PMC

Definition of SHP-1 and SHP-2 binding sites within the c-Kit juxtamembrane region. (A) GST-c–Kit–JUX fusion proteins containing either the wild-type (JUX) or mutated versions of the c-Kit juxtamembrane region were expressed in TKXI cells, and the tyrosine-phosphorylated proteins were then immobilized on gluathione-Sepharose beads and incubated with lysates prepared from 10⁸ ConA-treated EL4 cells. Complexes and lysate proteins were then subjected to SDS-PAGE and anti-SHP-1 immunoblotting analysis. The specific JUX domain mutations are indicated above the lanes and include phenylalanine replacement of tyrosine 567 and 569 individually (Y567F and Y569F, respectively) or together [DM (Y567, Y569F)], alanine replacement of valine 568, and alanine replacement of isoleucine 570. The position of SHP-1 is indicated on the left. (B) The filter shown in panel A was stripped and reblotted with anti-SHP-2 antibody. The position of SHP-2 is indicated on the left.

Deletion of c-Kit Y⁵⁶⁹ abrogates binding of SHP-1 to the c-Kit juxtamembrane region. Tyrosine-phosphorylated or unphosphorylated GST–c-Kit–JUX fusion proteins containing either wild-type (JUX) or mutated versions of the c-Kit juxtamembrane domain were immobilized on glutathione-Sepharose beads and incubated with cell lysates (1,800 μg) prepared from ConA-treated EL4 cells. Complexes and lysate protein (500 μg) were then resolved by SDS-PAGE and immunoblotted with anti-SHP-1 antibody. Sites of the JUX domain mutations are indicated above the lanes and include deletions of tyrosine residues at position 544, 567, or 569 and, in the left panel only, replacement of Tyr⁵⁵² with phenylalanine (Y552F). For each panel mobilities of molecular mass (MW) standards are shown on the right, and the position of SHP-1 is indicated on the left.

Increases in association and tyrosine phosphorylation of SHP-1 and c-Kit following SCF stimulation. Cell lysates were prepared from unstimulated (−) and SCF (100 ng/ml)-treated Mo7e, FMA3, and HEL cells and from Ba/F3 cells stably expressing the full-length wild-type c-Kit cDNA (Ba/F3-Kit). (A) Protein lysates (1,800 μg) prepared from unstimulated or SCF-stimulated Mo7e, Ba/F3-Kit FMA3, and HEL cells were immunoprecipitated (Ip) with anti-c-Kit antibody, and the immune complexes were subjected to SDS-PAGE, immunoblotting with anti-SHP-1 antibody, and visualization with ¹²⁵I-protein A. Ba/F3 total cell lysate protein (500 μg) was included as a control for SHP-1 expression. (B) Cell lysate proteins (1,800 μg) prepared from unstimulated or SCF-stimulated Mo7e and HEL cells were immunoprecipitated with anti-SHP-1 antibody, and the immunoprecipitated and cell lysate proteins (500 μg) were resolved by SDS-PAGE and immunoblotted with anti-c-Kit antibody. (C) SHP-1 immunoprecipitates prepared from unstimulated or SCF-treated Mo7e and HEL cells were analyzed by SDS-PAGE and immunoblotting with antiphosphotyrosine antibody. (D) Cell lysate proteins (500 μg) as well as c-Kit immunoprecipitates from unstimulated or SCF-stimulated HEL cells (1,800 μg of lysate protein) were analyzed by SDS-PAGE and antiphosphotyrosine immunoblotting. For each panel, mobilities of molecular mass (MW) standards are shown on the right and positions of SHP-1 and c-Kit are indicated by arrows.

The Kit receptor preferentially associates with the SHP-1 SH2-N and SH2-C′ domains. (A) Schematic diagram showing the structures of the 71- and 67-kDa splice variants of SHP-1 and the SHP-1 SH2 domain sequences present in the GST–SHP-1 fusion proteins used for in vitro binding assays. The shaded region represents a 39-amino-acid segment present in the C-terminal SH2 domain (SH2-C′) of the 71-kDa, but not the 67-kDa, SHP-1 species. (B and C) Cell lysates (1,800 μg) prepared from 10⁸ SCF-stimulated HEL cells were incubated for 2 h at 4°C with 5 μg of purified GST–SHP-1 fusion protein immobilized on glutathione-Sepharose beads. Complexes as well as lysate alone were fractionated by SDS-PAGE and subjected to immunoblotting with anti-c-Kit (B) or anti-GST (C) antibodies. Mobilities of molecular mass (MW) standards are shown on the right.

Identification of Tyr⁵⁶⁹ as the c-Kit binding site for SHP-1. (A) Phosphopeptides (12-mers) spanning the five tyrosine sites contained in the c-Kit juxtamembrane region were synthesized with tyrosines in phosphorylated or unphosphorylated states (upper diagram), and the individual phosphopeptides (10 μM) were then incubated with cell lysates (1,800 μg) from EL4 cells in the presence of 5 μg of glutathione-Sepharose–GST-JUX fusion protein. Complexes were washed four times, and the complexes and lysate protein were then subjected to SDS-PAGE followed by anti-SHP-1 immunoblotting analysis. (B) Lysates prepared from ConA-stimulated EL4 cells were incubated with glutathione-Sepharose–GST-JUX fusion proteins (5 μg) in the presence of various amounts (1, 5, or 10 μM) of phosphopeptide 4 and with 10 μM phosphopeptide 4 preincubated with anti-pTyr antibody (anti-pY). Following washing, complexes and lysate protein (500 μg) were subjected to SDS-PAGE and anti-SHP-1 immunoblotting analysis. (C) c-Kit phosphopeptides 3 and 4 were individually coupled to NHS-Sepharose beads and then incubated with ConA-treated EL4 cell lysates. Following washing, the complexes and lysate protein were resolved by SDS-PAGE and subjected to anti-SHP-1 immunoblotting analysis. (D) Glutathione-Sepharose–GST-JUX fusion protein (5 μg) was incubated with lysates from ConA-treated EL4 cells in the absence or presence of 10 μM phosphopeptide 4, 5, or 6, and the complexes and lysate proteins were subjected to SDS-PAGE and anti-SHP-1 immunoblotting analysis. In each panel, mobilities of molecular mass (MW) standards are shown on the right and the position of SHP-1 is indicated on the left.

Association of SHP-1 with the c-Kit juxtamembrane region. (A) Top, schematic diagram showing domain structure of the c-Kit cytosolic region, including the juxtamembrane (JUX), kinase insert (KI), and carboxy-tail (C-TAIL) domains and two kinase domains. Numbers indicate the positions of the amino acid residues bordering each domain. TM, transmembrane. Bottom, pGEX2-T–Kit constructs encoding GST–c-Kit–JUX, –KI, or –C-TAIL fusion proteins were used for transformation into TKX1 cells, and the bacterial cells were treated sequentially with IPTG and IAA. Tyrosine-phosphorylated GST fusion proteins were then purified by affinity chromatography with glutathione-Sepharose beads, fractionated by SDS-PAGE, and visualized by Coomassie blue staining (left) or anti-pTyr immunoblotting analysis (right). (B) Top, cell lysates (1,800 ng) prepared from ConA (20 μg/ml)-treated EL4 cells were either immunoprecipitated with anti-SHP-1 antibody (IP: SHP-1) or incubated for 2 h with 5 μg of tyrosine-phosphorylated GST or GST-JUX, -KI or –C-TAIL fusion proteins immobilized on glutathione-Sepharose beads. Complexes and lysate protein alone were fractionated by SDS-PAGE and subjected to immunoblotting analysis with anti-SHP-1 antibody. Bottom, cell lysates (1,800 μg) prepared from ConA-treated EL4 cells were incubated with various amounts (3, 6, 9, or 12 μl) of glutathione-Sepharose–GST-JUX fusion protein, and the complexes were then subjected to SDS-PAGE and anti-SHP-1 immunoblotting analysis. Mobilities of molecular mass (MW) standards are shown on the right, and the position of SHP-1 is indicated on the left.

Mutations at the SHP-1 or SHP-2 binding sites on c-Kit enhance SCF-driven proliferation of Ba/F3-Kit cells. Cell lysates were prepared from unstimulated (−) or SCF (100 ng/ml)-treated (+) Ba/F3 transfectants infected with a retroviral vector carrying the full-length wild-type (Ba/F3-Kit), Y567→F-mutated (Ba/F3-Kit Y567F), or Y569→F-mutated (Ba/F3-Kit Y569F) c-Kit cDNA. (A and B) Lysate proteins (1,800 μg) from the unstimulated and stimulated cells were immunoprecipitated (Ip) with anti-c-Kit antibody, and the immune complexes and lysate proteins were then subjected to SDS-PAGE and anti-SHP-1 (A) and anti-SHP-2 (B) immunoblotting analysis. (C) Top, Ba/F3-Kit wild-type (Wt), Kit Y567F, and Kit Y569F cells were suspended at 2.5 × 10⁵ cells/ml in culture medium in the presence of various concentrations (0-400 ng/ml) of SCF. Cultures were harvested at 48 h following a 6-h pulse with 1 mCi of [³H]thymidine, and proliferation was measured by beta scintillation counting. Results (means ± SD) represent averages for triplicate cultures and three independent experiments. Bottom, Ba/F3 c-Kit wild-type (WT), Y567F, and Y569F cells (10⁵) were stained with the ACK2 anti-Kit antibody and examined for expression of c-Kit by FACS analysis. (D) Ba/F3, Ba/F3-Kit wild-type (Wt), Kit Y567F, and Kit Y569F cells were suspended at 10⁴ cells/ml in culture medium, and IL-3 (50 ng/ml) was added at day zero and every second day thereafter. Proliferation was evaluated every 48 h by the Cell Titer assay and enzyme-linked immunosorbent assay at 570 nm. Results (means ± SD) represent averages of triplicate cultures. OD, optical density.