Restriction and DNA-modifying enzymes were obtained from New England Biolabs, Inc., Fermentas, or Promega. Big dye terminator cycle sequencing was from PE Applied Systems. Synthetic peptides were from Chiron Mimotopes. COS-7 cells were purchased from the American Type Culture Collection. M2 and A7 cell lines were a gift from Dr. T. Stossel (Brigham and Women's Hospital, Boston, MA). Oligonucleotides were obtained from Bresatec and the Department of Microbiology, Monash University, Melbourne, Australia. Partially purified rabbit polyclonal antibodies to human filamin B were a gift from Dr. Dominic Chung (Department of Biochemistry, University of Washington School of Medicine, WA). Goat polyclonal antibodies to chicken gizzard filamin and monoclonal antibodies to α-actinin (α-actinin) and FLAG were obtained from Sigma-Aldrich. Monoclonal antibodies to HA were obtained from Silenus, GFP antibodies were from Boehringer, β-actin antibodies were from Sigma-Aldrich, and antifilamin antibodies (raised to platelet filamin) were from Chemicon. Phalloidin stain was obtained from Molecular Probes. The GFP-PH/ARNO construct was a gift from Dr. Tamas Balla (National Institutes of Health, Bethesda, MD). The yeast two-hybrid system Matchmaker 3 and the pEGFP-C2 vector were obtained from CLONTECH Laboratories, Inc. and the pCGN vector was a gift from Dr. Tony Tiganis (Monash University, Melbourne, Australia). pEFBOS-Myc and pEFBOS-FLAG vectors were a gift from Dr. Tracey Willson (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). Filamin A and B cDNAs were a gift from Dr. Joe Trapani and Kylie Browne (Peter MacCallum Cancer Institute, Melbourne, Australia). All other reagents were from Sigma-Aldrich unless otherwise stated.
Production of antipeptide antibodies
SHIP-2 antipeptide antibodies were generated to a fusion peptide comprising the NH2-terminal seven amino acids of SHIP-2 fused to the COOH-terminal seven amino acids of SHIP-2 (MASACGADTLQLSK) (SHIP-2NC), or to the amino acid sequence, 1019–1030 (ITVPAPQLGHHRH) (SHIP-2P). SHIP-2NC antibodies were used for all experiments except indirect immunofluorescence of COS-7, M2, and A7 cells in which the SHIP-2P antibody was used. Peptide conjugated to diphtheria toxoid was injected subcutaneously into female New Zealand white rabbits. Affinity-purified antipeptide antibodies were obtained by chromatography of immune sera on the specific peptide coupled to thiopropyl-Sepharose 6B resin. After extensive washing, specific antibodies were eluted from the column with 0.1 M glycine-HCl, pH 2.5.
Generation of full-length SHIP-2 and SHIP-2 truncation mutants
Human SHIP-2 cDNA was generated by ligation of EST aa 279072 to the cDNA encoding INPPL-1, obtained from Dr. James Hejna (Oregon Health Sciences University, Portland, OR) (Hejna et al., 1995
). Several rounds of PCR amplification enabled prolongation of the 5′-end of the clone to encompass SHIP-2 nucleotides 257–3988, which correspond to aa 16–1258 plus a COOH-terminal hexa-His-tag. SHIP-2 cDNA was cloned in-frame into pCGN (XbaI site), pEGFP-C2 (EcoRI site) and pEFBOS (MluI site) vectors, that encode for HA, GFP, and FLAG-tagged SHIP-2 fusion proteins, respectively, using PCR amplification with the introduction of specific restriction sites. Truncation mutants of SHIP-2 were also generated and were subsequently cloned into the XbaI site of pCGN. The oligonucleotide sequences and a description of the constructs generated are listed in . Fidelity of all PCR products and the final constructs was confirmed by dideoxy sequencing.
Yeast two-hybrid analysis
The yeast two-hybrid Matchmaker III GAL4-based system was used for all yeast two-hybrid studies. The proline-rich domain of human SHIP-2 comprising nucleotides 3017–3989 (aa 936–1258), was cloned into the EcoRI site of pGBKT7, creating a GAL4 fusion protein, the “bait.” “Bait” protein-expressing yeast (AH109) were transformed with human skeletal muscle cDNA library (CLONTECH Laboratories, Inc.) according to the manufacturer's guidelines. Yeast plasmid was extracted from positive clones as described (Ausubel et al., 1991
SHIP-2 proline-rich domain–expressing yeast were transformed with filamin A and B isoforms cDNA (aa 2171–2647 and 2130–2602, respectively) to investigate an interaction. Specificity of the interactions with the SHIP-2 proline-rich domain was confirmed using a p53 “bait.” In addition, a “bait” lacking the proline-rich domain of SHIP-2, but containing the SH2 domain and 5-phosphatase domain (comprising nucleotides 210–3016) was constructed using a PCR based strategy and was also used as a negative control “bait.”
Immunoblot of endogenous SHIP-2, HA, and GFP-tagged SHIP-2 constructs
M2 and A7 cells were maintained as described (Cunningham et al., 1992
; Ohta et al., 1999
). COS-7 cells were maintained in DME, 10% (vol/vol) fetal calf serum containing 2 mM glutamine, and transfected using the DEAE-dextran procedure and allowed to grow for 2 d (Sambrook and Russel, 2001
). Cells were washed briefly with PBS and treated with lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA (ethylenediaminetetra-acetic acid DI-sodium salt), 1 mM benzamidine, 2 mM phenylmethylsulfonylfluoride, 2 μg/ml leupeptin, and 2 μg/ml aprotinin) for 2 h at 4°C. Lysates were centrifuged at 15,400 g
for 10 min to obtain the Triton X-100–soluble supernatant which was analyzed by immunoblot analysis using antibodies to the specific tag, affinity-purified SHIP-NC sera, or antifilamin antibodies.
Intracellular localization of SHIP-2 in COS-7 cells
COS-7 cells were transiently transfected with GFP–SHIP-2, Myc-filamin, HA–SHIP-2, HA-SHIP-2ΔPRD, HA–SHIP-2ΔSH2, or HA-PRD truncation mutants (), fixed/permeabilized, and stained. Alternatively, in some studies, 24 h after transfection, cells were placed in DME containing 0.1% FCS and 2 mM glutamate for a period of ~15 h and then stimulated with EGF (100 ng/ml). Cells expressing GFP-tagged proteins were gently washed with PBS and fixed with 3.7% formaldehyde. Cells expressing Myc and HA-tagged proteins were gently washed with PBS and then fixed and permeabilized for 10 min in PBS with 3.7% formaldehyde and 0.2% Triton X-100. Expression of Myc and HA-tagged proteins was localized using Myc and HA monoclonal antibodies and detected using tetramethylrhodamine isothiocyanate–conjugated TRITC anti–mouse IgG and fluorescein isothiocyanate–conjugated FITC anti–mouse IgG, respectively. Endogenous SHIP-2 was detected in nontransfected and Myc-filamin–transfected cells using the SHIP-2P antibody and FITC anti–rabbit IgG. Colocalization was performed using antibodies to filamin B detected with tetramethylrhodamine isothiocyanate–conjugated TRITC anti–rabbit IgG and specific actin markers, either phalloidin staining and/or antibodies to β-actin detected using TRITC anti–mouse IgG. Coverslips were mounted using SlowFade and visualized by confocal microscopy.
COS-7 cells were transfected via electroporation (Sambrook and Russel, 2001
) and either harvested in lysis buffer 48 h posttransfection or EGF stimulated for 1 or 5 min as outlined above and harvested. Transfected and nontransfected cells were harvested and Triton X-100 extracted as outlined above. Triton X-100–soluble lysates were immunoprecipitated with either 10 μg of monoclonal FLAG or HA antibody, 5 μg of polyclonal anti–SHIP-2NC sera or preimmune sera and 60 μl of 50% slurry of protein A–Sepharose. Immunoprecipitates were immunoblotted with either FLAG, Myc, or filamin monoclonal antibodies.
Intracellular localization of SHIP-2 in A7 and M2 cells
Human filamin–deficient melanoma cell line (M2) and full-length filamin replete cell line (A7) were maintained as described (Cunningham et al., 1992
; Ohta et al., 1999
). Endogenous SHIP-2 was localized in resting and EGF-treated A7 and M2 cells as described above for COS-7 cells.
Assessment of β-actin, phalloidin, or GFP-PH/ARNO staining
COS-7 cells were transiently transfected via DEAE dextran-chloroquine with HA–SHIP-2 or HA-PRD, stimulated for 5 min with EGF (100 ng/ml), and costained with HA- and β-actin–specific antibodies, as outlined above. Cells were assessed for β-actin staining at the plasma membrane as a percentage of the total transfected cells. COS-7 cells were transiently transfected with HA–SHIP-2 or empty vector HA, stimulated for 5 min with EGF (100 ng/ml), and costained with HA antibodies and phalloidin. Cells were scored for phalloidin staining. COS-7 cells were transiently cotransfected with HA–SHIP-2, HA-SHIP2ΔPRD, or HA-PRD, or myc-filamin and GFP fused to the PH domain of ARNO, (GFP-PH/ARNO), or empty vector HA and GFP-PH/ARNO, stimulated for 5 min with EGF (100 ng/ml), and stained with HA antibody as outlined above. Cells were assessed for GFP-PH/ARNO expression at the plasma membrane. Approximately 40 cells were scored by an independent observer for each experiment.
Generation of FLNC truncation mutants
A PCR-based strategy was employed to generate FLNC truncation mutants which were subcloned into the EcoRI site of pGADT7, creating HA-tagged GAL4 recombinant proteins. The oligonucleotide and construct descriptions are given in . Nucleotides 2,434–3,252 of FLNC was subcloned into the XbaI site and MluI site of pCGN and pEFBOS-Myc tagged, respectively. Fidelity of all PCR products and the final constructs were confirmed by dideoxy sequencing.
Oligonucleotides used for the generation of FLNC truncation mutants in pGADT7
Localization of SHIP-2 and filamin in murine heart and soleus muscle
Mice were killed humanely following National Health and Medical Research Council guidelines, Monash University animal ethics number BAM/B/2000/17. Murine heart and soleus were dissected from 12-wk-old male mice, C57B/6. Organs were snap frozen in isopentane chilled with liquid nitrogen and blocked in OCT (10.24% wt/wt polyvinyl alcohol, 4.26% wt/wt polyethylene glycol, and 85.5% wt/wt nonreactive ingredients) compound and stored at −70°C until used. Blocks were equilibrated to −20°C before sectioning. Cross-sections and longitudinal sections were cut 7-μm thick and placed on superfrost plus slides before staining. They were then fixed in PBS/4% paraformaldehyde for 5 min at room temperature, washed with PBS, then blocked and permeablized with PBS, 10% horse serum, and 0.1% Triton X-100 for 15 min at room temperature. Slides were washed and stained with anti–SHIP-2NC sera, and detected with FITC anti–rabbit IgG. Antifilamin was detected with TRITC anti–goat IgG and anti–α-actinin was detected with TRITC anti–rabbit IgG; overnight incubation at 4°C. Sections were washed with PBS, mounted using SlowFade, and visualized by confocal microscopy.