Research Article 965 Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain

Izumi Sato1,*, Yuuki Obata1,*, Kousuke Kasahara1, Yuji Nakayama1, Yasunori Fukumoto1, Takahito Yamasaki2, Kazunari K. Yokoyama2, Takashi Saito3 and Naoto Yamaguchi1,‡ 1Department of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan 2Gene Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan 3Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 24 November 2008 Journal of Cell Science 122, 965-975 Published by The Company of Biologists 2009 doi:10.1242/jcs.034843

Summary Src-family tyrosine kinases (SFKs), which participate in a traffic in the same way as Lyn and Yes. Moreover, construction variety of events, are known to localize to of Yes(S6C) and chimeric Lyn or Yes with the Fyn N-terminus the cytoplasmic face of the plasma membrane through lipid further substantiates the importance of the dual palmitoylation modification. Recently, we showed that Lyn, an SFK member, site for plasma membrane targeting. Taken together with our is exocytosed to the plasma membrane via the Golgi region along recent finding that Src, a nonpalmitoylated SFK, is rapidly the secretory pathway. We show here that SFK trafficking is exchanged between the plasma membrane and late specified by the palmitoylation state. Yes is also a endosomes/lysosomes, these results suggest that SFK trafficking monopalmitoylated SFK and is biosynthetically transported is specified by the palmitoylation state in the SH4 domain. from the Golgi pool of caveolin to the plasma membrane. This pathway can be inhibited in the trans-Golgi network (TGN)- to-cell surface delivery by temperature block at 19°Cor Supplementary material available online at dominant-negative Rab11 GTPase. A large fraction of Fyn, a http://jcs.biologists.org/cgi/content/full/122/7/965/DC1 dually palmitoylated SFK, is directly targeted to the plasma membrane irrespective of temperature block of TGN exit. Key words: Src-family tyrosine kinases, trafficking, palmitoylation, Fyn(C6S), which lacks the second palmitoylation site, is able to Lyn, Fyn, Yes Journal of Cell Science

Introduction acylation of SFKs has been shown to influence their interactions Src-family tyrosine kinases (SFKs), which are non-receptor-type with cell membranes (McCabe and Berthiaume, 1999; Resh, 1999) tyrosine kinases, consist of proto-oncogene products and structurally and as a consequence their intracellular distribution. related and include at least eight highly homologous It is generally thought that SFKs are predominantly located at proteins: Src, Lyn, Fyn, Yes, Fgr, Hck, and Blk (Brown and the cytoplasmic face of the plasma membrane through post- Cooper, 1996; Thomas and Brugge, 1997). SFKs are activated by translational myristoylation, usually with subsequent palmitoylation, various stimuli, including growth factors and adhesion proteins, and but in fact, appreciable fractions are found at in variety of are involved in a wide range of signaling events at the plasma intracellular locations, such as endosomes, secretory granules or membrane, resulting in cell proliferation, differentiation, migration, phagosomes and the Golgi complex (Kaplan et al., 1992; Mohn et and cell-shape changes. Src, Yes, Lyn and Fyn are widely expressed al., 1995; Brown and Cooper, 1996; Thomas and Brugge, 1997; in a variety of cell types, whereas Blk, Fgr, Hck and Lck are found Kasahara et al., 2004). Although distinctive localizations of SFK primarily in hematopoietic cells (Bolen and Brugge, 1997; Thomas members have been implicated in their specific functions, the and Brugge, 1997). mechanism that underlies the targeting of SFKs to their specific SFKs are composed of (1) an N-terminal Src homology (SH) 4 locations remains to be elucidated. domain that contains lipid modification sites; (2) a poorly conserved We recently showed that Lyn, a palmitoylated SFK, is exocytosed ‘unique’ domain; (3) an SH3 domain that can bind to specific to the plasma membrane via the Golgi region along the secretory proline-rich sequences; (4) an SH2 domain that can bind to specific pathway (Kasahara et al., 2004). More recently, we demonstrated sites of tyrosine phosphorylation; (5) an SH1 that Src, a non-palmitoylated SFK, rapidly moves between the catalytic domain; and (6) a negative regulatory tail for autoinhibition plasma membrane and late endosomes or lysosomes, and that of kinase activity (Brown and Cooper, 1996; Thomas and Brugge, mutation of Cys3 in Lyn allows Lyn to traffic in a similar manner 1997). All members of the Src family are cotranslationally to Src (Kasahara et al., 2007a), indicating the importance of myristoylated at Gly2 and, with the exception of Src and Blk, are palmitoylation for distinct trafficking between Lyn and Src. also post-translationally palmitoylated at Cys3, Cys5 or Cys6 In this study, we investigate the localization and trafficking of (Paige et al., 1993; Alland et al., 1994; Koegl et al., 1994; Resh, other ubiquitously expressed SFKs, such as Yes and Fyn. We 1994; Shenoy-Scaria et al., 1994; Kasahara et al., 2007a). Fatty demonstrate that Lyn and Yes, which are monopalmitoylated SFKs, 966 Journal of Cell Science 122 (7)

are transported to the plasma membrane via the Golgi region along (Kasahara et al., 2004). Similarly to Lyn, Yes was predominantly the secretory pathway, whereas a large fraction of Fyn, a dually found at the perinuclear region during the earlier phase of expression palmitoylated SFK, is directly targeted to the plasma membrane. and at the plasma membrane in the later phase (Fig. 1A,B, middle). Our findings suggest that the state of mono- or dual-palmitoylation By contrast, the majority of Fyn was found at the plasma membrane makes a difference in trafficking among palmitoylated SFKs. from the early phase (Fig. 1A,B, bottom), which is consistent with previous studies (van’t Hof and Resh, 1997). These results suggest Results that Yes, as well as Lyn, accumulates initially at the perinuclear Yes and Lyn localize to the Golgi region region and is then transported to the plasma membrane. Src, Lyn, Yes and Fyn are widely expressed in various cell types To produce a synchronous wave of synthesis and (Thomas and Brugge, 1997; Kuga et al., 2007). We recently showed trafficking, we used a HeLa cell clone (3-2) (Kasahara et al., 2007b) that Lyn is biosynthetically transported to the plasma membrane stably expressing the tetracycline repressor for an inducible via the Golgi pool of caveolin along the secretory pathway expression system. At 12 hours after transfection with Lyn, Yes or (Kasahara et al., 2004). To examine the localization of other Fyn, doxycycline (Dox) was added to medium to induce protein ubiquitously expressed SFKs, we transfected COS-1 cells with Lyn, expression. In the course of induction, most Lyn and Yes proteins Yes or Fyn and compared their localization. In the earlier phase of were initially observed in the perinuclear region and were expression, localization of Lyn was predominantly seen in the subsequently detected at the plasma membrane, whereas Fyn was perinuclear region and gradually changed to the plasma membrane predominantly present on the plasma membrane from the early in the later phase (Fig. 1A,B, top), consistent with our report phase of induction (supplementary material Fig. S1A,B). These Journal of Cell Science

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. Differential trafficking of Src kinases 967

results further support the possibility that Yes is transported from of the synthesized proteins in the Golgi region. Incubation with the perinuclear region to the plasma membrane. CHX at 19°C caused accumulation of Yes as well as Lyn in the Next, we characterized the perinuclear region using Golgi perinuclear region (Fig. 3A, middle), whereas perinuclear markers, such as β-1,4-galactosyltransferase (GalT), a trans-Golgi accumulation of Lyn and Yes was markedly reduced upon incubation protein and GM130, a cis-Golgi protein. A large fraction of with CHX for 3 hours at 37°C (Fig. 3A, left). When the cells perinuclear Yes was also partially colocalized with GalT (Fig. 1C), incubated at 19°C were warmed to 37°C, the perinuclear as also observed with inducible Yes and Lyn (supplementary accumulation of Lyn and Yes was rapidly dissipated (Fig. 3A, right). material Fig. S1C). In addition, Yes was precisely colocalized with In sharp contrast, incubation at 19°C did not allow Fyn to caveolin in the perinuclear region (supplementary material Fig. S2), accumulate in the perinuclear region (Fig. 3A, bottom). These results indicating that perinuclear Yes is localized to the Golgi pool of suggest that biosynthetic exocytic traffic is involved in the caveolin similarly to the localization of Lyn in the perinuclear region. recruitment of Yes to the plasma membrane in a manner similar to Perinuclear Yes and Lyn were dispersed by brefeldin A (BFA) (Fig. Lyn trafficking. 1D), further confirming their Golgi localization. Overexpression of the dominant-negative Rab11S25N mutant To examine whether localization of endogenous SFKs was (Rab11DN) causes inhibition of the TGN-to-cell surface delivery similar to those of overexpressed proteins, we used the human (Chen et al., 1998). To ascertain whether the plasma membrane monocytic cell line THP-1 in which endogenous Lyn and Fyn were delivery of a conventional exocytic cargo was mediated by Rab11, detectable by immunoblotting and immunostaining (Fig. 1E,F). we used green fluorescent protein (GFP)-tagged vesicular stomatitis- Although endogenous Yes was hardly visualized owing to its low virus-encoded glycoprotein (VSVG-GFP), whose export from the expression (data not shown), endogenous Lyn was localized to the perinuclear region and the plasma membrane. BFA treatment caused dispersal of endogenous Lyn from the perinuclear region (Fig. 1F). Endogenous Fyn was predominantly seen at the plasma membrane and showed no apparent redistribution in the presence of BFA. These results suggest that localization of endogenous SFKs mirror those of the overexpressed proteins.

Newly synthesized Yes initially accumulates in the Golgi region We next determined whether the presence of Yes in the Golgi region reflected newly synthesized protein in transit to the plasma membrane. COS-1 cells expressing Lyn, Yes or Fyn were treated with cycloheximide (CHX), a protein synthesis inhibitor. CHX treatment markedly reduced perinuclear Lyn within 60 minutes (Fig. 2A,B, top), which is consistent with an earlier report (Kasahara et al., 2004). Reduction of perinuclear Yes was also found within 60

Journal of Cell Science minutes of CHX treatment (middle), indicating that CHX treatment results in a time-dependent chase of newly synthesized Yes from the Golgi region to the plasma membrane. By contrast, CHX treatment did not affect Fyn localization to the plasma membrane (bottom). These results suggest that newly synthesized Yes but not Fyn is initially accumulated at the Golgi region and transported to the plasma membrane in a manner similar to Lyn trafficking. To further examine whether the localization of endogenous Lyn and Fyn was affected by CHX treatment, THP-1 cells were treated with CHX for 3 hours and stained for Lyn and Fyn. Perinuclear accumulation of endogenous Lyn diminished upon CHX treatment (Fig. 2C, top), whereas plasma membrane localization of endogenous Fyn was not affected (Fig. 2C, bottom). These results suggest that the trafficking of endogenous Lyn is different from that of endogenous Fyn, in agreement with the results shown in Fig. 2A,B.

Yes and Lyn traffic to the plasma membrane through the exocytic pathway Fig. 2. CHX treatment removes Lyn and Yes from the Golgi region. Since Yes is localized to the Golgi region and chased to the plasma (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. membrane by CHX treatment, we tested whether Yes, like Lyn, Cells were stained with anti-Lyn, anti-Yes or anti-Fyn antibody. (B) Cells was transported to the plasma membrane through the exocytic exhibiting the perinuclear localization of expressed proteins were quantified. pathway. We took advantage of the property of a reduced Results (%) are means ± s.d. (n=3-6). (C) THP-1 cells cultured in the absence temperature (19°C) to specifically block TGN membrane vesicle 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 exit (Griffiths et al., 1989; Watson et al., 2003). COS-1 cells hours reduced protein expression levels of endogenous Lyn and Fyn because expressing Lyn or Yes were incubated at 19°C for 3 hours in the of inhibition of protein synthesis. Arrows indicate the perinuclear region. Scale presence of CHX to block protein synthesis but allow accumulation bars: 20 μm. 968 Journal of Cell Science 122 (7)

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°Cr37°C). Arrows indicate the perinuclear region. Cells exhibiting the perinuclear localization of expressed proteins were quantified. Results (%) are means ± s.d. (n=3~5). **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.

minutes or for 6 hours, including CHX for the last 3 hours. In cells transfected with VSVG-GFP alone, VSVG-GFP was predominantly found in the Golgi region at 30 minutes after temperature shift, and most VSVG-GFP was transported to the plasma membrane after incubation for 6 hours (Fig. 3Ba, left), consistent with previous reports (Hirschberg et al., 1998; Hirose et al., 2004). Upon overexpression of Rab11DN, VSVG-GFP was largely retained in the Golgi region at 6 hours after temperature shift (Fig. 3Ba, right), in agreement with previous studies (Chen et al., 1998). We then examined whether overexpression of Rab11DN inhibited the transport of Lyn and Yes to the plasma membrane. COS-1 cells

Journal of Cell Science transfected with Lyn alone, Lyn plus Rab11DN, Yes alone or Yes plus Rab11DN were cultured for 24 hours. At this incubation period after transfection, perinuclear Lyn and Yes were largely transported to the plasma membrane and little accumulation of Lyn and Yes was seen in the perinuclear region (Fig. 3Bb, left; see Fig. 1B). Intriguingly, overexpression of Rab11DN strongly induced perinuclear accumulation of Lyn and Yes (Fig. 3Bb, right). These results indicate that overexpression of Rab11DN blocks the transport of Lyn and Yes to the plasma membrane, suggesting the involvement of Rab11 in the exocytic transport of Lyn and Yes. To examine whether the transport of endogenous Yes was also inhibited by temperature block, we used the human megakaryocytic cell line Dami, in which endogenous Yes as well as Lyn and Fyn was easily detected by immunoblotting and immunostaining (Fig. 3C-E). Despite low levels of perinuclear accumulation, endogenous Lyn and Yes were found in the plasma membrane (Fig. 3D, left), and incubation at 19°C for 3 hours increased levels of perinuclear Lyn and Yes in most cells (Fig. 3D, right; Fig. 3E). These results suggest that endogenous Yes as well as Lyn traffics to the plasma membrane through the exocytic pathway. By contrast, Fyn was primarily found at the plasma membrane in cells incubated at 37°C and no apparent redistribution was seen after incubation at 19°C (Fig. 3D, bottom), further confirming the finding that newly ER occurs in a temperature-dependent manner (Presley et al., 1997). synthesized Fyn is directly targeted to the plasma membrane (van’t COS-1 cells transfected with VSVG-GFP or VSVG-GFP plus HA- Hof and Resh, 1997). Rab11DN were incubated at 40°C (nonpermissive temperature) In addition, Dami cells were incubated at 15°C to examine the overnight and then shifted to 32°C (permissive temperature) for 30 involvement of ER membranes in Lyn and Yes transport. Incubation Differential trafficking of Src kinases 969

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. Journal of Cell Science Scale bars: 20 μm.

at 15°C, which impairs transport from the ER to the cis-Golgi to Lyn, newly synthesized Yes traffics from the Golgi region en (Presley et al., 1997), resulted in accumulation of Lyn and Yes in route to the plasma membrane. the Golgi region but not the ER (supplementary material Fig. S3), When we compared the trafficking of Fyn in cells cultured at suggesting that the trafficking pathways for Lyn, Yes and Fyn do 37°C and 19°C, we found that incubation at 19°C did not inhibit not involve ER membranes. the rapid recovery of Fyn-GFP in the non-perinuclear region after whole-cell photobleaching (Fig. 4C). Rather, this rapid recovery Newly synthesized Yes and Lyn, but not Fyn, traffic to the was similar to that observed at 37°C, and the recovery rates of plasma membrane through the Golgi region Fyn-GFP in the perinuclear region at 19°C and 37°C were To visualize the trafficking of Lyn, Yes and Fyn just after comparable with those in the non-perinuclear region. These biosynthesis in living cells, we added a GFP tag to the proteins, results suggest that most of the newly synthesized Fyn traffics which preserves their N-terminal lipid attachment sites (see directly to the plasma membrane and only a small fraction targets Materials and Methods) (Kasahara et al., 2004), and performed to the Golgi region. fluorescence recovery after photobleaching (FRAP) of Lyn-GFP, Yes-GFP and Fyn-GFP in COS-1 cells. When the whole-cell area Palmitoylation at Cys6 influences Fyn localization and was photobleached, rapid recovery of Lyn-GFP fluorescence at trafficking 37°C was observed in both the perinuclear region and the non- Palmitoylation of SFKs occurs on cysteine residues in the general perinuclear region that excludes the perinuclear region from whole motif Met-Gly-Cys, where Gly2 is myristoylated (Resh, 1999). cell area. Incubation at 19°C after whole-cell photobleaching Although Lyn and Yes are monopalmitoylated at Cys3 (Koegl et inhibited the recovery of Lyn-GFP fluorescence in the non- al., 1994; McCabe and Berthiaume, 1999; Kasahara et al., perinuclear region but not the perinuclear region (Fig. 4A). 2007a), Fyn is dually palmitoylated at Cys3 and Cys6 (Alland Intriguingly, the recovery rates of Yes-GFP fluorescence at 37°C et al., 1994). Since the trafficking pathway for Lyn and Yes is and 19°C were comparable with those of Lyn-GFP fluorescence different to that for Fyn (Figs 3 and 4), we asked whether the (compare Fig. 4B with 4A). These results suggest that, similarly lack of the second palmitoylation site in Fyn affected its 970 Journal of Cell Science 122 (7)

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 CysrSer 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

Journal of Cell Science 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.

trafficking. We generated a Fyn(C6S) mutant in which Cys6 in Localization of Fyn(C6S) to the Golgi region in the early phase Fyn was substituted for Ser (Fig. 5A), a substitution previously of expression prompted us to examine whether accumulation of shown to decrease palmitate incorporation of Fyn (Alland et al., Fyn(C6S) in the Golgi region was diminished by CHX. Upon 1994). The levels of protein expression and kinase activity were treatment with CHX, the number of cells exhibiting perinuclear comparable between wild-type Fyn (Fyn-wt) and Fyn(C6S) (Fig. Fyn(C6S) decreased (Fig. 5F), suggesting that following its initial 5B). Intriguingly, Fyn(C6S) significantly accumulated in the biosynthesis, Fyn(C6S) can enter the secretory membrane- Golgi region 12 hours after transfection compared with Fyn-wt trafficking system. We then examined the involvement of Rab11 (Fig. 5C,D), although the level of Fyn(C6S) was lower than that in transport of Fyn(C6S) to the plasma membrane. COS-1 cells for Lyn and Yes (compare with Fig. 1B). The number of cells transfected with Fyn-wt alone, Fyn-wt plus Rab11DN, Fyn(C6S) exhibiting the Golgi localization of Fyn(C6S) was gradually alone, or Fyn(C6S) plus Rab11DN were incubated for 24 hours. decreased after transfection (Fig. 5D), similar to the results for At this incubation period after transfection, most Fyn-wt and Lyn and Yes (Fig. 1B). Accumulation of Fyn(C6S) in the Golgi Fyn(C6S) were localized to the plasma membrane (Fig. 5G, left, region was dispersed by BFA treatment (Fig. 5E), and Control). Overexpression of Rab11DN induced perinuclear accumulation of inducible Fyn(C6S) was also observed in the accumulation of Fyn(C6S) (Fig. 5G, right), similar to the results Golgi region in the early phase of induction (supplementary for Lyn and Yes (Fig. 3Bb). These results suggest that Rab11 is material Fig. S4 and Fig. S1). involved in transport of Fyn(C6S). Differential trafficking of Src kinases 971

We noted that perinuclear accumulation of Fyn-wt was moderately induced by long-term coexpression of Rab11DN with Fyn-wt [~38% for Fyn-wt (Fig. 5G) and ~75% for Lyn or Yes (Fig. 3Bb)]. About 15% of cells expressing Fyn-wt showed perinuclear Fyn-wt, which partially colocalized with GM130, a Golgi marker (Fig. 5G; data not shown), and weak perinuclear fluorescence of Fyn-GFP could be detected at 19°C after whole-cell photobleaching (Fig. 4C). Also, a few percentages of Dami cells showed perinuclear staining for endogenous Fyn (supplementary material Fig. S3F). These results suggest that a small fraction of Fyn-wt might traffic from the Golgi region to the plasma membrane along the Rab11-mediated exocytic pathway. In addition, we transfected COS-1 cells with Fyn-wt, Fyn(C6S) or Lyn, and examined tyrosine phosphorylation at the early phase of expression when Golgi accumulation of Fyn(C6S) and Lyn but not Fyn-wt was visible. Similarly to results observed with Lyn, Fyn(C6S) but not Fyn-wt increased tyrosine phosphorylation of pp40 (supplementary material Fig. S5), supporting the finding that localization of Fyn(C6S) is similar to that of Lyn.

Palmitoylation at Cys6 in Fyn contributes to its plasma membrane targeting A Fyn-Src chimera exhibits by far the greatest incorporation of the palmitate analog, whereas mutation of Ser3 and Ser6 in viral Src to Cys shows mild incorporation (Alland et al., 1994). To examine whether palmitoylation at Cys 6 affected localization of Lyn and Yes, we first created a Fyn-Lyn chimera (Fyn24-Lyn), in which the SH4 domain of Lyn was substituted for that of Fyn-wt (Fig. 6A). Expression and kinase activity were confirmed in COS-1 cells transfected with each construct

Journal of Cell Science (Fig. 6B). Intriguingly, localization of Fyn24-Lyn was different from that of Lyn, despite containing 95% of the Lyn protein sequence, and Fyn24-Lyn was mainly localized at the plasma membrane in the same manner as Fyn (Fig. 6C). To assess whether the palmitoylation site at Cys6 was important for the targeting of Fyn24-Lyn to the plasma membrane, we introduced a mutation of Cys6 to Ser in Fyn24-Lyn and created Fyn24(C6S)-Lyn (Fig. 6A). Although Fyn24-Lyn was mainly localized to the plasma membrane, Fyn24(C6S)-Lyn localized to the perinuclear region and the plasma membrane (Fig. 6D), similarly to Lyn localization (Figs 1-3) (Kasahara et al., 2004). Additionally, perinuclear Fyn24(C6S)-Lyn was partially

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. 972 Journal of Cell Science 122 (7)

colocalized with GalT [Fig. 6D; see Fig. 5 and supplementary material S4 for Fyn(C6S)], which is consistent with the perinuclear localization of Lyn and Yes (Fig. 1C; Fig. 3E; supplementary material Fig. S1C and Fig. S3B,D). These results suggest that the N-terminus preserving the monopalmitoylation site is responsible for Golgi localization of SFKs. We then created a Fyn-Yes chimera (Fyn24-Yes) and confirmed its expression and kinase activity (Fig. 6B). Similarly to the results of Fyn24-Lyn, Fyn24-Yes was also localized at the plasma membrane (Fig. 6E), suggesting that the Fyn N-terminus, which contains one myristoylation site and two palmitoylation sites, allows Lyn and Yes to traffic directly to the plasma membrane. Furthermore, we generated a Yes(S6C) mutant in which Ser6 in Yes was substituted for Cys (Fig. 6F). The levels of protein expression were comparable between wild-type Yes and Yes(S6C) (Fig. 6F). Intriguingly, Yes(S6C) was mainly localized at the plasma membrane but not at the Golgi region, similarly to Fyn localization (Fig. 6F). Taken together, these results suggest that the Fig. 7. Model of three major pathways for SFK trafficking. (i) The cycling pathway between late endosomes/lysosomes and the plasma membrane for presence of the second palmitoylation site in the N-terminus has myristoylated but not palmitoylated SFKs. (ii) The secretory pathway from the an important role in the direct targeting of SFKs to the plasma Golgi region to the plasma membrane for myristoylated and membrane. monopalmitoylated SFKs. (iii) The direct targeting pathway for myristoylated and dually palmitoylated SFKs. Myr, myristate; Pal, palmitate; PM, plasma Discussion membrane; LE, late endosome; ER, endoplasmic reticulum. In the present study, we demonstrate that SFK trafficking is specified by its palmitoylation state. Lyn and Yes, which are both monopalmitoylated SFKs, are biosynthetically transported to the Importantly, the results for endogenous Lyn, Yes and Fyn in THP- plasma membrane via the Golgi pool of caveolin along the secretory 1 and Dami cells (Fig. 1F; Fig. 3D) agree with our proposed model. pathway, whereas Fyn, a dually palmitoylated SFK, is directly Although perinuclear staining for endogenous Lyn and Yes in Dami targeted to the plasma membrane. We further show that Fyn(C6S), cells is not so strong as that observed in THP-1 cells, build-up of which lacks the second palmitoylation site, behaves in a manner Lyn and Yes in the Golgi region after temperature block in Dami reminiscent of Lyn and Yes by accumulating in the Golgi region cells (Fig. 3D) allows us to assume that newly synthesized Lyn and early after biosynthesis, and that Yes(S6C), in which a second Yes can reach the plasma membrane rapidly in Dami cells compared palmitoylation site is created, becomes localized to the plasma with THP-1 cells. Furthermore, we found that like Lyn, Fyn(C6S), membrane. which lacks the second palmitoylation site, accumulated in the Golgi

Journal of Cell Science region (Fig. 5), and that the dually palmitoylated N-terminus of Three major pathways for SFK trafficking Fyn leads Lyn and Yes directly to the plasma membrane whereas Palmitoylation of SFKs has been shown to contribute to their the monopalmitoylated N-terminus of Fyn redirects Lyn to the Golgi localization (Bijlmakers et al., 1997; Carreno et al., 2000). Although region (Fig. 6). We also found that creation of the second Src is not palmitoylated, Lyn and Yes are monopalmitoylated at palmitoylation site in Yes enables its transfer directly to the plasma Cys3 (Koegl et al., 1994; McCabe and Berthiaume, 1999; Kasahara membrane (Fig. 6). These results suggest that the state of et al., 2007a) and Fyn is dually palmitoylated at Cys3 and Cys6 monopalmitoylation (Lyn and Yes) or dual palmitoylation (Fyn) (Alland et al., 1994). We recently showed that the trafficking of makes a difference in the trafficking pathways between Lyn-Yes Src (a nonpalmitoylated SFK) is different from that of Lyn (a and Fyn. Our model is also consistent with previous findings that monopalmitoylated SFK) because of the state of palmitoylation nonpalmitoylated p61Hck, like Src, is localized to lysosomes whereas (Kasahara et al., 2007a). monopalmitoylated p59Hck localizes to the Golgi and the plasma Here, we propose a model of three major trafficking pathways membrane (Carreno et al., 2000). Taken together, these results for SFKs (Fig. 7). (1) The cycling pathway for myristoylated but provide evidence that SFK trafficking is classified into three groups not palmitoylated SFKs: Src is rapidly exchanged between late depending on the state of palmitoylation, as depicted in our model. endosomes or lysosomes and the plasma membrane, possibly Notably, a very small fraction of Fyn was found to localize to through its cytosolic release (Kasahara et al., 2007a). (2) The the Golgi region (Fig. 4C, Fig. 5C, Fig. 5D; supplementary material secretory pathway from the Golgi region to the plasma membrane Fig. S3F) (van’t Hof and Resh, 1997). Given that palmitoylation for myristoylated and monopalmitoylated SFKs: newly synthesized of Fyn does not occur perfectly (Liang et al., 2004), it is plausible Lyn and Yes initially enter the Golgi system, where palmitoylation that mono- or non-palmitoylated Fyn can follow a pathway other probably occurs, providing entry into the membrane secretory than the direct targeting pathway to the plasma membrane. Imperfect transport pathway en route to the plasma membrane (Fig. 3A,D, palmitoylation, if any, might affect the trafficking of the other SFKs Fig. 4) (Kasahara et al., 2004). Rab11 is involved in exocytic including Lyn and Yes. transport of Lyn and Yes (Fig. 3B). (3) The direct plasma- Cys3 is considered to have a greater role in the membrane membrane-targeting pathway for myristoylated and dually localization of Fyn than Cys6 judging from biochemical data, palmitoylated SFKs: newly synthesized Fyn is targeted directly to although it would be difficult to detect localization changes among the plasma membrane (Fig. 2A, Fig. 3D, Fig. 4) (van’t Hof and various cell membranes (Shenoy-Scaria et al., 1994; Wolven et al., Resh, 1997). 1997). In the present study, we reveal that mutation at Cys6 Differential trafficking of Src kinases 973

influences intracellular localization of Fyn by accumulating the trisphosphate levels are increased to a larger extent at protein in the Golgi region (Fig. 5C). Taken together with the finding endomembranes than at the plasma membrane, which is triggered that Yes(S6C) becomes localized at the plasma membrane (Fig. 6), by endocytosed receptor tyrosine kinases (Sato et al., 2003). In we thus hypothesize that the second palmitoylation site at Cys6 is the present study, we demonstrate that each SFK member localizes important for plasma membrane targeting by keeping Fyn away to various intracellular organelles because of the distinct trafficking from the Lyn-type trafficking. The presence of the second cysteine pathway depicted in Fig. 7. Thus, our findings raise the intriguing residue in the SFK N-terminal domain might influence recognition possibility that the distinct localization brings about specific by proteins that are involved in protein palmitoylation or sorting functions of SFKs by allowing SFKs to interact with differently to the plasma membrane. This hypothesis may be also explained located substrates. by the results that Lck, which is dually palmitoylated at Cys3 and Indeed, we recently showed that Src is involved in targeting of Cys5 (Koegl et al., 1994; Yurchak and Sefton, 1995), accumulates macropinosomes to lysosomes and accumulation of lysosomes in the Golgi region by mutation of the second palmitoylation site (Kasahara et al., 2007b; Kasahara et al., 2008). It is also reported at Cys5 in transfected NIH-3T3 cells (Bijlmakers et al., 1997). that Src activation on RhoB-associated late endosomes leads to Src Since the level of Golgi-accumulated Fyn(C6S) was lower than translocation from the perinuclear region toward discrete cell that of Lyn and Yes (Fig. 1B; Fig. 5D), other factors besides peripheral structures (Sandilands et al., 2004). However, Lyn palmitoylation might influence localization of SFK. Given that phosphorylates annexin II on endomembranes including Golgi Fyn24-Lyn and Fyn24-Yes, which contain their SH3, SH2 and membranes (Matsuda et al., 2006). Moreover, p61Hck, but not kinase domains, tend to stay in the Golgi, unlike Fyn-wt (Fig. 6D- p59Hck, triggers the biogenesis of podosomes by exocytosis from E), we assume that the SH3 and SH2 domains of Lyn and Yes might lysosomes (Cougoule et al., 2005). These results strengthen our idea contribute to retention of Lyn and Yes in the Golgi region by protein- that specific functions among SFK members stem from their protein interactions with Golgi components. distinct intracellular localizations. In addition, we found that, like Palmitoylation is mediated by protein acyltransferases (PATs), Lyn, Fyn(C6S) but not Fyn-wt increased tyrosine phosphorylation which include a large family of integral membrane proteins that of pp40 (supplementary material Fig. S5), suggesting that an contain a DHHC cysteine-rich domain (DHHC PATs) (Linder and alteration in the subcellular localization of Fyn influences its access Deschenes, 2007). More than 20 DHHC PATs were isolated in to SFK substrates. Blk and Src, both of which are nonpalmitoylated mammals, and recent progress has shown that DHHC PATs have SFKs, phosphorylate coexpressed immunoglobulin-α, whereas exquisite substrate specificity (Fukata et al., 2004; Ohno et al., palmitoylated SFKs do not (Saouaf et al., 1997). Taken together, 2006; Linder and Deschenes, 2007). Most of the DHHC PAT the distinct trafficking of SFKs specified by the state of members are localized to the ER and/or Golgi, but some are in palmitoylation might be important for their specific roles and fact found at the plasma membrane (Ohno et al., 2006). Although functional compensation. DHHC PATs are likely to account for most palmitoylation events, Many signaling proteins are palmitoylated (Milligan et al., 1995; there might be substrate-specific PATs that are not members of Resh, 1999; Smotrys and Linder, 2004). The notion that the DHHC family (Linder and Deschenes, 2007). Taken together palmitoylation of SFKs is crucial for proper trafficking and with the hypothesis that Fyn is targeted directly to the plasma function reinforces the possibility that the trafficking of other

Journal of Cell Science membrane without a requirement for the exocytic machinery (van’t palmitoylated proteins, such as Ras GTPases and eNOS, is Hof and Resh, 1997; Bijilmakers and Marsh, 2003), it would be specified by their palmitoylation state and thus might lead to their reasonable to assume that PATs localizing to the Golgi region and specific functions. In fact, monopalmitoylation of H-Ras on the plasma membrane, although they have not yet been identified, Cys184 results primarily in a Golgi localization and impairs its are responsible for palmitoylation of Lyn-Yes and Fyn, ability to activate Raf, MEK and ERK (Roy et al., 2005). For respectively. eNOS, mutation of the palmitoylation sites blocks tight perinuclear Lyn and Yes are precisely colocalized with the Golgi pool of targeting and nitric oxide release from cells (Liu et al., 1996). caveolin (supplementary material Fig. S2) (Kasahara et al., 2004); Palmitoylation might be essential for many proteins as well as they also partially colocalize with GalT and GM130, which are SFKs, for proper trafficking and localization to fulfil their function. Golgi-resident proteins (Fig. 1C, Fig. 3E; supplementary material Furthermore, SFKs are abundantly expressed throughout the Fig. S1C and Fig. S3B,D). Inhibition of protein synthesis by CHX central nervous system (CNS) and regulate ion channel activity results in a chase of Lyn and Yes to the plasma membrane, with a and synaptic transmission in the developed CNS (Wang and Salter, concomitant chase of the Golgi pool of caveolin (this study) 1994; Thomas and Brugge, 1997). In neurons, palmitoylation has (Kasahara et al., 2004), but it does not allow GalT and GM130 to a key role in targeting proteins for transport to nerve terminals move from the Golgi complex to the plasma membrane (data not and for regulating trafficking at synapses (El-Husseini and Bredt, shown). Given that the Golgi pool of caveolin accounts for the 2002; Huang and El-Husseini, 2005). It is therefore attractive to intracellular pool of the protein in the presence of continual exit of speculate that SFK members might be deeply involved in neuronal caveolin in exocytic vesicles (Nichols, 2002), we presume that functions through their palmitoylation-regulated, spatiotemporal perinuclear Lyn and Yes localize to caveolin-positive exocytic trafficking. vesicles in the Golgi region. Considering the importance of protein trafficking in their proper localization and function, there is great interest in understanding Implications for roles of the distinct trafficking of SFKs the mechanisms that regulate palmitoylation-induced protein sorting Recent evidence provides a novel view that endomembranes, such and ‘cracking’ the palmitoylation codes within proteins to reveal as Golgi and ER membranes, serve as a platform of signaling how a palmitoylated peptide sequence relates to the final destination molecules. For example, Ras that is restricted to endomembranes of a protein in the cell. Like the search for palmitoylation consensus was shown to couple to the ERK-signaling cascade (Chiu et al., sequences, these questions are likely to present a significant 2002). It is also shown that phosphatidylinositol-3,4,5- challenge. 974 Journal of Cell Science 122 (7)

Materials and Methods fixed in 3% paraformaldehyde for 20 minutes. Fixed cells were permeabilized and Plasmids blocked in PBS containing 0.1% saponin and 3% bovine serum albumin for 30 minutes, cDNAs encoding human Lyn (Yamanashi et al., 1987), human Yes (Sukegawa et al., and then incubated with a primary and a secondary antibody for 1 hour each. After 1987), and human Fyn (Tezuka et al., 1999) were provided by Tadashi Yamamoto (The washing with PBS containing 0.1% saponin, cells were mounted with ProLong antifade University of Tokyo, Japan). Lyn and Yes were subcloned into the pcDNA4/TO vector reagent (Molecular Probes). Confocal images were obtained using an LSM510 (Carl (Invitrogen) as described (Kasahara et al., 2004; Kuga et al., 2007), and Fyn was also Zeiss) and a Fluoview FV500 (Olympus, Tokyo, Japan) laser-scanning microscope with subcloned into pcDNA4/TO. Fyn24-Lyn was generated by fusion of the N-terminal a ϫ40 0.75 NA or ϫ63 1.40 NA oil-immersion objective, or a ϫ40 1.00 NA oil or a sequence of Fyn (1-24; with 1 designating the initiator methionine) with Lyn lacking ϫ60 1.00 NA water-immersion objective. 200-400 cells were scored for each assay. the N-terminal sequence (26-512), and the linker sequence Gly-Arg-Ser-Ser was inserted For immunofluorescence of THP-1 and Dami cells, cells in suspension were directly between Fyn(1-24) and Lyn(26-512). The CysrSer mutation at position 6 in Fyn(C6S) fixed with 3% paraformaldehyde and then attached on coverslips by brief and Fyn24(C6S)-Lyn was created by PCR using Fyn or Fyn(24)-Lyn as a template cytocentrifugation. Composite figures were prepared using Photoshop 5.0 and Illustrator with the sense primer 5Ј-ACAGGATCCGCCACCATGGGCTGTGTGCAAAGTA - 9.0 software (Adobe). AGGATAAAG-3Ј and the respective antisense primer 5Ј-TTGCATATGAGTCC - ATTCCCCAC-3Ј or 5Ј-GGAGCGGCCGCCCTGTGCTCTAAGGCTGCTGCTG-3Ј. Temperature block The SerrCys mutation at position 6 in Yes(S6C) was created by PCR using Yes as a COS-1 cells transfected with Lyn or Yes were incubated at 37°C for 15 hours and template with the sense primer 5Ј-AACGGTACCGCCGCCACCATGGGCTGCAT - subsequently treated with 100 μg/ml cycloheximide (CHX) and incubated at 19°C or TAAATGTAAAG-3Ј and the antisense primer 5Ј-CATGGTCGGTCCTCTTTT - 37°C for 3 hours in medium supplemented with 25 mM HEPES (pH 7.4). For release AAATCCAGATCTCGT-3Ј. To construct Fyn24-Yes, Yes lacking the N-terminal from temperature block, the cells incubated at 19°C for 3 hours were warmed to 37°C sequence (25-543) was created by PCR using Yes as a template with the sense primer for further 2 hours. Dami cells were equilibrated in medium supplemented with 25 mM 5Ј-AACAGTCGACAGTCAGTACAAGTGTGAGCCA-3Ј and the antisense primer HEPES (pH 7.4) and then incubated at 15°C, 19°C or 37°C for 3 hours in suspension 5Ј-CGAAGATCTCGGTGAATATAGTT-3Ј, and Fyn(1-24) was fused with Yes(25-543), culture. and the linker sequence Gly-Arg-Ser-Thr was inserted between Fyn(1-24) and Yes(25- 543). Lyn-GFP was constructed as described previously (Kasahara et al., 2004). To VSVG-GFP transport assay construct Yes-GFP, Yes(1-543) was amplified by PCR using the sense primer COS-1 cells transfected with VSVG-GFP alone or VSVG-GFP plus HA-Rab11S25N 5Ј-AACGGTACCGCCGCCACCATGGGCTGCATTAAAAGTAAAG-3Ј and the were incubated at 40°C for 15 hours in medium supplemented with 25 mM HEPES antisense primer 5Ј-CATGGTCGGTCCTCTTTTAAATCCAGATCTCGT-3Ј. The (pH 7.4) and then shifted to 32°C for 30 minutes or 6 hours in the presence of 200 resulting Yes(1-543) was fused with GFP cDNA obtained from the pEGFP-C1 vector μg/ml CHX during the last 3 hours. (Clontech) at the terminus of Yes(1-543), and the linker sequence Gly-Leu-Glu-Arg- Pro-Ser-Asn-Ser-Cys-Ser-Pro-Gly-Asp-Pro-Leu-Val-Leu-Ala-Leu-Pro-Val-Ala-Thr was Photobleaching experiments inserted between Yes(1-543) and GFP. To construct Fyn-GFP, Fyn was amplified by Fluorescence recovery after photobleaching (FRAP) experiments were performed as PCR using the sense primer 5Ј-ACAGAATTCGCCGCCACC ATGGG CTGTG TG - described (Kasahara et al., 2004; Kasahara et al., 2007a) using a Fluoview FV500 laser CAATGTAAGG-3Ј and the antisense primer 5Ј-CATGGT TG GACCACTTTTGG - scanning microscope with a ϫ40 1.00 NA oil-immersion objective. COS-1 cells ACCCAAGTCCTTCGGACCTAGGCTT-3Ј. The resulting Fyn(1-536) was fused with transfected with Lyn-GFP, Yes-GFP, or Fyn-GFP were cultured for 20 hours. Cells GFP cDNA obtained from pEGFP-C1 at the C-terminus of Fyn(1-536), and the linker were maintained at 37°C or 19°C throughout photobleaching experiments. The whole sequence Gly-Ser-Gly-Ser-Leu-Asp-Pro-Leu-Val-Leu-Ala-Leu-Pro-Val-Ala-Thr was cell area was photobleached at 100% laser power and recovery of fluorescence was inserted between Fyn(1-536) and GFP. These constructs were subcloned into monitored by scanning the perinuclear area or whole cell area excluding perinuclear pcDNA4/TO. HA-Rab11S25N subcloned into the pcDNA3.1 vector (Invitrogen) was region at low laser power (3.0%). The mean fluorescence intensities within regions of provided by Stephen Ferguson (Dale et al., 2004). The plasmid for vesicular stomatitis interest were quantified using the ImageJ software (National Institutes of Health, USA). virus-encoded glycoprotein fused to green fluorescent protein (VSVG-GFP) was Composite figures were prepared using Photoshop 5.0 and Illustrator 9.0 software provided by Jennifer Lippincott-Schwartz (Presley et al., 1997) through Mitsuo Tagaya (Adobe). (Hirose et al., 2004). Western blotting Antibodies Cells were lysed in SDS-PAGE sample buffer at 4°C, and the samples were boiled for The following mouse monoclonal antibodies were used: Lyn (H6; Santa Cruz 3 minutes, subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Journal of Cell Science Biotechnology), Yes (clone 1; BD Biosciences, and 1B7; Wako Pure Chemicals, Osaka, electrotransferred onto polyvinylidene difluoride membranes (Millipore). Japan), Fyn (Fyn301; Wako Pure Chemicals, Osaka, Japan), HA epitope (F-7; Santa Immunodetection was performed by enhanced chemiluminescence (Amersham Cruz Biotechnology), actin (Chemicon International), phosphotyrosine [Tyr-P, 4G10; Biosciences) as described (Mera et al., 1999; Matsuda et al., 2006; Kuga et al., 2007; Upstate Biotechnology.; provided by Toshiki Tamura and Takayuki Yoshimoto (Tamura Kasahara et al., 2007c; Ikeda et al., 2008). Sequential reprobing of membranes with a et al., 2000)], and GM130 (clone 35, BD Transduction Laboratories). The following variety of antibodies was performed after the complete removal of primary and secondary rabbit polyclonal antibodies were used: Lyn (Lyn44, Santa Cruz Biotechnology), Fyn antibodies from membranes in stripping buffer or inactivation of HRP by 0.1% NaN3, (FYN3; Santa Cruz Biotechnology), HA epitope (Y11, Santa Cruz Biotechnology), according to the manufacturer’s instructions. Results were analyzed using an image calnexin (Stressgen Bioreagents, British Columbia, Canada), β-1,4-galactosyltransferase analyzer LAS-1000plus equipped with Science Lab software (Fujifilm, Tokyo, Japan). (GalT) [(Yamaguchi and Fukuda, 1995); provided by Michiko Fukuda], caveolin For detection of tyrosine phosphorylation (supplementary material Fig. S5), cells were (Transduction Laboratories), and Src[pY418] (Src-P-family; Cell Signaling Technology). lysed in SDS-PAGE sample buffer containing 10 mM unbuffered HEPES and 10 mM HRP-F(abЈ)2 of anti-mouse IgG and anti-rabbit Ig secondary antibodies were from Na3VO4 at 4°C, and the samples were subjected to SDS-PAGE. Amersham Biosciences; FITC-F(abЈ)2 of anti-rabbit IgG or of anti-mouse IgG, TRITC- anti-rabbit IgG, TRITC-anti-mouse IgG (Fc) secondary antibodies were from BioSource We are indebted to Tadashi Yamamoto, Stephen S. G. Ferguson, International and Sigma. Jennifer Lippincott-Schwartz, Mitsuo Tagaya, Toshiki Tamura, Takayuki Cells and transfection Yoshimoto, Atsushi Iwama and Michiko N. Fukuda for valuable plasmids, COS-1 cells were cultured in Iscove’s modified DME containing 5% fetal bovine serum antibodies, and cell lines. This work was supported in part by grants-in- at 37°C in a 5% CO2 incubator. Transient transfection was performed using TransIT aid for Scientific Research from the Japanese Ministry of Education, transfection reagent (Mirus) (Kasahara et al., 2007b; Kasahara et al., 2008) or linear Culture, Sports, Science, and Technology and a research grant from the polyethylenimine (25 kDa; Polysciences, Warrington, PA) (Durocher et al., 2002). 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