¹Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, Matsumoto, Japan 390-8621

²IRSP, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, MD 21702, USA

³Wlodawer's Program in Structural Biology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, MD 21702, USA

Correspondence to: T Kamata, Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan; E-mail: Kamatat@sch.md.shinshu-u.ac-jp

A 19 kDa protein was identified to associate with the Dbl oncogene homology domain of Sos1 (Sos-DH) and was purified from rat brains by GST-Sos-DH affinity chromatography. Peptide sequencing revealed that the protein is identical to light chain 3 (LC3), a microtubule-associated protein. LC3 coimmunoprecipitated with Sos1, and GST-LC3 was capable of forming complexes with Sos1 in in vitro GST-pull down assay. Furthermore, LC3 was colocalized with Sos1 in cells, as determined by immunohistochemistry. While Sos1 stimulated the guanine nucleotide exchange reaction on Rac1, LC3 suppressed the ability of Sos1 to activate Rac1 in in vitro experiments using COS cell lysates. Consistent with this, overexpression of LC3 decreased the level of active GTP-bound Rac1 in COS cells. Sos1 expression induced membrane ruffling, a downstream target for Rac1, but LC3 expression inhibited this biological effect of Sos1. These findings suggest that LC3 interacts with Sos1 and thereby negatively regulates the Sos1-dependent Rac1 activation leading to membrane ruffling

Oncogene (2002) 21, 7060-7066. doi:10.1038/sj.onc.1205790

Sos1; Rac1; light chain 3

Introduction

Sos1 was initially identified as a guanine nucleotide exchange factor for Ras which regulates cellular activities such as cell growth and differentiation (Downward, 1996). A carboxyl terminal domain homologous to a yeast CDC25 catalyzes the exchange of GDP bound to Ras for GTP. In addition, the amino terminal region of Sos1 possesses a Dbl homology (PH) domain in tandem with a pleckstrin homology (pH) domain. Both domains have been shown to be required for the transforming and growth stimulatory activities of Sos1 (Whitehead et al., 1997). Like the DH domain of other members of the Dbl family regulating the Rho GTPases, the DH domain of Sos1 has been demonstrated to serve as a GDP-GTP exchange factor for Rac1 and thereby activate the JNK activity and membrane ruffling (Nimnual et al., 1998). However, several lines of evidence suggest an additional regulatory role for the DH domain of Sos1. The overexpressed Sos1 NH₂-terminus, which contains the DH domain, appeared to compete with endogenous Sos1 for binding to a membrane protein(s), resulting in interference of both growth factor-induced DNA synthesis and MAP kinase activation (Qian et al., 1998). These observations suggest that the NH₂-terminal domain is required for physiological function of Sos1, and that the identification of a cellular component(s) interacting with the domain could be critical for understanding of the regulatory mechanism of Sos1 (Wang et al., 1995; Qian et al., 1998).

Light chain 3 (LC3) was identified as a subunit of the neuronal microtubule-associated proteins (MAPs), MAP1A and MAP1B (Mann and Hammarback, 1994). LC3 is abundant in neurons, but a low level of LC3 is expressed in other tissues. Although LC3 has been implicated in the regulation of the microtubule binding activity of MAP1A and MAP1B in neuronal cells, its exact role is unclear. Recently, LC3 was found to be homologous to Apg8p/Aut7p which is essential for yeast autophagy and associates with autophagosome membranes (Kirisako et al., 1999; Liang et al., 1999). LC3 was processed post-translationally into LC3-I, a cytosolic form and LC3-II, a membrane-bound form, and starvation of cells enriched LC3-II proteins into autophagic vesicles in mammalian cells (Kabeya et al., 2000). These findings suggest a vital role for LC3 in the autophagosome formation. The relationships between LC3 binding to microtubules and LC3 localization in phagosomes are currently unknown. Another LC3 homologue, -aminobutyric acid A receptor-associated protein (GABARAP) was identified and GABARAP seems to function in clustering of the receptors via tubulin molecules (Wang et al., 1999).

In this study, we analysed the proteins interacting with the Sos-DH. Our data indicate that LC3 binds to the Sos-DH, and that thereby inhibits the activity of Sos1 to promote the guanine nucleotide exchange reaction on Rac1. Furthermore, the data suggest that interaction of Sos1 with LC3 may serve for the negative regulation of the Sos1-Rac1 signaling involved in cytoskeleton rearrangement such as membrane ruffling.

Results and discussion

To identify the proteins interacting with the Sos-DH domain, the Sos-DH fused to GST (GST-Sos-DH) was immobilized to GST-sepharose beads, and the resins were incubated with cytosol or membrane fractions from NIH3T3 cells metabolically labeled with [³⁵S]cysteine/methionine. 19 kDa proteins in the membrane but not cytosol fraction were found associated with GST-Sos-DH affinity resins (Figure 1a). When membrane extracts prepared from rat brains were affinity purified with the resins, a higher level of 19 kDa proteins (hereafter indicated as p19) was detected compared to that in NIH3T3 cells. To further characterize p19, the proteins were purified from rat brain membrane fractions by GST-Sos-DH affinity chromatography (Figure 1b), and purified p19 peptides were subjected to amino acid sequencing. Amino acid sequences of three peptides were identical to residues 31-39, 42-48, and 51-62 of light chain 3 of the microtubule-associated protein 1A and 1B (Table 1). Moreover, the antibody directed to the NH₂-terminus of LC3 recognized with purified p19 in immunoblotting (data not shown). From these observations, we conclude that p19 is identical to LC3.

The data presented above imply the interaction of LC3 with Sos1 via the Sos-DH domain. To test this possibility, in vitro binding studies were performed by using GST-pull down assay. GST-LC3 fusion protein-coupled resins were prepared and incubated with lysates from Sos1-transfected COS cells. The analysis of proteins bound to the resins by immunoblotting with anti-Sos1 antibodies showed that Sos1 was associated with GST-LC3 but not GST alone (Figure 2a). To address a question of whether the LC3-Sos1 interaction is physiological, endogenous LC3 proteins in COS cell lysates were immunoprecipitated with rabbit antibodies against LC3, and the immunoprecipitates were probed with anti-Sos1 antibodies. As shown in Figure 2b, LC3 coimmunoprecipitated with Sos1. Reciprocally, the immunoprecipitates by anti-Sos1 antibodies were probed with antibodies against LC3, and Sos1 coimmunoprecipitated with LC3 (Figure 2b). The data suggest that LC3 is complexed with Sos1 in vivo. To examine whether LC3 binding to Sos1 is a consequence of direct interaction between the two proteins, Sos1 proteins were expressed in insect cells using baculovirus expression system and purified homogeneously. When purified Sos1 proteins were incubated with GST-LC3 protein-coupled resins, Sos1 bound to GST-LC3 but not GST (Figure 2c), indicating that LC3 is able to bind to Sos1 without cellular co-factors. To further assess the LC3-Sos1 interaction in untransfected cells, COS cells were double-stained with polyclonal anti-LC3 antibodies and monoclonal anti-Sos1 antibodies. LC3 was colocalized with Sos1 (Figure 3) at cytoplasmic and perinuclear regions. The data support the results obtained with above binding studies.

Since Sos1 serves as a potent nucleotide exchanger for Rac1 (Nimnual et al., 1998), interaction of LC3 with Sos1 might affect the ability of Sos1 to activate Rac1. To address this possibility, the effect of LC3 on the Sos1-catalysed nucleotide exchange on Rac1 was examined in vitro. COS cells were cotransfected with c-myc-LC3 and Sos1 expression vectors, cell lysates were reacted with [³H]GDP·Rac1 binary complexes, and the GDP release promoting activity was determined in time course experiments. While overexpression of Sos1 alone significantly stimulated the release of GDP bound to Rac1, coexpression of c-myc-LC3 with Sos1 decreased the amount of GDP release (Figure 4a), suggesting that LC3 inhibits the Sos1 nucleotide exchange promoting activity for Rac1. To detect the activation of Rac1 by transfected Sos1, the serum stimulation of cells was essential, which may indicate the involvement of an additional upstream signal(s) through a growth factor receptor(s). We next assessed the suppressive effect of LC3 on Rac1 activation in vivo. The activation state of Rac1 in cells was determined by measuring the amount of GTP-bound Rac1 associated to the Rac1-binding domain of p21-activated kinase (Pak). When Sos1 was transfected into COS cells, we were able to detect an increase in the GTP-bound form of Rac1. However, the level of GTP-bound Rac1 was significantly reduced when Sos1 was cotransfected with LC3 (Figure 4b). This indicates that LC3 can block the Sos1-mediated activation of Rac1 in cells, which is in agreement with the in vitro assay of LC3 effect on the Sos1 nucleotide exchange promoting activity for Rac1. When in vitro exchange reactions were performed with purified baculovirus Sos1 and GST-LC3 proteins, the Sos1 exchange activity was partially inhibited by LC3 (50% inhibition) (data not shown), which might reflect the requirement of some cellular cofactor for full inhibition.

Rac1 affects cytoskeleton organization, and activation of Rac1 induces membrane ruffling in fibroblast cells which is closely associated with early response of cells to mitotic stimulation (van Aelst and D'Souza-Schorey, 1997). Since LC3 inhibited the Rac1 activation by Sos1, we reasoned that LC3 might suppress membrane ruffling as a downstream cellular activity for the Rac1 signaling. To test this possibility, COS cells were transfected with c-myc-tagged LC3 and stimulated with 30% serum. While untransfected cells normally exhibited membrane ruffling in response to growth stimulation (72% of counted cells are membrane ruffling positive) (Figure 5c), cells overexpressing LC3 significantly reduced the production of membrane ruffles (14% of counted cells are membrane ruffling positive) (Figure 5d,e). Phalloidin staining patterns in COS cells not stimulated with serum showed no induction of membrane ruffling (Figure 5a) and LC3 overexpression had no essential effect on the actin distribution (Figure 5a,b). Control vector had no detectable effect (data not shown). We next examined the inhibitory effect of LC3 on the Sos1-induced membrane ruffling. HA-tagged Sos1 or c-myc-tagged LC3 was introduced into COS cells. Overexpression of Sos1 caused membrane ruffling (Figure 5f,g), but coexpression of Sos1 and LC3 reduced accumulation of polymerized F-actin at the focal contact (Figure 5h-j). Furthermore, when cells were cotransfected with Rac1Val12 (an active form of Rac1) and LC3, LC3 had no suppressive effect on Rac1Val12-induced membrane ruffling (Figure 5k-m), suggesting that inhibition of Sos1-induced membrane ruffling by LC3 depends on the event upstream of Rac1-inhibition of the Sos1 activity by LC3. Thus, LC3 has negative regulatory effects on the serum- and Sos1-induced membrane ruffling.

Sos1 is capable of controlling the activity of Rac1, acting as a guanine nucleotide exchange factor through the DH domain (Nimnual et al., 1998), but little is known about the precise molecular mechanism of the Sos1 regulation. The data presented here suggest that Sos1, via the DH domain, interacts with LC3, and that LC3 exerts a suppressive effect on the Sos1-dependent Rac1 signaling which leads to membrane ruffling. It is most likely that LC3 negatively regulates Rac1 by inhibiting the catalytic activity of Sos1 to promote the exchange of GDP for GTP on Rac1. An upstream activation signal(s) could release this repression in some way and trigger the Sos1-mediated Rac1 signal transduction. The structural basis for the suppression of the Sos1 activity by LC3 is not clear at present: LC3 binding to the Sos-DH domain might cause steric hindrance for Sos1-Rac1 interaction. The distribution of LC3 in cells that overexpress Sos1 (Figure 5h) appears to be different from that of LC3 in cells that do not co-express Sos1 (Figure 5d). The cause for this different distribution is not clear at present. We do not rule out the possibility that LC3 serves to sequester Sos1 in a compartment that prevents its association with Rac1, resulting in the inhibition of Sos1-Rac1 signaling.

Recently, another Dbl-related protein, Lfc, that mediates the activation of Rac1 signaling in cells, was found to localize to microtubules (Glaven et al., 1999). Lfc appears to bind to microtubules directly and its PH domain is responsible for this colocalization. In contrast, we were unable to establish clear localization of mSos1/LC3 complexes at microtubules in COS cells (data not shown) although LC3 was first reported as a neuronal microtubule-associated protein (Mann and Hammarback, 1994). Other researchers also reported that most of LC3 did not colocalize with microtubules (Kabeya et al., 2000). Our data show that LC3 distributed at cytoplasmic and perinuclear regions in COS cells, and that its staining pattern was distinct from microtubule networks (data not shown). Thus, our observation suggests that Sos1 regulates the Rac1 signaling in a manner different from Lfc. In conclusion, our study gives a clue about the functional role of LC3 and provides a new insight into the mechanism by which Sos1 regulates the Rac1 signaling pathway leading to cytoskeleton organization.

Materials and methods

GST-DH affinity chromatography

For identification of p19 interacting with the Sos-DH domain, NIH3T3 cells were metabolically labeled for 4 h with [³⁵S]cysteine/methionine (ICN, CA). The cells were disrupted in homogenization buffer A [10 mM Tris-HCl, pH 7.5, 5 mM EGTA, 5 mM MgCl_2, 1 mM DTT, 10% sucrose, 10 M phenylmethyl sulfonyl fluoride (PMSF) and 1 g/ml leupeptin] and centrifuged at 1000 r.p.m. for 5 min. After centrifugation at 40 000 r.p.m. for 10 min, the supernatant was saved as a cytosol fraction. The pellets were extracted with 2 M NaCl in buffer A. After centrifugation at 40 000 r.p.m. for 10 min, the supernatant was dialysed against buffer B (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 10 M PMSF and 1 g/ml leupeptin) and used as a membrane fraction. GST and GST-Sos-DH fusion proteins coupled to glutathione agarose beads (20 l) were incubated with the cytosol or membrane extracts at 4°C for 2 h. After extensive wash in buffer B plus 20 mM NaCl, proteins retained to the beads were resolved by SDS-PAGE and visualized by fluorography. For a large scale of purification, membrane extracts prepared from 30 rat brains were fractionated by GST-DH affinity chromatography. p19 proteins were eluted with 10 mM glutathione from the column and analysed with SDS-PAGE, followed by silverstaining as described.

Amino acid sequencing

Purified p19 proteins were electrotransferred to a polyvinylidene difluoride membrane and digested by endopeptidase LysC and the purified peptides were subjected to amino acid sequencing as described previously (Kamata et al., 1998).

GST pull down assay

GST-LC3 (BamHI-EcoRI site of pGEX-2T) fusion proteins were expressed in E. coli cells and immobilized to glutathione-S-transferase beads according to the company's protocol (Amersham Pharmacia, IL, USA). COS-1 cells were homogenized in buffer C (20 mM Tris-Cl, pH 7.5, 20 mM NaCl, 0.2% Nonidet P-40 (NP-40) and 1 mM PMSF), and cell lysates were prepared by centrifugation at 15 000 r.p.m. for 10 min. The extracts were incubated with GST-LC3 beads at 4°C for 4 h and after washing with PBS, the bound proteins were analysed by SDS-PAGE, followed by immunoblotting with rabbit anti-Sos1 antibodies (Santa Cruz, CA, USA). Alternatively, baculovirus recombinant Sos1 proteins were produced in SF-9 insect cells as described previously (Kubiseski et al., 1997) and purified by DE-52 column chromatography. Purified Sos1 (100 ng), that was more than 95% pure, was incubated with GST or GST-LC3 beads at 4°C for 2 h and bound proteins were subjected to immunoblotting analysis in the same way as described above.

Immunoprecipitation study

Cell extracts were immunoprecipitated with the indicated antibodies and the blots of the immunoprecipitates were further reacted with the indicated antibodies. Protein bands were visualized with ECL (Amersham Pharmacia, IL, USA).

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde and permeabilized as described (Kamata et al., 1998). Indirect immunostaining was performed with monoclonal mouse anti-c-myc or rabbit anti-HA epitope antibodies (Eastman Kodak, CT, USA), monoclonal mouse anti-mSos1 antibodies (Transduction Lab, MA, USA), or rabbit anti-LC3 antibodies (provided by Dr Y Oshima). Antibodies were visualized with anti-mouse IgG labeled with fluorescein FITC (Sigma, MO, USA) and anti-rabbit IgG labeled with Cy5 (Amersham Pharmacia, IL, USA) as described (Kamata et al., 1998). F-actin was stained with TRITC-labeled phalloidin (10 mg/ml) (Molecular Probe, OR) for 60 min. The stained cells were observed under a Zeiss confocal microscope (Carl Zeiss, Germany).

Guanine nucleotide exchange assay

Cells were transfected with pcDNA3.1-LC3 (EcoRI-BamHI site), pcDNA3.1-Sos1 or control vector by using lipofectamine/PLUS (GIBCO-BRL, MD, USA) and cultured for 36-48 h. Cells were then serum-starved for 12 h and stimulated with 30% serum for 10 min. Cell extracts were prepared by sonication in extraction buffer (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 1 mM PMSF and 0.2% NP-40), followed by high centrifugation. [³H]GDP-Rac1 complexes were prepared by incubating GST-Rac1 with [³H]GDP. GDP-GTP exchange assay was performed by filter binding assay as described (Li et al., 1992).

Rac1 activation assay

The p21 (Rac1) binding domain (PBD) of PAK1 fused to GST (GST-PBD) was immobilized to the resins and was used to precipitate activated Rac1·GTP complexes from transfected COS cells. Cell lysates were affinity precipitated at 4°C for 1 h according to the company's protocol (Upstate Biotechnology Inc., NY, USA). Bound proteins were analysed by immunoblotting with rabbit anti-Rac1 antibodies using ECL.

Acknowledgements

We thank Drs D Lowy, K Kaibuchi and M Tanaka for the generous gifts of plasmid DNAs. We thank Dr T Yoshimori for providing anti-LC3 antibodies and Dr K Hirose for valuable discussions and encouragement. We are grateful to Drs T Takenawa and H Yamaguchi for the excellent technical assistance with confocal microscopy. We also thank Miss C Wada for preparation of the manuscript. This research was supported by the National Cancer Institute, NIH, DBS and DHHS under contract with ABL and by Terumo Life Science Foundation.

Downward J. (1996). Cancer Surv., 27: 87-100. MEDLINE

Glaven JA, Whitehead I, Bagrodia S, Kay R, Cerione RA. (1999). J. Biol. Chem., 274: 2279-2285. Article MEDLINE

Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. (2000). EMBO J., 19: 5720-5728. Article MEDLINE

Kamata T, Subleski M, Hara Y, Yuhki N, Kung HF, Copeland NG, Jenkins NA, Yoshimura T, Modi W, Copeland TD. (1998). Mol. Brain Res., 54: 219-236. MEDLINE

Kirisako T, Baba M, Ishihara N, Miyazawa K, Ohsumi M, Yoshimori T, Noda T, Ohsumi Y. (1999). J. Cell Biol., 147: 435-446. Article MEDLINE

Kubiseski TJ, Chook YM, Parris WE, Rozakis-Adcock M, Pawson T. (1997). J. Biol. Chem., 272: 1799-1804. Article MEDLINE

Li BQ, Kaplan D, Kung HF, Kamata T. (1992). Science, 256: 1456-1459. MEDLINE

Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, Levine B. (1999). Nature, 402: 672-676. Article MEDLINE

Mann SS, Hammarback JA. (1994). J. Biol. Chem., 269: 11492-11497. MEDLINE

Nimnual AS, Yatsula BA, Bar-Sagi D. (1998). Science, 279: 560-563. Article MEDLINE

Qian X, Vass WC, Papageorge AG, Anborgh PH, Lowy DR. (1998). Mol. Cell. Biol., 18: 771-778. MEDLINE

Van Aelst L, D'Souza-Schorey C. (1997). Genes Dev., 11: 2295-2322. MEDLINE

Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW. (1999). Nature, 397: 69-72. Article MEDLINE

Wang W, Fisher EM, Jia Q, Dunn JM, Porfiri E, Downward J, Egan SE. (1995). Nature Genet., 10: 294-300. MEDLINE

Whitehead IP, Campbell S, Rossman KL, Der CJ. (1997). Biochem. Biophys. Acta, 1332: F1-F23.

Figure 1 Identification and purification of p19. (a) Binding of metabolically labeled p19 to GST-Sos-DH. NIH3T3 cells were metabolically labeled with [³⁵S]cysteine/methionine for 4 h. Extracts of the cytosol (cyto) and membrane (memb) fractions were prepared and incubated with GST- or GST-Sos-DH-immobilized glutathione agarose beads. Bound proteins were eluted with glutathione and resolved by SDS-PAGE, followed by flurography. Note that protein bands at the molecular mass region of 28 kDa were compressed because of the overloaded unlabeled GST proteins. (b) Purification of p19 from rat brains. Rat brain membrane fractions were loaded onto a GST-Sos-DH column (5 ml). Bound proteins were eluted with 200 mM NaCl after washing with 50 mM NaCl and analysed by SDS-PAGE, followed by silver staining. Number above each lane shows fraction number. The band right below p19 appears to be a proteolytic product of p19

Figure 2 Physical interaction of Sos1 with LC3. (a) Association of Sos1 with LC3 in cell free system. Cell lysates (30 g) prepared from COS-1 cells were incubated with 10 g of GST (lane 2) or GST-LC3 (lane 3) proteins-immobilized resins as described in Materials and methods. The proteins bound to the resins were analysed by SDS-PAGE, followed by immunoblotting with rabbit anti-mSos1 antibodies. Lane 1 shows immunoblotting of cell lysate alone with the anti-Sos1 antibodies. (b) Coimmunoprecipitation of Sos1 with LC3. Cell lysates prepared from COS-1 cells were immunoprecipitated with control IgG (lanes 1 and 3), rabbit anti-LC3 antibodies (lane 2) or rabbit anti-mSos1 antibodies (lane 4). Then, the immunoprecipitates were analysed by immunoblotting with rabbit anti-maasos1 antibodies (lanes 1 and 2) or rabbit anti-LC3 antibodies (lanes 3 and 4). (c) Binding of purified Sos1 to GST-LC3. Purified baculovirus mSos1 proteins (0.2 g) were incubated with GST (lanes 2 and 4)- or GST-LC3 (lanes 3 and 5)-immobilized resins, and bound proteins were analysed with immunoblotting with rabbit anti-mSos1 antibodies. Lane 1 shows immunoblotting of purified Sos1 proteins with the anti-Sos1 antibodies as a positive control

Figure 3 Localization of Sos1 and LC3 in COS cells. COS-1 cells were fixed and stained with indirect immunofluorescence. Pictures show confocal micrographs of double-stained cells. Sos1 was visualized with monoclonal mouse anti-mSos1 antibodies and FITC-labeled anti-mouse IgG (green). LC3 was detected with rabbit anti-LC3 antibodies and Cy3-labeled anti-rabbit IgG (red)

Figure 4 LC3 suppresses the Sos1 activity for Rac1. (a) Effect of LC3 expression on the Sos1 activity. COS cells (10 cm´1) were transfected with pcDNA3.1 (6 g) (open circles), pcDNA-Sos1 (1 g) plus pcDNA3.1 (5 g) (closed circles), or pcDNA-Sos1 (1 g) plus pcDNA-c-myc-LC3 (5 g) (triangles). Lysates were prepared and reacted with [³H]GDP-loaded GST-Rac1 as described in Materials and methods. The amounts of [³H]GDP retained to Rac1 were quantitated at the times indicated during the reaction. The expression of transfected Sos1 and c-myc-LC3 was determined by immunoblotting with -c-myc Ab (IP:). The data show the percentage of Rac1 in the GDP-bound form and represent the mean value of duplicate experiments. (b) Effect of LC3 on the Rac1 activity in vivo. COS cells (10 cm´1) were cotransfected with pcDNA-c-myc-LC3 (5 g) together with pcDNA-Sos1 (1 g) or a control vector. Cells were lysed at 48 h post-transfection and lysates were affinity precipitated with GST-PBD. Activated Rac1 bound to GST-PBD was analysed with immunoblotting with anti-Rac1 antibodies (1). The expression of transfected Sos1 and c-myc-LC3 was confirmed by immunoblotting with anti-Sos1 antibodies and anti-c-myc-antibodies (2). The expression level of endogenous Rac1 was examined by immunoblotting using anti-Rac1 antibodies (3)

Figure 5 LC3 inhibits induction of membrane ruffling. COS cells were transfected with c-myc-LC3 (a and b) and serum-starved for 16 h. Arrows in a and b indicate LC3 expressing cells. COS cells were transfected with (d and e) or without c-myc-LC3 (c), serum-starved for 16 h and stimulated with 30% serum for 15 min. Alternatively, HA-Sos1 alone was expressed (f and g) or both HA-Sos1 and c-myc-LC3 were coexpressed in COS cells (h, i and j). Arrows in h, i and j indicate the cells coexpressing both Sos1 and LC3. For studies on the LC3-effect on the activated Rac1-induced membrane ruffling, COS cells were cotransfected with the dominant active Rac1 (Rac1Val12) and c-myc-LC3 (k, l and m). a, c, e, g, j and m show phalloidin staining of actin. b, d, f, h, i, k and l show expression of exogenous LC3, LC3, Sos1, LC3, Sos1, Rac1 and LC3, respectively. c-myc-LC3 was stained with mouse monoclonal anti-c-myc antibodies, followed by FITC-conjugated anti-mouse IgG (b, d and h) staining. Alternatively, c-myc-LC3 was stained with rabbit anti-LC3 antibodies and Cy5-conjugated anti-rabbit IgG (l). HA-Sos1 was visualized with rabbit anti-HA antibodies, followed by FITC-conjugated anti-rabbit IgG (f) or Cy5-conjugated anti-rabbit IgG (i) staining. Rac1 was stained with mouse monoclonal anti-Rac1 antibodies, followed by FITC-conjugated anti-mouse IgG (k). Thirty-five cells per field were counted in several fields and 70~80% of counted cells exhibited the phenotype shown

Table 1 Amino acid sequences of p19 peptides