Portal de Periódicos da CAPES

SH2-containing 5′-Inositol Phosphatase, SHIP2, Regulates Cytoskeleton Organization and Ligand-dependent Down-regulation of the Epidermal Growth Factor Receptor

2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês

10.1074/jbc.m410289200

1083-351X

K. Nagendra Prasad, Stuart J. Decker,

Protein Kinase Regulation and GTPase Signaling

Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3′-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors. Phosphoinositide lipid second messengers are integral components of signaling pathways mediated by insulin, growth factors, and integrins. SHIP2 dephosphorylates phosphatidylinositol 3,4,5-trisphosphate generated by the activated phosphatidylinositol 3′-kinase. SHIP2 down-regulates insulin signaling and is present at higher levels in diabetes and obesity. SHIP2 associates with p130Cas and filamin, regulators of cell adhesion/migration and cytoskeleton, influencing cell adhesion/spreading. Type I collagen specifically induces Src-mediated tyrosine phosphorylation of SHIP2. To better understand SHIP2 function, we employed RNA interference (RNAi) approach to silence the expression of the endogenous SHIP2 in HeLa cells. Suppression of SHIP2 levels caused severe F-actin deformities characterized by weak cortical actin and peripheral actin spikes. SHIP2 RNAi cells displayed cell-spreading defects involving a notable absence of focal contact structures and the formation of multiple slender membrane protrusions capped by actin spikes. Furthermore, decreased SHIP2 levels altered distribution of early endocytic antigen 1 (EEA1)-positive endocytic vesicles and of vesicles containing internalized epidermal growth factor (EGF) and transferrin. EGF treatment of SHIP2 RNAi cells led to the following: enhanced EGF receptor (EGFR) degradation; increased EGFR ubiquitination; and increased association of EGFR with c-Cbl ubiquitin ligase. Taken together, these experiments demonstrate that SHIP2 functions in the maintenance and dynamic remodeling of actin structures as well as in endocytosis, having a major impact on ligand-induced EGFR internalization and degradation. Accordingly, we suggest that, in HeLa cells, SHIP2 plays a distinct role in signaling pathways mediated by integrins and growth factor receptors. SH2 1The abbreviations used are: SH2, Src homology 2; SHIP2, SH2-containing inositol phosphatase-2; PI, phosphatidylinositol; PI3K, PI 3′-kinase; PIP3, phosphatidylinositol 3,4,5-triphosphate; PI-3,4-P2, PI 3,4-bisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; EGF, epidermal growth factor; EGFR, EGF receptor; PH, pleckstrin homology; RNAi, RNA interference; siRNA, small interfering RNA; Cbl, c-Cbl; EEA1, early endocytic antigen 1; TRITC, tetramethylrhodamine isothiocyanate; RT, reverse transcription; E3, ubiquitin-protein isopeptide ligase; d.n., dominant negative.-containing inositol phosphatase-2 (SHIP2), also called INPPL1 (inositol polyphosphate 5′-phosphatase-like protein-1), is a lipid phosphatase that dephosphorylates phosphorylated phosphatidylinositol (PI) and inositol molecules on the 5′-position of the inositol ring (1.Woscholski R. Parker P.J. Trends Biochem. Sci. 1997; 22: 427-431Abstract Full Text PDF PubMed Scopus (71) Google Scholar). In particular, phosphatidylinositol 3,4,5-trisphosphate (PIP3), produced by PI 3′-kinase (PI3K), is an important substrate of SHIP2 (2.Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar, 3.Wisniewski D. Strife A. Swendeman S. Erdjument-Bromage H. Geromanos S. Kavanaugh W.M. Tempst P. Clarkson B. Blood. 1999; 93: 2707-2720Crossref PubMed Google Scholar). PI3K products are critical second messengers in cell signal transduction pathways involving growth factors (e.g. EGF, vascular endothelial growth factor), hormones (e.g. insulin), and cell-cell (cadherins) and cell-matrix interactions (integrins) (4.Cantrell D.A. J. Cell Sci. 2001; 114: 1439-1445Crossref PubMed Google Scholar). PI lipids interact with the pleckstrin homology (PH) domain containing proteins, leading to their membrane recruitment and/or allosteric activation (5.Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1372) Google Scholar). Examples of PH domain-containing enzymes include protein kinase B/Akt, phosphoinositide-dependent kinase 1, Tec family tyrosine kinases, and guanine nucleotide exchange factors for Ras and Rho family GTPases (e.g. Sos1 and Vav1). PIP3 is converted to phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2) by SHIP2 action. The role of PI-3,4-P2 in signaling is unclear as some suggest a lack of function for this intermediate perhaps due to its rapid rate of turnover (6.Taylor V. Wong M. Brandts C. Reilly L. Dean N.M. Cowsert L.M. Moodie S. Stokoe D. Mol. Cell. Biol. 2000; 20: 6860-6871Crossref PubMed Scopus (158) Google Scholar), whereas some evidence supports that PI-3,4-P2 may have a regulatory role in the signaling pathways because it binds to several PH domain proteins including Akt, which it activates in vitro (7.Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (447) Google Scholar, 8.Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1305) Google Scholar). Moreover, Akt cannot be fully activated by PIP3 when PI-3,4-P2 is absent in vivo (9.Scheid M.P. Huber M. Damen J.E. Hughes M. Kang V. Neilsen P. Prestwich G.D. Krystal G. Duronio V. J. Biol. Chem. 2002; 277: 9027-9035Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In addition to PIP3, SHIP2 substrates include phosphatidylinositol 4,5-bisphosphate (PIP2) and a soluble inositol phosphate, inositol 1,3,4,5-tetrakiphosphate, suggesting that SHIP2 may play a role in processes other than PI3K signaling (6.Taylor V. Wong M. Brandts C. Reilly L. Dean N.M. Cowsert L.M. Moodie S. Stokoe D. Mol. Cell. Biol. 2000; 20: 6860-6871Crossref PubMed Scopus (158) Google Scholar, 10.Chi Y. Zhou B. Wang W.Q. Chung S.K. Kwon Y.U. Ahn Y.H. Chang Y.T. Tsujishita Y. Hurley J.H. Zhang Z.Y. J. Biol. Chem. 2004; 279: 44987-44995Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). SHIP2 mRNA is expressed ubiquitously (2.Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar). SHIP2 is thought to down-regulate insulin signaling (11.Baumgartener J.W. Curr. Drug Targets Immune Endocr. Metabol. Disord. 2003; 3: 291-298Crossref PubMed Scopus (10) Google Scholar). The genetic knockout of SHIP2 in mice led to neonatal lethality due to hypoglycemia caused by insulin hypersensitivity (12.Clement S. Krause U. Desmedt F. Tanti J.F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2001; 409: 92-97Crossref PubMed Scopus (317) Google Scholar). However, Clement et al. (12.Clement S. Krause U. Desmedt F. Tanti J.F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2001; 409: 92-97Crossref PubMed Scopus (317) Google Scholar) have acknowledged recently that this SHIP2 knock-out mouse strain had inadvertent deletion of the adjacent Phox2a gene. Thus, they were unsure whether the observed insulin hypersensitivity phenotype can be attributed solely to the absence of SHIP2 function (13.Clement S. Krause U. Desmedt F. Tanti J.-F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2004; 431: 878Crossref Scopus (12) Google Scholar). However, studies in adipocytes and skeletal myocytes indicate that SHIP2 down-regulates insulin signaling (14.Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell. Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar, 15.Sasaoka T. Hori H. Wada T. Ishiki M. Haruta T. Ishihara H. Kobayashi M. Diabetologia. 2001; 44: 1258-1267Crossref PubMed Scopus (80) Google Scholar). Other evidence suggests a possible link between increased SHIP2 levels and high fat diet, obesity, and diabetes (16.Hori H. Sasaoka T. Ishihara H. Wada T. Murakami S. Ishiki M. Kobayashi M. Diabetes. 2002; 51: 2387-2394Crossref PubMed Scopus (80) Google Scholar, 17.Marion E. Kaisaki P.J. Pouillon V. Gueydan C. Levy J.C. Bodson A. Krzentowski G. Daubresse J.C. Mockel J. Behrends J. Servais G. Szpirer C. Kruys V. Gauguier D. Schurmans S. Diabetes. 2002; 51: 2012-2017Crossref PubMed Scopus (106) Google Scholar, 18.Murakami S. Sasaoka T. Wada T. Fukui K. Nagira K. Ishihara H. Usui I. Kobayashi M. Endocrinology. 2004; 145: 3215-3223Crossref PubMed Scopus (15) Google Scholar, 19.Kaisaki P.J. Delepine M. Woon P.Y. Sebag-Montefiore L. Wilder S.P. Menzel S. Vionnet N. Marion E. Riveline J.P. Charpentier G. Schurmans S. Levy J.C. Lathrop M. Farrall M. Gauguier D. Diabetes. 2004; 53: 1900-1904Crossref PubMed Scopus (70) Google Scholar). The effect of SHIP2 overexpression on Akt activation and cell cycle progression remains contentious, because two reports describe conflicting results (6.Taylor V. Wong M. Brandts C. Reilly L. Dean N.M. Cowsert L.M. Moodie S. Stokoe D. Mol. Cell. Biol. 2000; 20: 6860-6871Crossref PubMed Scopus (158) Google Scholar, 20.Choi Y. Zhang J. Murga C. Yu H. Koller E. Monia B.P. Gutkind J.S. Li W. Oncogene. 2002; 21: 5289-5300Crossref PubMed Scopus (88) Google Scholar). Another report (21.Sasaoka T. Kikuchi K. Wada T. Sato A. Hori H. Murakami S. Fukui K. Ishihara H. Aota R. Kimura I. Kobayashi M. Endocrinology. 2003; 144: 4204-4214Crossref PubMed Scopus (25) Google Scholar) describes a dual role for SHIP2 in vascular smooth muscle cells: 1) an anti-apoptotic function dependent on the SH2 domain; and 2) a growth inhibitory function dependent on the catalytic activity. Thus, the functions of endogenous SHIP2 in different cell types and its role in signaling pathways that are mediated by growth factors, integrins, and cadherins remain poorly understood. SHIP2 contains an amino-terminal SH2 domain, a carboxyl-terminal proline-rich region, and a central inositol phosphatase domain. In addition, SHIP2 contains a sterile α-motif domain at the carboxyl terminus and a NPXY motif. Previously, we showed that SHIP2 is tyrosine-phosphorylated in response to treatment of cells with growth factors and insulin and is associated with Shc adaptor protein when treated with EGF and platelet-derived growth factor but not when treated with insulin (22.Habib T. Hejna J.A. Moses R.E. Decker S.J. J. Biol. Chem. 1998; 273: 18605-18609Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). We have also demonstrated an essential role for SHIP2 in adhesion and spreading of HeLa cells and have established a role for Src family tyrosine kinases in the regulation of SHIP2 during cell adhesion and spreading (23.Prasad N. Topping R.S. Decker S.J. Mol. Cell. Biol. 2001; 21: 1416-1428Crossref PubMed Scopus (120) Google Scholar, 24.Prasad N. Topping R.S. Decker S.J. J. Cell Sci. 2002; 115: 3807-3815Crossref PubMed Scopus (51) Google Scholar). Others (25.Dyson J.M. O'Malley C.J. Becanovic J. Munday A.D. Berndt M.C. Coghill I.D. Nandurkar H.H. Ooms L.M. Mitchell C.A. J. Cell Biol. 2001; 155: 1065-1079Crossref PubMed Scopus (140) Google Scholar) have reported a role for SHIP2 in submembrane actin regulation through interaction with an actin-binding protein, filamin. In this report, we employed RNA interference (RNAi) to suppress the endogenous SHIP2 levels in HeLa cells to further elucidate SHIP2 function. SHIP2 knockdown in these cells caused marked actin abnormalities and cell-spreading defects. Also, alterations in the endocytic vesicular distribution were evident upon SHIP2 silencing, having major impact on the ligand-dependent EGF receptor (EGFR) internalization and degradation pathway. Accordingly, we suggest that, in HeLa cells, SHIP2 functions to regulate actin remodeling and receptor endocytosis, specifically that of EGFR. Materials—For RNAi experiments, single-stranded or duplex RNA oligonucleotides were purchased from Dharmacon Research. We obtained RNA Extraction kit from Qiagen, DNA-Free kit and 18 S rRNA primers from Ambion, and RT-PCR first-strand cDNA synthesis kit and high fidelity PCR mixture from Invitrogen. Generation of rabbit polyclonal antibodies against SHIP2 and EGFR were described earlier (22.Habib T. Hejna J.A. Moses R.E. Decker S.J. J. Biol. Chem. 1998; 273: 18605-18609Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 26.Decker S.J. J. Biol. Chem. 1989; 264: 17641-17644Abstract Full Text PDF PubMed Google Scholar). Antibodies to c-Cbl (Cbl), EEA-1, and ubiquitin were from BD Transduction Laboratories. Anti-FLAG (M2) antibody and TRITC or fluorescein isothiocyanate conjugates of phalloidin were from Sigma. We obtained antibody against Akt and horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG from Cell Signaling Technologies, rhodamine-tagged EGF and Oregon Green-conjugated anti-mouse IgG from Molecular Probes, and rhodamine-conjugated transferrin from Rockland, Inc. Cell Culture and RNA Interference Experiments—HeLa and human embryonic kidney 293/T cells were routinely cultured in Dulbecco's modified Eagle's medium (with high glucose, pyridoxine hydrochloride, and l-glutamine and without sodium pyruvate) containing 7.5% fetal bovine serum. Single-stranded RNA oligomers were annealed for 1 h at 37°C in 100 mm potassium acetate, 30 mm Hepes, pH 7.4, 2 mm magnesium acetate. Synthetic duplex or laboratory-annealed duplex RNA molecules (50 nm) were transfected using Oligofectamine (Invitrogen) reagent according to the manufacturer's instructions. At the indicated intervals following transfection, cell lysates were routinely assayed for silencing effect by Western blots. Unique SHIP2-targeted oligomers (confirmed through BLAST analyses) contained the following sequences: S2 (nucleotides 2058–2076), 5′-TCA TGG TGT GAC CGG ATT C-3′; S2-a (nucleotides 191–209), 5′-GCA TGT GCA CAC GTA TCG C-3′; and MS2-a, 5′-GCA TGT GTT CAC GTA TCG C-3′ where the underlined nucleotides were mismatches in the sequence S2-a. A sequence (denoted by Ds), 5′-AGC CCA GCC TTC GAT CAA C-3′, with no significant homology to any published gene sequences is used as control. For RT-PCR, total RNA was prepared at the indicated intervals and was rendered free of DNA. First-strand reverse transcription reactions were carried out using random hexamer primers. PCR reactions were carried out using SHIP2-specific primers and high fidelity PCR mixture along with a 1:9 ratio of 18 S rRNA primers. Generation of expression constructs of FLAG-tagged wild type SHIP2 and mutant SHIP2 (R47G and 986YY987FF) were described previously (23.Prasad N. Topping R.S. Decker S.J. Mol. Cell. Biol. 2001; 21: 1416-1428Crossref PubMed Scopus (120) Google Scholar, 24.Prasad N. Topping R.S. Decker S.J. J. Cell Sci. 2002; 115: 3807-3815Crossref PubMed Scopus (51) Google Scholar). Transient transfection of 293/T cells was performed using modified calcium phosphate method (Stratagene) as described previously (23.Prasad N. Topping R.S. Decker S.J. Mol. Cell. Biol. 2001; 21: 1416-1428Crossref PubMed Scopus (120) Google Scholar). Immunofluorescence Staining—F-actin organization was examined by staining the cells with 1 μg/ml F-actin-binding phalloidin (TRITC- or fluorescein isothiocyanate-conjugated) for 15 min. Endocytic vesicles were stained with an antibody against EEA1 (1 μg/ml for 60 min) followed by goat anti-mouse IgG-Oregon Green (0.75 μg/ml) for 30 min. After three 5-min phosphate-buffered saline washes, cells were mounted and fluorescent images were captured using a laser confocal microscope. F-actin staining of spreading cells was done 60 min after freshly plating on type I collagen-coated surfaces (3 μg/cm2). The procedures for harvesting cells and coating plates as well as for fixing, blocking, and washing were all described previously (24.Prasad N. Topping R.S. Decker S.J. J. Cell Sci. 2002; 115: 3807-3815Crossref PubMed Scopus (51) Google Scholar). Immunoprecipitation and Western Blot Analyses—Preparation of whole cell lysates and immunoprecipitation experiments using Hepes-NaCl-Triton X-100-glycerol buffer as well as protein analyses by SDS-PAGE and Western blot assays were performed as described earlier (24.Prasad N. Topping R.S. Decker S.J. J. Cell Sci. 2002; 115: 3807-3815Crossref PubMed Scopus (51) Google Scholar, 27.Prasad N. Topping R.S. Zhou D. Decker S.J. Biochemistry. 2000; 39: 6929-6935Crossref PubMed Scopus (74) Google Scholar). Conditions for co-immunoprecipitations using low salt Nonidet P-40 buffer were also described previously (24.Prasad N. Topping R.S. Decker S.J. J. Cell Sci. 2002; 115: 3807-3815Crossref PubMed Scopus (51) Google Scholar). RNAi-mediated Silencing of SHIP2—To better understand SHIP2 function, we employed RNAi to suppress endogenous SHIP2 levels in HeLa cells. Small interfering RNA (siRNA) duplexes of the sequence specific to SHIP2 (denoted S2′ and S2-a in Figs. 1, 5, 6, and 7) significantly reduced SHIP2 levels at both mRNA and protein levels (Fig. 1, A and B). Among three different sequences tested, two caused easily detectable gene silencing (Fig. 1B, bottom panel). Sequence S2 consistently produced a target protein suppression of nearly 70–80%, an efficiency of silencing commonly achieved by this approach (Fig. 1, top and middle panels). Gene knockdown lasted between 70 and 100 h post-transfection in a reproducible and consistent manner as shown with the samples from five independent experiments in Fig. 1B. A duplex siRNA oligomer with two mismatches in the sequence S2-a (depicted as MS2-a in Fig. 1B, bottom panel) was completely inactive. SHIP2 protein levels returned to pre-transfection levels in 6–8 days post-transfection (data not shown). The silencing was evident at concentrations as low as 1 nm specific siRNA duplexes, whereas an siRNA for firefly luciferase (GL3) did not alter SHIP2 protein levels at concentrations of 50 nm (see supplementary data). Data from experiments with S2 siRNA are described below, whereas similar results were also obtained with S2-a siRNA as well (see supplementary data).Fig. 5Altered endocytic vesicular distribution after SHIP2 RNAi.A–F, 48 h after transfection with double-stranded (Ds) control siRNA (panels A, C, and E) or SHIP2-specific siRNA (S2) (panels B, D, and F), cells were serum-starved for an additional 24 h. Cells then were either left untreated (panels A and B) or treated with 25 ng/ml EGF for 5 min (panels C and D) or for 30 min (panels E and F). Early endocytic vesicles are visualized by anti-EEA1 immunostaining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6SHIP2 knockdown modulates endocytic uptake and routing of EGF.A—F, 48 h after transfection with double-stranded (Ds) control siRNA (panels A, C, and E) or SHIP2-specific siRNA (S2) (panels B, D, and F), cells were serum-starved for an additional 24 h. All of the samples then were treated with rhodamine-tagged EGF (25 ng/ml) for 5 min. Following the washing of cells to remove tagged EGF, cells were chased for the indicated durations (panels A and B, 0 h/no chase; panels C and D, 1 h; and panels E and F, 3 h) in serum-free medium. Cells then were fixed and examined under fluorescence microscope for the distribution of internalized EGF. Panel G, localization of internalized TRITC-conjugated transferrin. Three days post-transfection with indicated 50 nm duplex siRNA oligomers, serum-starved HeLa cells were treated with TRITC-transferrin (5 μg/ml) for 20 min on ice and examined after 30 min of chase in the absence of conjugated transferrin. siRNA oligomers used are as follows: siRNA specific to firefly luciferase (GL3) and two siRNAs specific to SHIP2 (S2 and S2-a) (two fields are shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7A, silencing of SHIP2 enhances ligand-induced EGFR degradation. HeLa cells were transfected with double-stranded control siRNA (Ds) or SHIP2-specific siRNA (S2). 48 h post-transfection, cells were serum-starved for 24 h prior to the treatment with EGF (as indicated in the figure). Anti-EGFR immunoblots of whole cell lysates are shown. B, enhanced Cbl-mediated ubiquitination of EGFR upon SHIP2 RNAi. Anti-EGFR or control pre-immune (Pre) immunoprecipitations (IP) from HeLa cells 72 h post-RNAi were blotted with anti-ubiquitin or anti-EGFR antibodies (loading control). Cells were treated as described above for A, but EGF treatment lasted for 5 min. The numbers reflect the quantitation of relative densities of signals in the respective blots. C, increased binding of Cbl to EGFR after SHIP2 silencing. The experiment was performed similar to B with the exception that the anti-EGFR IPs were blotted with anti-c-Cbl antibody. Treatment with low (5 ng/ml) and high (50 ng/ml) concentrations of EGF was carried out for 2 or 5 min as indicated in the figure.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Actin Cytoskeleton Abnormalities upon SHIP2 Knockdown— One of the striking results of SHIP2 knockdown was alteration in cell morphology. Given our earlier findings of an important role for SHIP2 in cell adhesion and spreading events (23.Prasad N. Topping R.S. Decker S.J. Mol. Cell. Biol. 2001; 21: 1416-1428Crossref PubMed Scopus (120) Google Scholar, 24.Prasad N. Topping R.S. Decker S.J. J. Cell Sci. 2002; 115: 3807-3815Crossref PubMed Scopus (51) Google Scholar), we reasoned that we should examine these changes at the level of actin cytoskeleton organization by staining for F-actin. Staining with phalloidin showed profound abnormalities in actin cytoskeleton following SHIP2 knockdown (Fig. 2). In the presence of serum, ∼10–15% SHIP2 RNAi cells displayed long transversely oriented actin filaments or stress fibers along with depletion of submembranous cortical actin (Fig. 2B). A majority of cells (nearly 75%) also presented long filamentous fascicles of actin that protruded at the periphery and at the extremities. These actin filamentous "spikes" became quite prominent (thick arrows), whereas stress fibers disappeared after 24 h of serum starving (Fig. 2D). The weakening of submembranous cortical actin upon suppression of SHIP2 was clearly evident in the absence of stress fibers (thin arrows point to cortical actin in all of the panels; Fig. 2D). Actin abnormalities were induced by SHIP2-specific siRNAs at low concentrations (10 nm), whereas the siRNA for luciferase (GL3) did not cause overt defects in the actin cytoskeleton (see supplementary data). Rho family small G proteins are central regulators of actin cytoskeleton dynamics. Important among them are RhoA, Rac1, and Cdc42. Together, these enzymes play a coordinated role in cell migration process (28.Schmitz A.A. Govek E.E. Bottner B. Van Aelst L. Exp. Cell Res. 2000; 261: 1-12Crossref PubMed Scopus (511) Google Scholar). F-actin spikes and spreading defects are commonly observed in experimental dysfunction of one or more of these proteins in vitro (29.Raftopoulou M. Hall A. Dev. Biol. 2004; 265: 23-32Crossref PubMed Scopus (1147) Google Scholar). Stress fibers are induced by activated Rho A, whereas actin spikes are formed upon activation of Rac1 or Cdc42. Formation of lamellipodia is determined by the actions of PI3K and Rac1. Therefore, morphological changes seen after SHIP2 silencing suggest that SHIP2 may regulate coordinated functioning of Rho family members. We tested this notion by expressing dominant negative (d.n.) forms of Rac1 and Cdc42 in SHIP2 RNAi cells (a gift from Dr. A. Hall, University College, London, United Kingdom). Expression of d.n. Rac1 reduced the severity of F-actin deformities to a large extent in nearly 90% of the cells, whereas d.n. Cdc42 was somewhat less effective in this assay, which rescued the actin abnormalities in ∼40% cells (Fig. 2E). In control cells (transfected with Ds siRNA), expression of d.n. Rac1 caused no overt phenotypic change with the exception of a modest decrease in the F-actin network (data not shown). In HeLa cells, a phosphatase-inactive mutant of SHIP2 prevents cell spreading and mutations of the tyrosines in the NPXYY motif of SHIP2 disrupts lamellipodia extension on collagen I (23.Prasad N. Topping R.S. Decker S.J. Mol. Cell. Biol. 2001; 21: 1416-1428Crossref PubMed Scopus (120) Google Scholar, 24.Prasad N. Topping R.S. Decker S.J. J. Cell Sci. 2002; 115: 3807-3815Crossref PubMed Scopus (51) Google Scholar). Therefore, we tested whether suppression of SHIP2 similarly affected the spreading ability of HeLa cells. Fig. 3 depicts an experiment where control or SHIP2 siRNA-transfected cells were allowed to spread on type I collagen for 60 min. Although there were no significant differences between control and SHIP2 RNAi in the number of cells attached to the collagen-coated surface, striking actin deformities and spreading defects were seen following SHIP2 silencing. After SHIP2 RNAi, newly spreading cells showed a lack of formation of "focal contacts" (also called "contact points," initial transitory structures formed upon cell contact with matrix proteins; see thin arrows in control cells in panel A) and failed to extend lamellipodia (see thick arrows in control cells in panel A). Instead, SHIP2 RNAi cells displayed numerous long membrane protrusions capped at their extremities by a fascicle of actin filaments. As many as 65–70% SHIP2 RNAi cells displayed these actin deformities. The cells maintained this phenotype for as long as 3 h after plating and eventually were able to spread but with a somewhat distorted morphology. Together, these results demonstrate an essential function for SHIP2 in the maintenance and dynamic remodeling of actin structures in HeLa cells. Cytoplasmic Vesicles following SHIP2 Silencing—Another striking feature of cells undergoing silencing of SHIP2 via RNAi was the accumulation of phase-bright cytoplasmic vesicular structures, indicative of disruption of vesicular trafficking (Fig. 4). These vesicles were easily detected in the perinuclear area in <10% cells (Fig. 4, middle panel, denoted by the arrow). Serum starvation did not cause significant alteration in these vesicles; but treatment with EGF (50 ng/ml) for 24 h markedly enhanced the extent of vesicular accumulation in some cells and increased the total number of cells displaying the phenotype as well (Fig. 4, bottom panel, denoted by the arrow). Some but not all of these enlarged vesicles were positive for the early endosome marker, EEA1, whereas markers for late endosomes and the lysosomes (LAMP1, LysoTracker dye, respectively) failed to co-localize to these enlarged vesicles (data not shown). Interestingly, after SHIP2 knockdown, endocytic vesicles identified by the presence of EEA1 markedly differed in their size and the distribution when compared with the control cells (Fig. 5). EEA1-positive vesicles appeared significantly larger than those in control cells and were distributed randomly in the cytoplasm of SHIP2 siRNA-transfected cells, in contrast to the perinuclear accumulations seen in a majority of control cells (Fig. 5, A and B). Increased numbers of small (pin-point) EEA1-positive vesicles were seen in the peripheral cytoplasm of SHIP2 RNAi cells immediately after treatment with EGF (50 ng/ml) for 5 min (Fig. 5D). Control cells displayed similar changes in response to EGF but to a reduced extent (Fig. 5C). EEA1-positive vesicles remained distributed in the cytoplasm in nearly 50–60% SHIP2 RNAi cells, whereas these vesicles re-accumulated at the perinuclear region in control cells when treated for 30 min with EGF (Fig. 5, E and F). Defective Receptor Endocytosis upon SHIP2 RNAi—The accumulation of cytosolic vesicle