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First published online January 26, 2005
doi: 10.1242/10.1242/jcs.01632

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A role for Mer tyrosine kinase in vß5 integrin-mediated phagocytosis of apoptotic cells

¹ Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA
² Department of Neuro-Oncology and Molecular Genetics, MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

View larger version (41K): [in a new window]   Fig. 1. Mer stimulates tyrosine phosphorylation of p130CAS, complex formation of p130CAS-CrkII-Dock180 and Rac1 activation. (A) (upper panel) HEK-293T cells were transfected with 1.0 µg plasmid encoding empty vector (lane 1), wild-type Mer (lanes 2 and 3), CDMer (lane 4) and CDMer/KD (lane 5). Mer-expressing cells were stimulated with Gas6 (150 nM) for 10 minutes (lane 3), although at this level of receptor expression, Mer activation was saturated. The cell lysates were separated by SDS-PAGE, and analyzed by immunoblotting with anti-phosphotyrosine mAb. (Lower panel) The blot was stripped and reprobed with anti-Mertk Ab, the arrows indicate Mer, CDMer or CDMer/KD. The lysates were immunoprecipitated with anti-p130CAS mAb and immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine Ab (inset). (B) (upper panel) Gas6 stimulates p130CAS phosphorylation in Mer-expressing HEK-293 T cells. HEK-293 T cells were transfected with 40 ng plasmid DNA encoding empty vector, wild-type Mer or Mer/KD as in A. The cells were starved for 18 hours, and stimulated with or without 150 nM Gas6 for 10 minutes as indicated. The cell lysates were immunoprecipitated with anti-p130CAS mAb, and the immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine mAb. The blot was reprobed with anti-p130CAS mAb to show equal recovery of the proteins (lower panel). (C) Mer induces CrkII binding to p130CAS and Dock180. HEK-293 T cells were transfected with empty vector (lane 1), CDMer (lane 2) and CDMer/KD (lane 3). After 48 hours, the cell lysates were immunoprecipitated with anti-CrkII Ab, and the immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine mAb. The blots were then cut and appropriate sections were reprobed with antibodies against p130CAS, Dock180 and CrkII (shown on right). (D) Effects of CDMer and Mer-Gas6 on Rac1 activation. Empty vector, CDMer and CDMer/KD were coexpressed with HA-Rac1 in HEK-293T cells. The detergent lysates were precipitated with 5 µg/ml GST-PAK CRIB Sepharose beads. The levels of Rac1 GTP loading were determined by immunoblotting with anti-HA antisera (i). A similar experiment was performed in Mer-expressing cells stimulated with Gas6 (ii).

View larger version (31K): [in a new window]   Fig. 2. Mer-mediated tyrosine phosphorylation of p130CAS is dependent on FAK activation. (A) HEK-293T cells were transfected with FAK-expressing plasmid plus empty vector, CDMer, or CDMer/KD as indicated. After 48 hours, cell lysates were immunoprecipitated with anti-FAK mAb, and the immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine Ab (i) or antibodies specific for Tyr 397/FAK or Tyr861/FAK (ii). FAK immunoprecipitates were subjected to an in vitro kinase assay to measure FAK activity and the autophosphorylation level of FAK was analyzed by densitometry (iii). (B) Effect of mutant FAK on p130CAS phosphorylation. HEK-293T cells were co-transfected with vector encoding p130CAS (lanes 1-4), CDMer (lanes 2-4), in combination with 0 (lane 1), 0 (lane 2), 1 (lane 3) and 3 (lane 4) µg of plasmid encoding FAK/KD (kinase negative mutant). p130CAS was immunoprecipitated from extracts containing equal amounts of total proteins and subjected to immunoblotting with mAb against phosphotyrosine. (C) Tyrosine phosphorylation of FAKTyr861 is Gas6-inducible. Mer- or Mer/KD-expressing HEK-293T cells were prepared as in Fig. 1B, and stimulated with or without Gas6, and the cell lysates were immunoprecipitated with anti-FAK mAb, and the immunoprecipitates were analyzed as in A.

View larger version (33K): [in a new window]   Fig. 3. FAKTyr861 associates with vß5 integrin upon Mer activation. (A) Cells were transfected with plasmid DNA encoding FAK and ß5 integrin as well as empty vector, CDMer or CDMer/KD as indicated. Subsequently, cell lysates were subjected to immunoprecipitation with anti-vß5 mAb (P1F6), and immunoblotting with anti-FAK Ab to detect FAK/vß5 integrin complex. The blot was stripped and reprobed with anti-phospho-FAK at Tyr861. (B) FAKTyr861 binding to vß5 integrin is linked to the p130CAS/CrkII/Dock180 complex. ß5CS-1 cells were transfected with empty vector, CDMer or CDMer/KD. CS-1 cells without transfection serve as control (-). The cell lysates were immunoprecipitated with anti-p130CAS and analyzed by immunoblotting with anti-phosphotyrosine mAb. The blot was stripped and reprobed with anti-phospho-FAK at Tyr861 to detect active FAK-p130CAS complex. (C) Gas6 induces a complex between FAKTyr861 and vß5 integrin. Empty vector (lane 1), wild-type Mer (lanes 2 and 3), or Mer/KD (lane 4) was expressed with FAK in control CS-1 (lane 1) or ß5CS-1 cells (lanes 2-4) for 48 hours. After starvation for 18 hours, the cells were stimulated with 150 nM Gas6 for 10 minutes (lanes 3 and 4). The cell lysates were analyzed by immunoblotting with antibodies specific for Tyr397/FAK and Tyr861/FAK (i). The lysates were also immunoprecipitated with anti-vß5 (P1F6) mAb, and immunoblotted with anti-FAK mAb or anti-phospho-FAK at Tyr861 to detect the phosphorylated and total FAK recovered with vß5 integrin immunoprecipitates (ii).

View larger version (48K): [in a new window]   Fig. 6. Activation of Mer enhances vß5 integrin-directed actin cytoskeletal remodeling. (A) The surface expression of integrin vß5 complex on control CS-1 cells (shaded) and ß5CS-1 (unshaded) was analyzed using anti-vß5 integrin mAb (P1F6) by flow cytometry (i). Control CS-1 or ß5CS-1 cells were allowed to attach to vitronectin-coated culture dishes at 37°C for 60 minutes and were washed with PBS twice to remove non-adherent cells. The adherent cells were visualized by microscopy (ii). (B) ß5CS-1 cells were transfected with empty vector or CDMer for 48 hours, cells were then fixed (i). Subsequently, cells were permeabilized by 0.2% Triton X-100 in PBS and stained with Rhodamine-Phalloidin (ii). (C) Gas6 induces Rac1 activation and morphological changes in cells plated on vitronectin. After starvation for 18 hours, DC2.4 cells were pretreated with or without 150 nM Gas6 at 37°C for 10 minutes, and 20,000 cells were seeded onto vitronectin-coated 96-well culture plate. After incubation at 37°C for 30 minutes, the cells were washed twice with PBS, fixed and visualized (i). Cell spreading was assessed by enumerating cells that displayed membrane filopodia and/or lamellipodia. Cells in four fields were counted on each plate (ii). After starvation for 18 hours, the DC 2.4 cells were stimulated with 150 nM Gas6 at 37°C for 10 minutes, and the lysates were precipitated with GST-PAK CRIB Sepharose beads. The levels of Rac1 GTP loading were determined by immunoblotting with anti-Rac1 mAb (iii). Bar, 20 µm.

View larger version (29K): [in a new window]   Fig. 4. Collaboration of Mer and vß5 integrin in phagocytosis of apoptotic cells. (A) HEK-293T cells were transfected with bicistronic pIRES-EGFP (GFP), pIRES-EGFP-ß5 (ß5), pIRES2-EGFP-Mer (Mer) or pIRES-EGFP-ß5 plus pIRES2-EGFP-Mer (ß5+Mer) plasmids. After 48 hours, transfected HEK-293T cells were co-cultured with red-labeled apoptotic T cells. (B) Phagocytosis was assayed 2 hours post co-culture, whereby double positive cells were scored from GFP-expressing cells. Data represent the mean±s.e.m. of triplicate measurements.

View larger version (24K): [in a new window]   Fig. 5. Src family kinases are required for Mer-mediated downstream tyrosine phosphorylation. (A) Challenge of Mer-expressing cells with apoptotic cells. Mer-expressing cells starved for 18 hours, were co-cultured with apoptotic T cells (AC) at a ratio of 1:10 (Mer-expressing cells: apoptotic cells). Mer was immunoprecipitated from extracts for subsequent in vitro kinase assay. The arrow represents Mer. (B) Mer-mediated FAKTyr861 and p130CAS phosphorylation is dependent on SFKs. Control NIH3T3 cells and SYF cells were transfected with empty vector or CDMer. Cell lysates were analyzed by immunoblotting with anti-phosphotyrosine mAb, and with anti-Mertk Ab to show equal Mer protein recovered (i). Kinase activity of active CDMer immunoprecipitated with anti-Mertk Ab from NIH 3T3 and SYF cells was compared by in vitro kinase assay to show Mer kinase is functional (ii). (C,D) Cell lysates were immunoblotted with anti-phospho-FAK at Tyr861 (C), or immunoprecipitated with anti-p130CAS antibody to show the level of tyrosine phosphorylation (D). (E) Mouse NIH3T3 or SYF cells were expressed with pCx-IRES-EGFP or pCx-IRES-EGFP-ß5 using retrovirus infection method as described in the Materials and Methods. When coexpressed with or without Mer, the NIH3T3 and SYF cells were analyzed for phagocytosis assay as described in the legend for Fig. 4. As indicated, phagocytotic ability of NIH3T3 or SYF cells expressing control vector pCx-IRES-EGFP was arbitrarily set as 100% (grey) with cells coexpressing ß5 and Mer shown in black.

View larger version (42K): [in a new window]   Fig. 7. Integrin vß5 is essential for Mer-mediated p130CAS phosphorylation, Rac1 activation, and phagocytosis. (A) ß5 null CS-1 cells expressing empty vector, ß5 or ß5C were expressed with or without CDMer cells. The lysates were immunoprecipitated with anti-p130CAS Ab, and analyzed by immunoblotting with anti-phosphotyrosine Ab (i). Alternatively, the lysates were precipitated with GST-PAK CRIB Sepharose beads, and the levels of Rac1 GTP loading were determined by immunoblotting with anti-Rac1 mAb (ii). ß5/ß5C expression was comparable between cells co-overexpressed with and without CDMer, as analyzed with anti-ß5 immunoblotting in RIPA buffer lysates (A, panel i) and P1F6 staining by FACScan (B). Unshaded, control CS-1 cells; shaded, CS-1 cells expressing ß5. (C) Phagocytosis assay. CS-1 cells were transfected with bicistronic pIRES-EGFP, pIRES-EGFP-ß5, or pIRES-EGFP-ß5C, with empty vectors or Mer. After 48 hours, transfected cells were co-cultured with red-labeled apoptotic T cells (1:10), and phagocytosis assay was performed as described in the legend for Fig. 4. Data represent the mean±s.e.m. percentage of phagocytosing cells from four separate experiments (i and ii). (D) CS-1 cells were transfected with bicistronic pIRES-EGFP or pIRES-EGFP-ß5, with control vector (-) or mutant Mer/KD and phagocytosis assay was performed as described above. Data represent the mean±s.e.m. percentage of phagocytosing cells from three separate experiments.

View larger version (21K): [in a new window]   Fig. 8. Schematic model for convergence of PS-dependent receptors vß5 integrin and Mer signaling. We propose that Mer activates a Src family kinase, resulting in FAKTyr861 phosphorylation and subsequent recruitment of FAK to the cytoplasmic tail of ß5, thereby vß5 integrin and Mer functionally cross-talk to amplify internalization signals.

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