Article |
Address correspondence to Christina A. Mitchell, Dept. of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800 Australia. Tel.: (61) 3990-53790. Fax: (61) 3990-54699. E-mail: christina.mitchell{at}med.monash.edu.au
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: SHIP-2; inositol polyphosphate 5-phosphatase; filamin; cytoskeleton; phosphatidylinositol 3,4,5-trisphosphate
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PtdIns(3,4,5)P3 is metabolized by the removal of either the 5- or 3-position phosphate by specific 5- or 3-lipid phosphatases, respectively. The product of the tumor suppressor gene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a PtdIns(3,4,5)P3 3-phosphatase which hydrolyses PtdIns(3,4,5)P3, forming PtdIns(4,5)P2. The lipid 3-phosphatase activity of PTEN is critical for its tumor suppressor function (for review see Cantley and Neel, 1999).
The 5-phosphatases hydrolyze the 5-position phosphate from both inositol phosphates and phosphoinositides and share the same catalytic mechanism to the apurinic/apyrimidinic endonucleases (Majerus, 1996; Whisstock et al., 2000; Tsujishita et al., 2001). SHIP-2 is a widely expressed 5-phosphatase which plays a significant role in negatively regulating insulin signaling (Ishihara et al., 1999; Clement et al., 2001). SHIP-2 contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal prolinerich domain and bears significant sequence identity with the 5-phosphatase, SHIP-1, except in the proline-rich domain. SHIP-2 hydrolyses the 5-position phosphate from PtdIns(3,4,5)P3 and PtdIns(4,5)P2, and in some, but not all, studies has been shown to hydrolyze the soluble inositol phosphate Ins(1,3,4,5)P4 (Pesesse et al., 1997; Wisniewski et al., 1999; Taylor et al., 2000). In contrast to SHIP-1, which has a restricted hematopoietic expression, SHIP-2 is widely expressed. SHIP-2 undergoes cytokine-, growth factor, and insulin-stimulated phosphorylation in a number of cell lines (Habib et al., 1998; Wisniewski et al., 1999). In addition, SHIP-2 is constitutively tyrosine phosphorylated and associated with Shc in chronic myeloid leukemic progenitor cells, suggesting a role for SHIP-2 in 210bcr/abl-mediated myeloid expansion (Wisniewski et al., 1999). SHIP-2, like PTEN, regulates both PtdIns(3,4,5)P3-mediated Akt activation and the induction of cell cycle arrest associated with increased stability of expression of the cell cycle inhibitor p27KIPI (Taylor et al., 2000). Recent studies have demonstrated SHIP-2 negatively regulates insulin signaling. Homozygous mice lacking SHIP-2 develop severe neonatal hypoglycemia and prenatal death. Adult SHIP-2 heterozygous mutant mice demonstrate insulin sensitivity associated with increased translocation of GLUT4 to the plasma membrane in response to insulin treatment (Clement et al., 2001).
In this study we examine the intracellular location of the 5-phosphatase SHIP-2 and demonstrate the enzyme is located at membrane ruffles mediated via its proline-rich domain. SHIP-2 forms a complex with the actin binding protein, filamin, and thereby regulates PtdIns(3,4,5)P3 and actin at the leading edge of the cell. This may represent a mechanism for the tight spatial regulation of PtdIns(3,4,5)P3 at specific sites after growth factor or insulin stimulation.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Identification of SHIP-2 binding partners using yeast two-hybrid analysis
The SHIP-2 COOH-terminal prolinerich domain contains numerous "PXXP" motifs which conform to consensus sequences for SH3 binding domains, 1 WW binding domain motif (PPLP) which may bind to WW domain-containing proteins, and one EVH1 binding domain motif (E/DFPPPPXD/E) which may link the cytoskeletal network to signal transduction pathways (Fedorov et al., 1999). The SHIP-2 prolinerich domain sequence demonstrates no significant sequence homology over the extreme COOH-terminal 322 amino acids with SHIP-1. We searched for proteins that specifically interact with the proline-rich domain using yeast two-hybrid analysis. The entire SHIP-2 proline-rich domain (amino acids [aa] 9361258) was expressed in yeast cells with a library of proteins expressed as fusions with the GAL4 transcription activation domain. Several rounds of screening a human skeletal muscle library (4 x 106 clones) identified a number of interacting clones in which growth on selective media suggested the presence of bona fide interactors for the proline-rich domain. Sequence analysis demonstrated that one clone, an 818 bp fragment, encoded aa 24342705 of the last COOH-terminal two and a half immunoglobulin repeats of the cytoskeletal actin-binding protein filamin C (FLNC), which were in frame with the GAL4 activation domain (Fig. 2 A). Filamin is located in the cortical cytoplasm subjacent to the plasma membrane, and binds actin, promoting orthogonal branching of actin filaments and thereby cell migration and membrane stability (reviewed by Stossel et al., 2001). Filamin forms a complex with a variety of cell surface receptors including FcRI, the platelet von Willebrand factor receptor, glycoprotein Ib-IX-V, ß1 and ß2 integrin receptors, and intracellular proteins involved in various signaling cascades including Traf 2, granzyme B, caveolin-1, and the stress-activated protein kinase (reviewed by Stossel et al., 2001).
|
|
Identification of FLNC sequences mediating interaction with SHIP-2
To determine the region of FLNC specifically interacting with SHIP-2, a series of wild-type and mutant FLNC constructs comprising the COOH-terminal immunoglobulin repeat regions R22R24 (aa 24342705), which include a "hinge II region" between R23 and R24, were cloned into the activation domain and cotransformed with the DNA-BDPRD bait and interactions scored as strong (+++), or weak (+) (Fig. 2 C). The fragment containing FLNC repeats 2224 demonstrated the strongest binding to the proline-rich domain bait. FLNC repeats 23 and 24, either alone or in combination, did not interact with SHIP-2. Repeats 22 and 23 in combination, with or without the hinge II region, interacted with SHIP-2; however, this was weaker than repeats 2224 (Fig. 2 C). All FLNC truncation mutants were expressed in the yeast strain AH109 and were soluble (unpublished data).
We took several approaches to verify whether SHIP-2 interacted with filamin in vivo and thereby regulated the membrane localization of SHIP-2. First, we investigated using cotransfection and coimmunopreciptation assays if filamin interacted with SHIP-2 in COS-7 cells. We also determined association of endogenous proteins using immunoprecipitation and immunoblot analysis. Second, we demonstrated colocalization of SHIP-2 and filamin at membrane ruffles in resting and EGF-stimulated COS-7 cells. Third, we showed colocalization of filamin and SHIP-2 in mouse heart and skeletal muscle sections using immunolocalization of both species. Finally, we showed the membrane localization of SHIP-2 is dependent on filamin by determining the intracellular localization of SHIP-2 in cells which do not express filamin.
Association of SHIP-2 and filamin was demonstrated in COS-7 cells, which were cotransfected with FLAG-tagged SHIP-2 and HA-tagged filamin (encoding aa 24342705 of FLNC), followed by immunoprecipitation and immunoblot analysis using antibodies to each tag. FLAGSHIP-2 was detected in HA immunoprecipitates of HA-filamintransfected cells, but not cells transfected with HA empty vector (Fig. 3 A). We determined the effect of EGF stimulation on association between recombinant SHIP-2 and filamin. COS-7 cells were cotransfected with myc-filamin and FLAGSHIP-2, and after EGF stimulation for 5 min, Triton X-100soluble lysates were immunoprecipitated using FLAG antibody and immunoblotted with myc antibody. The level of filamin in SHIP-2 immunoprecipitates was unchanged after 5 min EGF stimulation, compared with nonstimulated cells (Fig. 3 B). These studies were repeated expressing SHIP-2 and filamin as fusion proteins with different tags with similar results (unpublished data).
|
|
|
Colocalization of filamin and SHIP-2 in heart and skeletal muscle
FLNC is highly expressed in striated muscle, where it is predominantly localized in myofibrillar Z-discs, with a minor fraction of the protein showing subsarcolemma localization (van der Ven et al., 2000). SHIP-2 is also expressed in skeletal muscle, although its intracellular location in this tissue has not been reported. SHIP-2 homozygous null mice demonstrate increased sensitivity to insulin (Clement et al., 2001). One of the major sites of insulin action is skeletal muscle, where insulin stimulates the translocation of the glucose transporter GLUT4 in a PI-3 kinasedependent manner to the sarcolemma (Khan et al., 2000). We investigated whether SHIP-2 and filamin colocalized in striated muscle. Mouse heart striated muscle sections were isolated, fixed, and probed with specific affinity purified SHIP-2 antibodies (Fig. 6 A). Soleus muscle showed similar localization (unpublished data). In longitudinal sections SHIP-2 antibodies stained intensively in an alternate banding pattern at areas that resembled Z-lines. No staining of any structure was observed using preimmune serum (Fig. 6 B). Counter-staining sections using antibodies to filamin and the Z-linespecific protein -actinin demonstrated both colocalized with SHIP-2 (Fig. 6, CH). Cross-sectional analysis of skeletal muscle demonstrated both filamin and SHIP-2 localized to the sarcolemma, the site of insulin-stimulated GLUT4 translocation (Fig. 6, IL).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inositol polyphosphate 5-phosphatases and the cytoskeleton
Increasing evidence indicates that both mammalian and yeast 5-phosphatase isoforms, via hydrolysis of PtdIns(4,5)P2 and or PtdIns(3,4,5)P3, play a significant role in regulating cytoskeletal reorganization. The 5-phosphatases comprise 10 mammalian and 4 yeast isoforms with many spliced variants described. Null mutation of any two yeast Sac-1 domain containing 5-phosphatases results in a phenotype which includes disorganization of polymerized actin and delocalization of actin patches from the growing yeast bud to the mother cell (for review see Hughes et al., 2000). The yeast 5-phosphatases, Inp52p and Inp53p, translocate to actin patches upon osmotic stress, the site of plasma membrane invaginations. In addition, overexpression of Inp52p and Inp53p, but not catalytically inactive Inp52p, results in a significant reduction in the repolarization time of actin patches after osmotic stress (Ooms et al., 2000). The mammalian 5-phosphatase, synaptojanin-1, hydrolyses Ptd- Ins(4,5)P2 bound to the actin regulatory proteins, -actinin, vinculin, gelsolin, and profilin, and decreases the number of stress fibers in the cell (Sakisaka et al., 1997). Synaptojanin-2 directly interacts with Rac1 in a GTP-dependent manner, resulting in translocation of the 5-phosphatase to membrane ruffles and inhibition of endocytosis (Malecz et al., 2000). Overexpression of SKIP (skeletal muscle and kidney-enriched inositol phosphatase) results in loss of actin stress fibers in areas of SKIP expression (Ijuin et al., 2000). The recently identified proline-rich inositol polyphosphate 5-phosphatase (PIPP) localizes to membrane ruffles, but unlike SHIP-2 does not appear to regulate the actin cytoskeleton (Mochizuki and Takenawa, 1999). SHIP-2 regulation of submembraneous actin levels is most probably mediated via localized regulation of PtdIns(3,4,5)P3. However, both SHIP-1 and SHIP-2 also hydrolyze PtdIns(4,5)P2, forming PtdIns(4)P (Kisseleva et al., 2000; Taylor et al., 2000).
Role of SHIP-2 association with FLNC in insulin signaling
The recent characterization of SHIP-2 homozygous null mice has demonstrated that this 5-phosphatase plays a significant role in regulating insulin sensitivity (Clement et al., 2001). Although the signaling pathways mediating the phenotype of insulin hypersensitivity have yet to be fully determined, insulin-stimulated GLUT4 translocation to the plasma membrane appears to be enhanced in mice lacking SHIP-2. In addition, overexpression of SHIP-2, but not catalytically inactive SHIP-2, in 3T3-L1 adipocytes results in negative regulation of insulin-induced signaling (Wada et al., 2001). The submembraneous actin microfilament network links various signaling proteins, including IRS-1 and PI-3 kinase, that regulate GLUT4 translocation to the plasma membrane. Insulin-induced reorganization of the subplasma membrane actin filaments may allow exocytic GLUT4 vesicles to fuse with the plasma membrane during stimulation by insulin (Khayat et al., 2000). GLUT4 expression is restricted to muscle and adipose tissue. Insulin-stimulated glucose disposal in skeletal muscle accounts for 80% of glucose uptake postprandial (Khayat et al., 2000), localizing GLUT4 to the sarcolemma. Filamin may provide a scaffold for the juxtaposition of SHIP-2 to the sarcolemma, localizing the enzyme to PtdIns(3,4,5)P3 after insulin treatment and thereby regulating GLUT4 translocation.
Filamin forms a scaffold for the binding of Rho GTPases, including Rac1, Cdc42, Rho A, and RalA, and their activators such as Trio, a Rho guanine nucleotide exchange factor (GEF) (Ohta et al., 1999). The localization of both Rho GTPases and their activators on filamin may allow spatial coordination of actin nucleation at sites where newly assembled actin filaments are cross-linked. It is noteworthy that many filamin-binding proteins, including Trio and SHIP-2, bind to the extreme COOH-terminal repeats 2124 of filamin, by a variety of interacting modules, including proline-rich domains, as is the case for SHIP-2 and PH domains as shown for Trio. This would therefore provide close proximity between all these signaling proteins, including SHIP-2, that regulate actin polymerization on a filamin scaffold.
Several recent studies in fibroblasts and the neutrophil cell line HL60 have demonstrated using the PH domain of PtdIns(3,4,5)P3-binding proteins fused to GFP, that PI-3 kinase signals are generated at the leading edge of the cell (Blomberg et al., 1999; Watton and Downward, 1999; Balla et al., 2000; Servant et al., 2000). These PH domains function as an accurate probe for the localized agonist-dependent accumulation of PtdIns(3,4,5)P3. It has been proposed, but not previously shown, that this asymmetric distribution of PtdIns(3,4,5)P3 may result from its localized synthesis or degradation (Rickert et al., 2000). The results of the study reported here are consistent with this contention. We have shown the spatially controlled synthesis of PtdIns(3,4,5)P3 at membrane ruffles is regulated by the Ptd- Ins(3,4,5)P3 5-phosphatase SHIP-2, which also localizes to membrane ruffles.
We have demonstrated SHIP-2 localization to membrane ruffles is mediated by its COOH-terminal proline-rich domain binding to the actin binding protein filamin. The expression of SHIP-2 in filamin deficient cells is exclusively cytosolic. In addition, SHIP-2 membrane localization appears to contribute to localized PtdIns(3,4,5)P3 hydrolysis. SHIP-2 COOH-terminal truncation mutants were not as efficient at regulating PtdIns(3,4,5)P3 at membrane ruffles as intact SHIP-2. Furthermore, displacement of endogenous SHIP-2 by overexpression of the SHIP-2 filamin binding domain (myc filamin aa 24342705) lead to the marked enhancement of the PI-3 kinase signal PtdIns(3,4,5)P3. Several recent reports have shown the membrane localization of the highly related SHIP-2 homologue SHIP-1 is also important for PtdIns(3,4,5)P3 hydrolysis (Phee et al., 2000). Enforced plasma membrane localization of SHIP-1, mediated by overexpression of a human CD8SHIP-1 chimera, decreased the total cellular levels of PtdIns(3,4,5)P3. Membrane targeting of SHIP-1 mediated by the COOH-terminal proline-rich domain appears to be important in B cell inhibitory function (Aman et al., 2000). In addition, the SHIP-1 COOH terminus is essential for PtdIns(3,4,5)P3 hydrolysis and inhibition of mast cell degranulation (Damen et al., 2001). Collectively, these studies show membrane targeting of these two 5-phosphatases mediated by their respective COOH-terminal proline-rich domains plays an important functional role in regulating PI-3 kinase signals.
SHIP-2 associates with the p130Cas adaptor protein at focal adhesions and regulates cell spreading, which is dependent on the SHIP-2 SH2 domain and is enhanced by tyrosine phosphorylation and cell adhesion (Prasad et al., 2001). We have shown SHIP-2 and filamin also form a functionally significant complex both in the resting cells and after cellular activation at membrane ruffles. We therefore propose the binding of SHIP-2 to filamin provides a mechanism for exquisite localized hydrolysis of PtdIns(3,4,5)P3 in resting, growth factor and insulinstimulated cells at the leading edge of cell.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 10191030 (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 2573988, which correspond to aa 161258 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 Table I. 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 30173989 (aa 9361258), 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 domainexpressing yeast were transformed with filamin A and B isoforms cDNA (aa 21712647 and 21302602, 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 2103016) 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-100soluble 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 GFPSHIP-2, Myc-filamin, HASHIP-2, HA-SHIP-2PRD, HASHIP-2
SH2, or HA-PRD truncation mutants (Table I), 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 isothiocyanateconjugated TRITC antimouse IgG and fluorescein isothiocyanateconjugated FITC antimouse IgG, respectively. Endogenous SHIP-2 was detected in nontransfected and Myc-filamintransfected cells using the SHIP-2P antibody and FITC antirabbit IgG. Colocalization was performed using antibodies to filamin B detected with tetramethylrhodamine isothiocyanateconjugated TRITC antirabbit IgG and specific actin markers, either phalloidin staining and/or antibodies to ß-actin detected using TRITC antimouse IgG. Coverslips were mounted using SlowFade and visualized by confocal microscopy.
Immunoprecipitations
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-100soluble lysates were immunoprecipitated with either 10 µg of monoclonal FLAG or HA antibody, 5 µg of polyclonal antiSHIP-2NC sera or preimmune sera and 60 µl of 50% slurry of protein ASepharose. Immunoprecipitates were immunoblotted with either FLAG, Myc, or filamin monoclonal antibodies.
Intracellular localization of SHIP-2 in A7 and M2 cells
Human filamindeficient 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 ß-actin, phalloidin, or GFP-PH/ARNO staining
COS-7 cells were transiently transfected via DEAE dextran-chloroquine with HASHIP-2 or HA-PRD, stimulated for 5 min with EGF (100 ng/ml), and costained with HA- and ß-actinspecific antibodies, as outlined above. Cells were assessed for ß-actin staining at the plasma membrane as a percentage of the total transfected cells. COS-7 cells were transiently transfected with HASHIP-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 HASHIP-2, HA-SHIP2PRD, 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 Table II. Nucleotides 2,4343,252 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.
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 antiSHIP-2NC sera, and detected with FITC antirabbit IgG. Antifilamin was detected with TRITC antigoat IgG and anti-actinin was detected with TRITC antirabbit IgG; overnight incubation at 4°C. Sections were washed with PBS, mounted using SlowFade, and visualized by confocal microscopy.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
Cindy O'Malley was funded by an Anti-Cancer Council of Victoria scholarship. This work was funded by the National Health and Medical Research Council of Australia.
Submitted: 2 April 2001
Revised: 28 September 2001
Accepted: 19 October 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aman, M.J., S.F. Walk, M.E. March, H.P. Su, D.J. Carver, and K.S. Ravichandran. 2000. Essential role for the C-terminal noncatalytic region of SHIP in FcRIIB1-mediated inhibitory signaling. Mol. Cell. Biol. 20:35763589.
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1991. Current protocols in molecular biology. John Wiley and Sons, Inc., New York.
Balla, T., T. Bondeva, and P. Varnai. 2000. How accurately can we image inositol lipids in living cells? Trends Pharmacol. Sci. 21:238241.[Medline]
Blero, D., F. De Smedt, X. Pesesse, N. Paternotte, C. Moreau, B. Payrastre, and C. Erneux. 2001. The SH2 domain containing inositol 5-phosphatase SHIP2 controls phosphatidylinositol 3,4,5-trisphosphate levels in CHO-IR cells stimulated by insulin. Biochem. Biophys. Res. Commun. 282:839843.[Medline]
Blomberg, N., E. Baraldi, M. Nilges, and M. Saraste. 1999. The PH superfold: a structural scaffold for multiple functions. Trends Biochem Sci. 24:441445.[Medline]
Cantley, L.C., and B.G. Neel. 1999. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl. Acad. Sci. USA. 96:42404245.
Clement, S., U. Krause, F. Desmedt, J.-F. Tanti, J. Behrends, X. Pesesse, T. Sasaki, J. Penninger, M. Doherty, W. Malaisse, et al. 2001. The lipid phosphatase SHIP2 controls insulin sensitivity. Nature. 409:9297.[Medline]
Corvera, S., A. D'Arrigo, and H. Stenmark. 1999. Phosphoinositides in membrane traffic. Curr. Opin. Cell Biol. 11:460465.[Medline]
Cunningham, C.C., J.B. Gorlin, D.J. Kwiatkowski, J.H. Hartwig, P.A. Janmey, H.R. Byers, and T.P. Stossel. 1992. Actin-binding protein requirement for cortical stability and efficient locomotion. Science. 255:325327.[Medline]
Damen, J.E., M.D. Ware, J. Kalesnikoff, M.R. Hughes, and G. Krystal. 2001. SHIP's C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation. Blood. 97:13431351.
Datta, S.R., A. Brunet, and M.E. Greenberg. 1999. Cellular survival: a play in three Akts. Genes Dev. 13:29052927.
Fedorov, A.A., E. Fedorov, F. Gertler, and S.C. Almo. 1999. Structure of EVH1, a novel proline-rich ligand-binding module involved in cytoskeletal dynamics and neural function. Nat. Struct. Biol. 6:661665.[Medline]
Gorlin, J.B., R. Yamin, S. Egan, M. Stewart, T.P. Stossel, D.J. Kwiatkowski, and J.H. Hartwig. 1990. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring. J. Cell Biol. 111:10891105.[Abstract]
Habib, T., J.A. Hejna, R.E. Moses, and S.J. Decker. 1998. Growth factors and insulin stimulate tyrosine phosphorylation of the 51C/SHIP2 protein. J. Biol Chem. 273:1860518609.
Hejna, J.A., H. Saito, L.S. Merkens, T.V. Tittle, P.M. Jakobs, M.A. Whitney, M. Grompe, A.S. Friedberg, and R.E. Moses. 1995. Cloning and characterization of a human cDNA (INPPL1) sharing homology with inositol polyphosphate phosphatases. Genomics. 29:285287.[Medline]
Hughes, W.E., F.T. Cooke, and P.J. Parker. 2000. Sac phosphatase domain proteins. Biochem. J. 350:337352.[Medline]
Ijuin, T., Y. Mochizuki, K. Fukami, M. Funaki, T. Asano, and T. Takenawa. 2000. Identification and characterization of a novel inositol polyphosphate 5-phosphatase. J. Biol. Chem. 275:1087010875.
Ishihara, H., T. Sasaoka, H. Hori, T. Wada, H. Hirai, T. Haruta, W.J. Langlois, and M. Kobayashi. 1999. Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling. Biochem. Biophys. Res. Commun. 260:265272.[Medline]
Khan, A.H., D.C. Thurmond, C. Yang, B.P. Ceresa, and J.E. Pessin. 2000. Munc18c regulates insulin-stimulated GLUT4 translocation to the transverse tubules in skeletal muscle. J. Biol. Chem. 38:12.
Khayat, Z.A., P. Tong, K. Yaworsky, R.J. Bloch, and A. Klip. 2000. Insulin-induced actin filament remodeling colocalizes actin with phosphatidylinositol 3-kinase and GLUT4 in L6 myotubes. J. Cell Sci. 113:279290.
Kisseleva, M.V., M.P. Wilson, and P.W. Majerus. 2000. The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase. J. Biol. Chem. 275:2011020116.
Maestrini, E., C. Patrosso, M. Mancini, S. Rivella, M. Rocchi, M. Repetto, A. Villa, A. Frattini, M. Zoppe, P. Vezzoni, et al. 1993. Mapping of two genes encoding isoforms of the actin binding protein ABP-280, a dystrophin like protein, to Xq28 and to chromosome 7. Hum. Mol. Genet. 2:761766.[Abstract]
Majerus, P.W. 1996. Inositols do it all. Genes Dev. 10:10511053.[Medline]
Malecz, N., P.C. McCabe, C. Spaargaren, R. Qiu, Y. Chuang, and M. Symons. 2000. Synaptojanin 2, a novel rac1 effector that regulates clathrin-mediated endocytosis. Curr. Biol. 10:13831386.[Medline]
Martin, T.F. 1997. Phosphoinositides as spatial regulators of membrane traffic. Curr. Opin. Neurobiol. 7:331338.[Medline]
Mochizuki, Y., and T. Takenawa. 1999. Novel inositol polyphosphate 5-phosphatase localizes at membrane ruffles. J. Biol. Chem. 274:3679036795.
Ohta, Y., N. Suzuki, S. Nakamura, J.H. Hartwig, and T.P. Stossel. 1999. The small GTPase RalA targets filamin to induce filopodia. Proc. Natl. Acad. Sci. USA. 96:21222128.
Ooms, L.M., B.K. McColl, F. Wiradjaja, A.P. Wijayaratnam, P. Gleeson, M.J. Gething, J. Sambrook, and C.A. Mitchell. 2000. The yeast inositol polyphosphate 5-phosphatases np52p and np53p translocate to actin patches following hyperosmotic stress: mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations. Mol. Cell. Biol. 20:93769390.
Pesesse, X., S. Deleu, F. De Smedt, L. Drayer, and C. Erneux. 1997. Identification of a second SH2-domain-containing protein closely related to the phosphatidylinositol polyphosphate 5-phosphatase SHIP. Biochem. Biophys. Res. Commun. 239:697700.[Medline]
Pesesse, X., V. Dewaste, F. De Smedt, M. Laffargue, S. Giuriato, C. Moreau, B. Payrastre, and C. Erneux. 2001. The SH2 domain containing inositol 5-phosphatase SHIP2 is recruited to the EGF receptor and dephosphorylates phosphatidylinositol 3,4,5-trisphosphate in EGF stimulated COS-7 cells. J. Biol. Chem. 10:10.
Phee, H., A. Jacob, and K.M. Coggeshall. 2000. Enzymatic activity of the Src homology 2 domain-containing inositol phosphatase is regulated by a plasma membrane location. J. Biol. Chem. 275:1909019097.
Prasad, N., R.S. Topping, and S.J. Decker. 2001. SH2-containing inositol 5'-phosphatase SHIP2 associates with the p130(Cas) adapter protein and regulates cellular adhesion and spreading. Mol. Cell. Biol. 21:14161428.
Rickert, P., O.D. Weiner, F. Wang, H.R. Bourne, and G. Servant. 2000. Leukocytes navigate by compass: roles of PI3Kgamma and its lipid products. Trends Cell Biol. 10:466473.[Medline]
Sakisaka, T., T. Itoh, K. Miura, and T. Takenawa. 1997. Phosphatidylinositol 4,5-bisphosphate phosphatase regulates the rearrangement of actin filaments. Mol. Cell. Biol. 17:38413849.[Abstract]
Sambrook, J., and D.W. Russel. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Servant, G., O.D. Weiner, P. Herzmark, T. Balla, J.W. Sedat, and H.R. Bourne. 2000. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science. 287:10371040.
Stossel, T.P., J. Condeelis, L. Cooley, J.H. Hartwig, A. Noegel, M. Schleicher, S. Shapiro. 2001. Filamin as integrators of cell mechanics and signalling. Nat. Rev. 2:138145.
Takafuta, T., G. Wu, G.F. Murphy, and S.S. Shapiro. 1998. Human beta-filamin is a new protein that interacts with the cytoplasmic tail of glycoprotein Ibalpha. J. Biol. Chem. 273:1753117538.
Taylor, V., M. Wong, C. Brandts, L. Reilly, N.M. Dean, L.M. Cowsert, S. Moodie, and D. Stokoe. 2000. 5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells. Mol. Cell. Biol. 20:68606871.
Tsujishita, Y., S. Guo, L.E. Stolz, J.D. York, and J.H. Hurley. 2001. Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase. Cell. 105:379389.[Medline]
van der Ven, P.F., W.M. Obermann, B. Lemke, M. Gautel, K. Weber, and D.O. Furst. 2000. Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation. Cell Motil. Cytoskeleton. 45:149162.[Medline]
Wada, T., T. Sasaoka, M. Funaki, H. Hori, S. Murakami, M. Ishiki, T. Haruta, T. Asano, W. Ogawa, H. Ishihara, and M. Kobayashi. 2001. Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity. Mol. Cell. Biol. 21:16331646.
Watton, S.J., and J. Downward. 1999. Akt/PKB localisation and 3' phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction. Curr. Biol. 9:433436.[Medline]
Whisstock, J.C., S. Romero, R. Gurung, H. Nandurkar, L.M. Ooms, S.P. Bottomley, and C.A. Mitchell. 2000. The inositol polyphosphate 5-phosphatases and the Apurinic/Apyrimidinic base excision repair endonucleases share a common mechanism for catalysis. J. Biol. Chem. 275:3705537061.
Wisniewski, D., A. Strife, S. Swendeman, H. Erdjument-Bromage, S. Geromanos, W.M. Kavanaugh, P. Tempst, and B. Clarkson. 1999. A novel SH2-containing phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells. Blood. 93:27072720.
Xie, Z., W. Xu, E.W. Davie, and D.W. Chung. 1998. Molecular cloning of human ABPL, an actin-binding protein homologue. Biochem Biophys. Res. Commun. 251:914919.[Medline]
Xu, W., Z. Xie, D.W. Chung, and E.W. Davie. 1998. A novel human actin-binding protein homologue that binds to platelet glycoprotein Ibalpha. Blood. 92:12681276.