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First published online 18 January 2005
doi: 10.1242/jcs.01643

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Integrin-dependent PLC-1 phosphorylation mediates fibronectin-dependent adhesion

Denis Tvorogov^1,*,, Xue-Jie Wang^1,*,, Roy Zent^2,3 and Graham Carpenter^1,¶

¹ Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
² Departments of Medicine and Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
³ Veterans Affairs Hospital, Nashville, TN 37232, USA

View larger version (31K): [in a new window]   Fig. 1. Expression of different integrin subunits in Null and Null+ cell lines. Null and Null+ cells were subjected to FACS analysis for different integrin subunits.

View larger version (15K): [in a new window]   Fig. 2. PLC-1 is required for maximal adhesion to fibronectin. (A) Null (white) and Null+ (gray) cells were allowed to adhere for 30 minutes on plates coated with the indicated concentrations of fibronectin. (B) Null () and Null+ () cells were incubated for the indicated times with plates coated with the indicated concentrations of fibronectin.

View larger version (40K): [in a new window]   Fig. 3. Influence of PLC-1 on spreading and migration behavior. (A) Null and Null+ cells were allowed to adhere to cover glasses coated with the indicated concentration of fibronectin for the indicated times. Actin distribution was then visualized by staining with phalloidin-TRITC. (B) Null (white) and Null+ (gray) cells were applied to fibronectin-coated transwell chambers and allowed to migrate for 3 hours. Subsequently, the chambers were processed to remove all cells from the top and the remaining cells were fixed and stained with crystal violet and counted as migrating cells.

View larger version (70K): [in a new window]   Fig. 4. Influence of PLC-1 on integrin ß1 clustering, FAK phosphorylation and ERK1,2 phosphorylation following adhesion to fibronectin. (A) Equal numbers of Null and Null+ cells were allowed to adhere for the indicated times to cover glasses coated with 10 µg ml-1 or 2.5 µg ml-1 fibronectin. Integrin ß1 distribution was visualized by staining with anti-integrin-ß1 antibody. (B) Equal numbers of Null and Null+ cells were allowed to adhere to 10 µg ml-1 fibronectin-coated dish for the indicated times. The cells were then lysed and each lysate was blotted with antibodies against phosphorylated or unphosphorylated FAK. (C) Equal numbers of Null and Null+ cells were allowed to adhere to 10 µg ml-1 fibronectin-coated dish for the indicated times. Then cells were lysed and each lysate was blotted with antibodies phosphorylated or unphosphorylated Erk1,2. Cells maintained in suspension (Susp) were used as a control.

View larger version (35K): [in a new window]   Fig. 5. Fibronectin-stimulated tyrosine phosphorylation of PLC-1. (A) Null+ cells were allowed to adhere to fibronectin-coated dishes (10 µg ml-1) in serum-free medium for the indicated times. The cells were then lysed and each lysate was precipitated with anti-PLC-1 antibody followed by blotting with site-specific antibodies against tyrosine-phosphorylated-PLC-1 or PLC-1 as indicated. (B) Null+ cells expressing wild-type PLC-1 or an N+C-SH2-domain loss-of-function PLC-1 mutant (N+C SH2-) (Ji et al., 1999) were allowed to adhere to fibronectin (10 µg ml-1) for the indicated times. The cells were than lysed and the lysate were precipitated with anti-PLC-1 antibody and then blotted with antibody against tyrosine-783-phosphorylated PLC-1 or PLC-1 as indicated. Cells maintained in suspension (Susp) were used as a control for time 0.

View larger version (28K): [in a new window]   Fig. 6. Influence of PLC-1 mutants on adhesion to fibronectin. (A) Null cells were infected for 8 hours with wild-type vaccinia virus or virus containing point mutations Y771F, Y783F or Y1253F, or wild-type PLC-1, as described elsewhere (Sekiya et al., 2004). Null+ cells were used as control. Equal numbers of cells from each cell line were allowed to adhere on fibronectin-coated plates for 30 minutes at 37°C. , Null cells with control virus; , Null cells with wild-type PLC-1 virus; , Null cells with Y771F virus; , Null cells with Y783F virus; , Null cells with Y1253F virus. (B) Null cells or Null cells expressing either wild-type PLC-1 or a PLC-1 mutant that contains loss of function mutations in the N- and C-terminal SH2 domains [N+C SH2- (Ji et al., 1999)] were allowed to adhere to fibronectin-coated plates for 30 minutes at 37°C. , Null cells; , Null+ cells expressing wild-type PLC-1; , Null+ cells expressing N+C SH2- PLC-1.

View larger version (33K): [in a new window]   Fig. 7. Src-kinase activity is necessary for fibronectin-induced PLC-1 phosphorylation. Null+ cells were allowed to adhere to fibronectin-coated dishes (10 µg ml-1) in serum-free medium containing 50 µM sodium pervanadate and in the presence of (A) EGF-receptor tyrosine-kinase inhibitor AG1478 or (B) Src-kinase inhibitor PP2. Then, the cells were lysed and each lysate was precipitated with anti-PLC-1 antibodies followed by blotting with antibodies against tyrosine-783-phosphorylated PLC-1 or PLC-1 as indicated. In each experiment, 25 ng ml-1 EGF was added for comparative purposes.

View larger version (37K): [in a new window]   Fig. 8. Fibronectin-dependent PLC-1 phosphorylation in SYF-/- and FAK-/- cells. (A) SYF-/- and Null+ cells were allowed to adhere to fibronectin-coated dish (10 µg ml-1) in serum-free media containing 50 µM sodium pervanadate. The cells were then lysed and each lysate was precipitated with anti-PLC-1 antibodies followed by blotting with site-specific antibodies against tyrosine-783-phosphorylated PLC-1 or PLC-1 as indicated. Cells maintained in suspension (Susp) were used as a control. (B) TFW-46 cells were grown in the presence of tetracycline (Tet+) to maintain an uninduced state. To induce FAK expression, cells were incubated in tetracycline-free growth medium. Cells were allowed to adhere on fibronectin-coated dish (10 µg ml-1) in serum-free medium containing 50 µM sodium pervanadate. Then, the cells were lysed and each lysate was precipitated with PLC-1 antibodies, followed by blotting with site-specific antibodies against tyrosine-783-phosphorylated PLC-1 or PLC-1 as indicated.

View larger version (55K): [in a new window]   Fig. 9. Fibronectin-induced PLC-1 association with Src or FAK. (A) Null+ cells were allowed to adhere for the indicated times on fibronectin-coated (10 µg ml-1) dish in serum-free medium. The cells were then lysed and each lysate was precipitated with anti-Src-kinase antibody followed by blotting with PLC-1, FAK or Src antibodies. Whole-cell lysate was loaded as a marker for migration pattern of each protein. (B) Null+ cells expressing wild-type (wt) PLC-1 or N+C SH2- PLC-1 mutant were allowed to adhere for the indicated times on fibronectin-coated dishes (10 µg ml-1) in serum-free medium. The cells were then lysed and each lysate was precipitated with anti-Src-kinase antibodies followed by blotting with anti-PLC-1 or anti-Src antibodies. (C) Null+ cells were allowed to adhere to fibronectin-coated dishes (10 µg ml-1) for the indicated times. Cells were lysed and each lysate was precipitated with anti-FAK antibody followed by blotting with anti-PLC-1 or anti-FAK antibodies.

View larger version (44K): [in a new window]   Fig. 10. PLC-1 association with Src and FAK in TFW-46 and SYF-/- cells. (A) TFW-46 cells were grown in the presence of tetracycline (Tet+) to keep them in the uninduced state. To induce FAK expression, the cells were incubated in tetracycline-free growth medium for the indicated periods of time, then lysed and blotted with anti-FAK and anti-PLC-1 antibody. (B) TFW-46 cells with uninduced (Tet+) and induced (Tet-) FAK expression were allowed to adhere for the indicated times on fibronectin(10 µg ml-1)-coated dishes in serum-free medium. Then, cells were lysed and each lysate was precipitated with anti-Src-kinase antibodies followed by blotting with anti-PLC-1 or anti-Src antibodies. PDL, cells that were allowed to adhere on a poly-D-lysine-coated dish. (C) SYF-/- and Null+ cells were allowed to adhere for the indicated times on fibronectin-coated dishes in serum-free medium. Then, cells were lysed and each lysate was precipitated with anti-FAK antibodies followed by blotting with anti-PLC-1 or anti-FAK antibodies. PDL, cells that were allowed to adhere on a poly-D-lysine-coated dish.

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