Limits...
The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.

Dyson JM, O'Malley CJ, Becanovic J, Munday AD, Berndt MC, Coghill ID, Nandurkar HH, Ooms LM, Mitchell CA - J. Cell Biol. (2001)

Bottom Line: SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation.SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2.Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800 Australia.

ABSTRACT
SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

Show MeSH

Related in: MedlinePlus

SHIP-2 interacts with filamin A, B, and C in the yeast two-hybrid system. (A) Optimized alignment of the predicted amino acid sequences of human filamin A, B, and C. ↓ denotes the first amino acid of each domain; an asterisk represents identical amino acids in all three sequences; a colon represents conservative substitutions. Modified from Xie et al. (1998). The FLNC sequence shown in bold represents that encoded by the clone isolated in skeletal muscle library screening, using the SHIP-2 proline-rich domain as a bait. (B) Yeast expressing DNA-BDPRD bait were transformed with filamin A, B, or C cDNA (1, 3, and 5, respectively), and yeast expressing DNA-BDSHIP2ΔPRD were transformed with filamin A, B, or C (2, 4, and 6, respectively) and plated on medium lacking Tryptophan, Leucine, Histidine, and Adenine. (C) FLNC wild-type (aa 2434–2705 pFLNC) or truncation mutants (Table II) were prepared as described in Materials and methods. Yeast strain AH109 expressing the DNA-BDPRD “bait” were transformed with each FLNC mutant individually. The transformants were plated onto media lacking Tryptophan, Leucine, Adenine, and Histidine and assessed for LacZ. A strong or weak interaction is indicated by +++ or +, respectively. No interaction is indicated by (−).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2150887&req=5

fig2: SHIP-2 interacts with filamin A, B, and C in the yeast two-hybrid system. (A) Optimized alignment of the predicted amino acid sequences of human filamin A, B, and C. ↓ denotes the first amino acid of each domain; an asterisk represents identical amino acids in all three sequences; a colon represents conservative substitutions. Modified from Xie et al. (1998). The FLNC sequence shown in bold represents that encoded by the clone isolated in skeletal muscle library screening, using the SHIP-2 proline-rich domain as a bait. (B) Yeast expressing DNA-BDPRD bait were transformed with filamin A, B, or C cDNA (1, 3, and 5, respectively), and yeast expressing DNA-BDSHIP2ΔPRD were transformed with filamin A, B, or C (2, 4, and 6, respectively) and plated on medium lacking Tryptophan, Leucine, Histidine, and Adenine. (C) FLNC wild-type (aa 2434–2705 pFLNC) or truncation mutants (Table II) were prepared as described in Materials and methods. Yeast strain AH109 expressing the DNA-BDPRD “bait” were transformed with each FLNC mutant individually. The transformants were plated onto media lacking Tryptophan, Leucine, Adenine, and Histidine and assessed for LacZ. A strong or weak interaction is indicated by +++ or +, respectively. No interaction is indicated by (−).

Mentions: The SHIP-2 COOH-terminal proline–rich 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 proline–rich 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] 936–1258) 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 × 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 2434–2705 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 FcγRI, the platelet von Willebrand factor receptor, glycoprotein Ib-IX-V, β1 and β2 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).


The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin.

Dyson JM, O'Malley CJ, Becanovic J, Munday AD, Berndt MC, Coghill ID, Nandurkar HH, Ooms LM, Mitchell CA - J. Cell Biol. (2001)

SHIP-2 interacts with filamin A, B, and C in the yeast two-hybrid system. (A) Optimized alignment of the predicted amino acid sequences of human filamin A, B, and C. ↓ denotes the first amino acid of each domain; an asterisk represents identical amino acids in all three sequences; a colon represents conservative substitutions. Modified from Xie et al. (1998). The FLNC sequence shown in bold represents that encoded by the clone isolated in skeletal muscle library screening, using the SHIP-2 proline-rich domain as a bait. (B) Yeast expressing DNA-BDPRD bait were transformed with filamin A, B, or C cDNA (1, 3, and 5, respectively), and yeast expressing DNA-BDSHIP2ΔPRD were transformed with filamin A, B, or C (2, 4, and 6, respectively) and plated on medium lacking Tryptophan, Leucine, Histidine, and Adenine. (C) FLNC wild-type (aa 2434–2705 pFLNC) or truncation mutants (Table II) were prepared as described in Materials and methods. Yeast strain AH109 expressing the DNA-BDPRD “bait” were transformed with each FLNC mutant individually. The transformants were plated onto media lacking Tryptophan, Leucine, Adenine, and Histidine and assessed for LacZ. A strong or weak interaction is indicated by +++ or +, respectively. No interaction is indicated by (−).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150887&req=5

fig2: SHIP-2 interacts with filamin A, B, and C in the yeast two-hybrid system. (A) Optimized alignment of the predicted amino acid sequences of human filamin A, B, and C. ↓ denotes the first amino acid of each domain; an asterisk represents identical amino acids in all three sequences; a colon represents conservative substitutions. Modified from Xie et al. (1998). The FLNC sequence shown in bold represents that encoded by the clone isolated in skeletal muscle library screening, using the SHIP-2 proline-rich domain as a bait. (B) Yeast expressing DNA-BDPRD bait were transformed with filamin A, B, or C cDNA (1, 3, and 5, respectively), and yeast expressing DNA-BDSHIP2ΔPRD were transformed with filamin A, B, or C (2, 4, and 6, respectively) and plated on medium lacking Tryptophan, Leucine, Histidine, and Adenine. (C) FLNC wild-type (aa 2434–2705 pFLNC) or truncation mutants (Table II) were prepared as described in Materials and methods. Yeast strain AH109 expressing the DNA-BDPRD “bait” were transformed with each FLNC mutant individually. The transformants were plated onto media lacking Tryptophan, Leucine, Adenine, and Histidine and assessed for LacZ. A strong or weak interaction is indicated by +++ or +, respectively. No interaction is indicated by (−).
Mentions: The SHIP-2 COOH-terminal proline–rich 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 proline–rich 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] 936–1258) 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 × 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 2434–2705 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 FcγRI, the platelet von Willebrand factor receptor, glycoprotein Ib-IX-V, β1 and β2 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).

Bottom Line: SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation.SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2.Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800 Australia.

ABSTRACT
SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline-rich domain. To identify proteins that bind to the SHIP-2 proline-rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

Show MeSH
Related in: MedlinePlus