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

Fas ligand (FasL), a potent mediator of apoptosis expressed by CTL and NK cells, is sorted into the inner vesicles of secretory lysosomes for release via exosome-like vesicles. Previous studies identified a proline-rich domain in the cytoplasmic tail required for sorting FasL to secretory lysosomes, but the mechanisms by which this occurs have not been identified. Here we demonstrate that the PRD of FasL binds Fgr, Fyn and Lyn tyrosine kinases, leading to phosphorylation of FasL. Loss of phosphorylation reduces internalisation of FasL into multivesicular bodies. FasL is also directly mono-ubiquitylated at lysines flanking the PRD and mutation of these lysines reduces MVB localisation of FasL. Phosphorylation is not required for ubiquitylation because FasL lacking all tyrosines undergoes mono-ubiquitylation. These studies show that phosphorylation and ubiquitin signals regulate the sorting of FasL to secretory lysosomes by controlling entry into multivesicular bodies.

[1]  J. Slot,et al.  Improving structural integrity of cryosections for immunogold labeling , 1996, Histochemistry and Cell Biology.

[2]  Veronica Huber,et al.  Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles , 2002, The Journal of experimental medicine.

[3]  A. Ullrich,et al.  Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body , 1990, Cell.

[4]  R. Perlmutter,et al.  Expression of lineage‐restricted protein tyrosine kinase genes in human natural killer cells , 1991, European journal of immunology.

[5]  Giulio Superti-Furga,et al.  Dynamic Coupling between the SH2 and SH3 Domains of c-Src and Hck Underlies Their Inactivation by C-Terminal Tyrosine Phosphorylation , 2001, Cell.

[6]  L. Page,et al.  Sorting out the multiple roles of Fas ligand. , 2000, European journal of cell biology.

[7]  P. Krammer,et al.  Regulation of cell surface APO‐1/Fas (CD95) ligand expression by metalloproteases , 1995, European journal of immunology.

[8]  G. Griffiths,et al.  Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells , 1999, Nature Medicine.

[9]  S. Emr,et al.  Receptor downregulation and multivesicular-body sorting , 2002, Nature Reviews Molecular Cell Biology.

[10]  Peter J. Peters,et al.  Molecules relevant for T cell‐target cell interaction are present in cytolytic granules of human T lymphocytes , 1989, European journal of immunology.

[11]  K. Yoshino,et al.  Metalloproteinase-mediated Release of Human Fas Ligand , 1995 .

[12]  Jun Zhang,et al.  Microtubule‐Dependent Transport of Secretory Vesicles in RBL‐2H3 Cells , 2003, Traffic.

[13]  M. Martínez-Lorenzo,et al.  Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles. , 1999, Journal of immunology.

[14]  J. Kuriyan,et al.  Structures of Src-family tyrosine kinases. , 1997, Current opinion in structural biology.

[15]  S. Nagata,et al.  TCR/CD3 coupling to Fas-based cytotoxicity , 1995, The Journal of experimental medicine.

[16]  Y. Yarden,et al.  A mutant EGF‐receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling , 2002, The EMBO journal.

[17]  J. Tschopp,et al.  Interaction of peptides derived from the Fas ligand with the Fyn‐SH3 domain , 1995, FEBS letters.

[18]  P. Schur,et al.  Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. , 1996, The Journal of clinical investigation.

[19]  B. Howard,et al.  The SV40 large T antigen and adenovirus E1a oncoproteins interact with distinct isoforms of the transcriptional co‐activator, p300. , 1996, The EMBO journal.

[20]  K. Helin,et al.  Multiple autophosphorylation sites of the epidermal growth factor receptor are essential for receptor kinase activity and internalization. Contrasting significance of tyrosine 992 in the native and truncated receptors. , 1992, Journal of Biological Chemistry.

[21]  Xuejun Jiang,et al.  Epidermal Growth Factor Receptor Internalization through Clathrin‐Coated Pits Requires Cbl RING Finger and Proline‐Rich Domains But Not Receptor Polyubiquitylation , 2003, Traffic.

[22]  Sheila M. Thomas,et al.  Cellular functions regulated by Src family kinases. , 1997, Annual review of cell and developmental biology.

[23]  L. Mincheva-Nilsson,et al.  Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level. , 2005, Molecular human reproduction.

[24]  M. Martínez-Lorenzo,et al.  Differential Secretion of Fas Ligand- or APO2 Ligand/TNF-Related Apoptosis-Inducing Ligand-Carrying Microvesicles During Activation-Induced Death of Human T Cells1 , 2001, The Journal of Immunology.

[25]  C. Willman,et al.  Differential expression and regulation of the c-src and c-fgr protooncogenes in myelomonocytic cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Tschopp,et al.  Conversion of Membrane-bound Fas(CD95) Ligand to Its Soluble Form Is Associated with Downregulation of Its Proapoptotic Activity and Loss of Liver Toxicity , 1998, The Journal of experimental medicine.

[27]  D. Link,et al.  The proto-oncogene c-fgr is expressed in normal mantle zone B lymphocytes and is developmentally regulated during myelomonocytic differentiation in vivo. , 1995, Blood.

[28]  D. Black.  Activation of c-src neuron-specific splicing by an unusual RNA element in vivo and in vitro , 1992, Cell.

[29]  L. Mincheva-Nilsson,et al.  gammadelta T cells of human early pregnancy decidua: evidence for cytotoxic potency. , 2000, International immunology.

[30]  S. Ju,et al.  CD95 (Fas) Ligand-Expressing Vesicles Display Antibody-Mediated, FcR-Dependent Enhancement of Cytotoxicity1 , 2000, The Journal of Immunology.

[31]  M. Zvelebil,et al.  Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail. , 2001, Journal of cell science.

[32]  S. Nagata,et al.  Downregulation of Fas ligand by shedding , 1998, Nature Medicine.