demonstrate that microdomain assembly occurs normally in both cases; higher (z=2 μm) sections demonstrate presence or absence of CD59 and flotillin-positive endosomes (arrowed) in the region of cell fusion. Scale bars: 5 μm.
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