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doi: 10.1242/10.1242/jcs.00070

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Src family tyrosine kinases regulate adhesion-dependent tyrosine phosphorylation of 5'-inositol phosphatase SHIP2 during cell attachment and spreading on collagen I

Nagendra Prasad, Robert S. Topping and Stuart J. Decker*

Department of Cancer Molecular Sciences, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA



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  		Fig. 8. SHIP2 associates with Shc via the NPXY motif. (A) Anti-SHIP2 or control (Pre) IPs from HeLa cells that were detached (D) or replated for 60 minutes on collagen I (6 µg/cm2; C.I) were blotted with anti-Shc (monoclonal) or anti-SHIP2 antibodies. (B) Anti-SHIP2 or control (Pre) IPs, from HeLa cells that were re-plated for 60 minutes on collagen I (6 µg/cm2; C.I), fibronectin (5 µg/cm2; F), or poly-L-lysine (0.01% solution, 0.5 ml/25 cm2; P-Ly) were blotted with anti-Shc (monoclonal) or anti-SHIP2 antibodies as indicated. Cells were plated in the presence of vehicle control DMSO (-) or 1 µM Src inhibitor compound PD180970 (+). Whole cell lysate was used as a control (WCL). (C) HeLa cells were transiently transfected with wild-type or YY-FF (986-987) mutant SHIP2 expression constructs. 48 hours post-transfection, serum-starved cells were re-plated on collagen I (6 µg/cm2) for 60 minutes. Anti-FLAG (rabbit polyclonal) IPs from these samples were blotted with anti-Shc (monoclonal) or monoclonal anti-FLAG (M2) antibodies. Whole cell lysate was used as a control (WCL). (D) Anti-Shc (monoclonal) or control mouse IgG (MIg) IPs from HeLa cells re-plated for 60 minutes on collagen I (6 µg/cm2; C.I), fibronectin (5 µg/cm2; F), or poly-L-lysine (0.01% solution, 0.5 ml/25 cm2; P-Ly) were blotted with anti-SHIP2 or anti-Shc (polyclonal) antibodies as indicated. Cells were plated in the presence of either vehicle control DMSO (-) or 1 µM Src inhibitor compound PD180970 (+). Whole cell lysate was used as a control (WCL). Small arrows point to the three forms of Shc proteins. The large arrowhead points to SHIP2 protein.

 


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  		Fig. 1. Collagen I induces SHIP2 tyrosine phosphorylation. Anti-SHIP2 or control pre-immune (Pre) immunoprecipitates (IP) from HeLa cells were blotted with anti-phosphotyrosine ({alpha}-PY). Anti-SHIP2 blots show amounts of SHIP2 protein in respective IP samples. The arrows point to tyrosine-phosphorylated SHIP2. (A) Time course: adherent (Ad), detached (D) or cells that were freshly plated on a collagen-I-coated (6 µg/cm2) surface (C.I) at indicated post-plating intervals were used for IP. (B) Coating density dependency: IPs were carried out as above. Cells were re-plated for 60 minutes on a polystyrene surface coated with increasing concentrations of collagen I (0.1 µg/cm2 to 6 µg/cm2).

 


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  		Fig. 2. SHIP2 was not tyrosine phosphorylated when plated on other ECM proteins. IPs and blots were carried out as described in Fig. 1. IPs were from HeLa cells that were adherent (Ad), detached (D) or re-plated (RP) for 60 minutes on surfaces coated with collagen I [6 µg/cm2; C.I, in the presence (+) or absence (-) of 1 µM Src inhibitor PD173956], fibronectin (5 µg/cm2; F), laminin (2 µg/cm2; L), collagen IV (6 µg/cm2; C.IV), or poly-L-lysine (0.01% solution, 0.5 ml/25cm2; P-Ly). Serum-starved adherent cells (Ad) treated with EGF (50 ng/ml for 5 minutes; E) or left untreated (control) are also shown. The arrow points to tyrosine-phosphorylated SHIP2.

 


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  		Fig. 3. Inhibitors of Src family kinases blocked collagen-I-induced SHIP2 tyrosine phosphorylation. (A) IPs were carried out from HeLa cells that were detached (D) or freshly re-plated (RP) on collagen-I-coated dishes (6 µg/cm2) in the presence of vehicle dimethyl sulfoxide (DMSO; —) or Src-kinase-selective Pyrido (2,3-d) pyrimidine tyrosine kinase inhibitors PD173956 (#56), PD173958 (#58) and PD180970 (#70) at 1 µM for 60 minutes. Anti-SHIP2 or pre-immune IPs were immunoblotted with anti-phosphotyrosine ({alpha}-PY) or anti-SHIP2 antibodies. (B) As described above except HeLa cells were re-plated in the presence of DMSO (-) or increasing concentrations (0.05-1.0 µM) of PD173956 (similar results were obtained with PD180970). The arrows point to tyrosine-phosphorylated SHIP2.

 


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  		Fig. 4. Integrin activation caused Src-dependent SHIP2 phosphorylation in other cell types. Anti-SHIP2 or control preimmune (Pre) IPs were blotted with anti-phosphotyrosine ({alpha}-PY) or anti-SHIP2 antibodies. Samples were from cells that were adherent (Ad), detached (D) or freshly re-plated (RP) on collagen-I-coated dishes (6 µg/cm2) in the presence of DMSO (-) or 1 µM Src inhibitor compound PD173956 (+) for 60 minutes. The arrows point to tyrosine-phosphorylated SHIP2. (A) SH-SY5Y neuroblastoma cells. (B) MDCK cells.

 


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  		Fig. 5. Involvement of Src family kinases in collagen I, but not in EGF- or insulin-induced SHIP2 phosphorylation. (A) IPs were carried out from serum-starved HeLa cells that were adherent (Ad), detached (D) or freshly re-plated (RP) on collagen-I-coated dishes (6 µg/cm2) as indicated. Adherent cells were pre-treated with DMSO (-) or Src inhibitor PD173956 at 1 µM (+) for 60 minutes followed by addition of EGF (50 ng/ml for 5 minutes; E) or IGF-1 (25 ng/ml for 10 minutes; I). Adherent samples without any growth factor treatment (Control) are shown as well. Similarly, re-plating of cells was done in the presence of DMSO (-) or 1 µM Src inhibitor PD173956 (+). Anti-SHIP2 or pre-immune IPs were immunoblotted with anti-phosphotyrosine ({alpha}-PY) or anti-SHIP2 antibodies. PD173958 and PD173956 yielded similar results. (B) Anti-SHIP2 IPs from 3T3-L1 adipocytes treated with insulin (50 nM for 15 minutes) or vehicle (V) were blotted with anti-phosphotyrosine ({alpha}-PY). Cells were pre-treated with 1 µM of Src inhibitor compounds, PD173956 (#56) or PD180970 (#70), for 60 minutes prior to insulin treatment. The arrows point to tyrosine-phosphorylated SHIP2.

 


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  		Fig. 6. Tyrosines in the 983NPAYY987 motif are important sites of phosphorylation. (A) Wild-type and YY-FF mutant of FLAG-tagged SHIP2 were transiently expressed in HeLa cells and anti-FLAG immunoprecipitates from adherent control (-), adherent EGF-treated (50 ng/ml for 5 minutes; E), detached (D) or re-plated on collagen I (6 µg/cm2 for 60 minutes; C.I) were blotted with anti-phosphotyrosine ({alpha}-PY) or anti-FLAG (M2) antibodies. (B) Wild-type and three Y-F (497, 747 and 1135) point mutant SHIP2 proteins expressed in HeLa cells were analyzed as above. (C) In vitro phosphorylation reactions were carried out using recombinant Src and immunopurified wild-type or YY-FF mutant SHIP2. Samples were blotted with anti-phosphotyrosine and anti-FLAG (M2) antibodies. The arrows point to tyrosine-phosphorylated SHIP2.

 


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  		Fig. 7. Transient transfection studies with exogenous Src corroborate its role in SHIP2 phosphorylation. (A) IPs (anti-SHIP2 and pre-immune) from HeLa cells transiently transfected with expression constructs encoding constitutively active Src (CA-Src) or dominant-negative Src (DN-Src) were blotted with anti-phosphotyroisne ({alpha}-PY). Mock-transfected cells were included as a control. Cells were either adherent on plastic (Ad), detached (D) or freshly re-plated on collagen-I-coated surface (6 µg/cm2) for 60 minutes. The arrow points to tyrosine-phosphorylated SHIP2. The bottom panel shows an anti-SHIP2 blot of the above immunoprecipitate samples. (B) Whole cell lysates prepared from the cells transfected as described above were blotted with anti-Src antibody. (C) Densitometric analyses of SHIP2 tyrosine phosphorylation levels observed in mock-transfected or DN-Src-transfected HeLa cells that were re-plated on a collagen-I-coated surface (6 µg/cm2) for 60 minutes.

 


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  		Fig. 9. Expression of YY-FF (986-987) mutant SHIP2 impairs lamelliodia formation. HeLa cells expressing wild-type SHIP2 (A,B,E,F) or YY-FF (986-987; C,D,G,H) mutant proteins were plated on collagen-I-coated (A-D) or fibronectin-coated (E-H) surface for 60 minutes. Cells were stained with anti-FLAG (M2) antibody to reveal expression of exogenous proteins (green) and counterstained with phalloidin-TRITC (red). Bar, 10 µm.

 

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