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First published online 12 December 2006
doi: 10.1242/jcs.03315

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Sorting of Fas ligand to secretory lysosomes is regulated by mono-ubiquitylation and phosphorylation

Elisabetta Zuccato^1,*, Emma J. Blott^1,*, Oliver Holt¹, Sara Sigismund², Michael Shaw¹, Giovanna Bossi¹ and Gillian M. Griffiths^1,

¹ Sir William Dunn School of Pathology, South Parks Rd, Oxford, OX1 3RE, UK
² IFOM, The FIRC Institute for Molecular Oncology, Via Adamello 16, 20139 Milano, Italy

View larger version (44K): [in this window] [in a new window]   Fig. 1. Fgr binds FasL proline-rich domain (PRD) residues 41-60. (A) Amino acid sequences of the cytoplasmic tails of WT, -Pro, KKR and 3Y FasL. All constructs are tagged with GFP at the amino terminus and have wild-type transmembrane and extracellular domains at the carboxy-terminus. Amino acid numbering is shown with the PRD shaded and asterisks marking the positions of tyrosine and lysine residues. Amino acid substitutions are underlined for each construct and the deleted region in -Pro is shown. Please note that Figure 2a in a related paper (Blott et al. 2001) contains a typographical error, showing the sequence of C32 in FasL cytoplasmic tail as L. The correct sequence is shown here (A), and all constructs used in both studies corresponded to this sequence. (B) Western blots of pull downs using Fgr-SH3-GST incubated with cell lysates from untransfected RBL (-) or RBL expressing wild-type (WT) or mutant FasL GFP-tagged constructs lacking the PRD (-Pro), with K71, K72 and R73 all mutated to glutamic acid (KKR) or Y7, Y9, Y13 all mutated to alanine (3Y). Controls of the total lysate used in each pull down are shown. All blots were probed with anti-FasL, G247-4. Molecular mass markers are shown (kDa). (C) Different concentrations [shown above peaks (µM)] of monomeric Fgr-SH3 were passed over a random peptide (solid line), peptide 1-20 (light dotted line) and peptide 61-80 (bold dotted line) immobilised on a BIAcoreTM chip at 37°C. Protein injection points are indicated by horizontal bars. (D) Different concentrations [shown above peaks (µM)] of monomeric Fgr-SH3 were passed over peptides corresponding to amino acids 1-20 (dotted line) and 41-60 (bold line) immobilised on a BIAcoreTM chip at 37°C. Protein injection points are indicated by horizontal bars. (E) Specific equilibrium binding values were plotted (squares) and the KD calculated by nonlinear curve fitting (line) and Scatchard analysis (see inset graph).

View larger version (65K): [in this window] [in a new window]   Fig. 2. FasL interacts with members of the Src family of tyrosine kinases. (A) GST alone or GST-fusion proteins of the SH3 domains of Lyn, Lck, Fyn or Fgr were used to pull-down FasL from cells lysates of RBL expressing FasL-GFP. The western blot was probed with anti-FasL, G247-4. Molecular mass markers are shown (kDa) and the arrow indicates the position of FasL-GFP. The 40 kDa band is a degradation product. (B) Agarose gel separation of RT-PCR products made with primers specific for rat Fgr, Fyn, Lyn and Lck mRNA isolated from RBL, YT and a mixed lymphocyte reaction (MLR) containing activated CD4+ and CD8+ T lymphocytes. Products were verified by sequencing. DNA ladder markers are shown on the left.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Overexpression of Fgr, but not Lck, leads to mis-sorting of FasL. (A-F,J,K) Confocal images of transiently transfected RBL expressing FasL-GFP (A,B), Fgr-GFP (C-E), Fgr-YFP (F) and Lck-GFP (J,K) or untagged FasL (E,K) and stained with antibodies against Lgp120 (A,C), EEA1 (B,D,F,J) or FasL (E,K). Scale bars represent 10 µm (C,D,E,J,K) or 25 µm (A,B,F). (G-I) FACS plots showing cell surface expression of FasL (G,I) or CD63 in RBL expressing equal levels of FasL-GFP or CD63-GFP. Surface staining was measured using antibodies against the extracellular domains of FasL, or CD63 in the presence of the metalloprotease inhibitor BB3013. RBL expressing FasL-GFP alone are shown as red histograms and RBL expressing FasL-GFP and either Fgr (G,H) or Lck (J) are shown as blue histograms.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Cell surface expression of FasL mutants in RBL cells. (A) Cell surface expression of Fas ligand as a percentage of total FasL expression in cells expressing equal levels of GFP (protein). (B) FACS plot showing histograms of cell surface expression of FasL in RBL transiently transfected with WT (shaded) and 3Y (unshaded) FasL-GFP constructs, gated for equal expression of GFP and stained with the NOK-1 antibody against the extracellular domain of FasL, in the presence of metalloprotease inhibitor BB3013.

View larger version (31K): [in this window] [in a new window]   Fig. 5. FasL is phosphorylated by Fgr binding to the PRD. (A) Western blots of lysates from stably transfected RBL clones expressing equivalent levels of FasL-GFP (WT) and the GFP-tagged FasL mutants (KKR, -Pro and 3Y), immunoprecipitated (IP) with anti-FasL antibody MIKE 1 and Protein-G-Sepharose or Protein-G-Sepharose alone (-). The blot was probed with anti-phosphotyrosine (PY) antibody 4G10 (upper panel) and stripped and re-probed with anti-FasL antibody G247-4 (lower panel). Arrows denote the positions of bands corresponding to phosphorylated FasL-GFP (P-FasL-GFP), FasL-GFP and the -Pro mutant as well as IgG heavy chain (IgGHc). (B) Immunotyrosine blot of HeLa cells expressing untagged WT and -Pro FasL and Fgr-YFP (+) as indicated probed with anti-phosphotyrosine (PY) antibody 4G10 (upper panel) and stripped and re-probed with anti-FasL antibody G247-4 (lower panel). Arrows denote the positions of bands corresponding to phosphorylated Fgr (P-Fgr-YFP), FasL-GFP (P-FasL-GFP), FasL-GFP and the -Pro mutant. Positions of molecular masses are shown (kDa).

View larger version (22K): [in this window] [in a new window]   Fig. 6. FasL is monoubiquitylated in vivo. Western blots of cell lysates from stable transfectants of RBL expressing equivalent levels of WT or FasL-GFP mutants (KKR, -Pro and 3Y) or 1/10 of the immunoprecipitates both probed with anti-FasL, G247-4 and 9/10 of the same immunoprecipitate probed with anti-Ub, P4D1. Arrows denote the position of bands corresponding to ubiquitylated FasL (Ub-FasL-GFP) and IgG heavy chain (IgGHc). Note that both KKR and -Pro constructs lack K72 and K73 (Fig. 1). Positions of molecular masses are shown (kDa).

View larger version (81K): [in this window] [in a new window]   Fig. 7. Phosphorylation and ubiquitylation modifications are required for FasL sorting into MVBs. (A) Immunogold labelling of cryo-sections from stable transfectants of RBL expressing equivalent levels of WT, -Pro, KKR and 3Y GFP-tagged constructs of FasL, detected with NOK-1 labelling of the extracellular domain of FasL and Protein A-gold. Bars, 100 nm. (B) Quantification of number of gold particles in MVBs when WT, -Pro, KKR and 3Y FasL are expressed in RBL. Equivalent expression levels of FasL were confirmed by western blotting.

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