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First published online 3 March 2009
doi: 10.1242/jcs.034843

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Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain

Izumi Sato^1,*, Yuuki Obata^1,*, Kousuke Kasahara¹, Yuji Nakayama¹, Yasunori Fukumoto¹, Takahito Yamasaki², Kazunari K. Yokoyama², Takashi Saito³ and Naoto Yamaguchi^1,

¹ Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan
² Gene Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
³ Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan

View larger version (58K): [in this window] [in a new window]   Fig. 1. Localization of Lyn, Yes and Fyn. (A,B) COS-1 cells transfected with Lyn, Yes or Fyn were cultured for the indicated periods and stained with anti-Lyn, anti-Yes or anti-Fyn antibody. (A) Representative cells exhibiting predominant perinuclear (PeriN) and plasma membrane (PM) localization. Arrows indicate the perinuclear localization. (B) The graph shows the percentage of cells with predominant perinuclear staining. Results (%) are means ± s.d. (n=3-6). The results for Lyn at 18 and 24 hours after transfection were obtained from a representative experiment. (C) COS-1 cells transfected with Lyn or Yes cultured for 18 hours and double stained with anti-Lyn (green) or anti-Yes (green) antibody together with anti-galactosyltransferase (anti-GalT; red) antibody. Boxed areas are magnified in the insets. (D) COS-1 cells transfected with Lyn, Yes or Fyn were cultured for 15 hours in the absence or presence of 5 µg/ml brefeldin A (BFA) during the last 1 hour, and the distribution of expressed proteins were examined with anti-Lyn, anti-Yes or anti-Fyn antibody. (E) Endogenous Lyn (p56/p53) and Fyn (p59) expressed in THP-1 cells immunoblotted with anti-Lyn and anti-Fyn antibodies. (F) THP-1 cells cultured in the absence or presence of 5 µg/ml BFA for 2 hours. Endogenous Lyn and Fyn were visualized with anti-Lyn or anti-Fyn antibody. Arrows indicate the perinuclear region. N, nucleus. Scale bars: 20 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 2. CHX treatment removes Lyn and Yes from the Golgi region. (A,B) COS-1 cells transfected with Lyn, Yes or Fyn cultured for 12 hours and then treated with 100 µg/ml cycloheximide (CHX) for 30 and 60 minutes. Cells were stained with anti-Lyn, anti-Yes or anti-Fyn antibody. (B) Cells exhibiting the perinuclear localization of expressed proteins were quantified. Results (%) are means ± s.d. (n=3-6). (C) THP-1 cells cultured in the absence or presence of 200 µg/ml CHX for 3 hours. Endogenous Lyn and Fyn were detected with anti-Lyn or anti-Fyn antibody. Note that CHX treatment for 3 hours reduced protein expression levels of endogenous Lyn and Fyn because of inhibition of protein synthesis. Arrows indicate the perinuclear region. Scale bars: 20 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Temperature block of TGN membrane transport results in perinuclear accumulation of Lyn and Yes. (A) COS-1 cells transfected with Lyn, Yes or Fyn were incubated for 15 hours, subsequently treated with 100 µg/ml CHX for 3 hours at 19°C or 37°C and stained with anti-Lyn, anti-Yes or anti-Fyn antibody. The transfected cells incubated at 19°C for 3 hours in the presence of 100 µg/ml CHX were warmed to 37°C for further 2 hours (19°C37°C). Arrows indicate the perinuclear region. Cells exhibiting the perinuclear localization of expressed proteins were quantified. Results (%) are means ± s.d. (n=35). **P<0.01, Student's t-test. NS, not significant. Scale bars: 20 µm. (B) COS-1 cells transfected with VSVG-GFP or VSVG-GFP plus HA-Rab11S25N (+Rab11DN) were incubated overnight at 40°C and then shifted to 32°C for 30 minutes or for 6 hours, including 200 µg/ml CHX during the last 3 hours. Expressed proteins were detected with GFP fluorescence (green) or anti-HA antibody (red) (a). COS-1 cells transfected with Lyn, Lyn plus Rab11DN, Yes or Yes plus Rab11DN, cultured for 24 hours. Cells were doubly stained with anti-Lyn (green) or anti-Yes (green) antibody plus anti-HA antibody (red) (b). Insets show fluorescence images of Rab11DN (red). Cells exhibiting the perinuclear localization of Lyn or Yes were quantified. Results (%) are means ± s.d. (n=3-6). ***P<0.001, Student's t-test. N, nucleus. Scale bars: 20 µm. (C) Endogenous Lyn (p56/p53), Yes (p62) and Fyn (p59) expressed in Dami cells were immunoblotted with anti-Lyn, anti-Yes and anti-Fyn antibodies. (D) Dami cells incubated for 3 hours at 37°C or 19°C and stained with anti-Lyn, anti-Yes or anti-Fyn antibody. Scale bars: 20 µm. (E) Cells incubated at 19°C for 3 hours and doubly stained for Lyn or Yes (green), and GalT (red). Arrows indicate the perinuclear region. Scale bars: 10 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 4. Trafficking of Lyn and Yes just after biosynthesis is different from that of Fyn in living cells. COS-1 cells transfected with Lyn-GFP (A), Yes-GFP (B) or Fyn-GFP (C) cultured for 20 hours. Whole cell area was bleached and monitored at 1-minute intervals at 37°C or 19°C for the next 20 minutes. Times are shown after photobleaching. Mean fluorescence intensities of recovery after photobleaching in the perinuclear area (PeriN) and whole cell area excluding perinuclear region (Non-PeriN) plotted versus time. A representative result (A-C) is shown from at least 2-3 independent experiments. N, nucleus. Scale bars: 20 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Mutation of the second palmitoylation site of Fyn redirects the protein to the Golgi region. (A) Sequences of the first 16 amino acids of SFKs. The underlined amino acids at position 6 indicate a Cys residue and a CysSer mutation. Myristoylated glycines are highlighted in blue, and palmitoylated cysteines in red. (B) Equal amounts of lysates from COS-1 cells transfected with Fyn or Fyn(C6S) analyzed by western blotting with anti-Fyn, anti-Src[pY418] and anti-actin antibodies. (C,D) COS-1 cells transfected with Fyn or Fyn(C6S) cultured for the indicated periods and stained with anti-Fyn (green) and anti-GM130 (red) antibodies. (D) Cells exhibiting the perinuclear localization of expressed proteins were quantified. Data represent means ± s.d. (n=3-6). **P<0.01, Student's t-test. (E) COS-1 cells transfected with Fyn(C6S) cultured for 15 hours in the absence or presence of 5 µg/ml BFA for the last 1 hour and doubly stained for Fyn (green) and GM130 (red). (F) COS-1 cells transfected with Fyn(C6S) cultured for 12 hours, then treated with 100 µg/ml CHX for the indicated periods, and stained with anti-Fyn antibody. Cells exhibiting the perinuclear localization of Fyn(C6S) were quantified. Results (%) are means ± s.d. (n=3). **P<0.01, Student's t-test. (G) COS-1 cells transfected with Fyn alone, Fyn plus Rab11DN, Fyn(C6S) alone and Fyn(C6S) plus Rab11DN cultured for 24 hours. Cells were double stained with anti-Fyn (green) and anti-HA (red) antibodies. Cells exhibiting the perinuclear localization of Fyn or Fyn(C6S) were quantified. Results (%) are means ± s.d. (n=6-7). **P<0.01, Student's t-test. Arrows indicate the perinuclear region. N, nucleus. Scale bars: 20 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Dually palmitoylated N-terminus of Fyn leads Lyn and Yes to the plasma membrane localization. (A,F) Schematic representations of the constructs used in this study are shown with the Src homology (SH) domains and the kinase domain. Fyn24-Lyn, Fyn24-Yes and Fyn24(C6S)-Lyn contain the first 24 amino acids of Fyn-wt or Fyn(C6S). (B-F) COS-1 cells transiently transfected with the indicated constructs cultured for 15 hours. (B,F) Cell lysates analyzed by western blotting with anti-Lyn, anti-Yes, anti-Fyn, anti-Src[pY418] and anti-actin antibodies. (C-F) Expressed proteins visualized with anti-Lyn, anti-Yes or anti-Fyn antibody. Cells expressing Fyn24-Lyn or Fyn24(C6S)-Lyn doubly stained with anti-Lyn (green) and anti-GalT (red) antibody. N, nucleus. Scale bars: 20 µm. Cells exhibiting the perinuclear localization of expressed proteins were quantitated. Data represent means ± s.d. (n=3-4). **P<0.01, *** P<0.001, Student's t-test. NS, not significant.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Model of three major pathways for SFK trafficking. (i) The cycling pathway between late endosomes/lysosomes and the plasma membrane for myristoylated but not palmitoylated SFKs. (ii) The secretory pathway from the Golgi region to the plasma membrane for myristoylated and monopalmitoylated SFKs. (iii) The direct targeting pathway for myristoylated and dually palmitoylated SFKs. Myr, myristate; Pal, palmitate; PM, plasma membrane; LE, late endosome; ER, endoplasmic reticulum.

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