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SHP-2 complex formation with the SHP-2 substrate-1 during C2C12 myogenesis

Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066, USA

View larger version (62K): [in a new window]   Fig. 1. Tyrosyl phosphorylation and SHP-2 protein expression during myogenic differentiation in the murine C2C12 cell line. C2C12 myoblasts were cultured either in the presence of growth medium (GM) or in differentiation medium (DM) for the indicated times as described in Materials and Methods. (A) (Top) Total cell lysates were prepared, separated by SDS-PAGE and immunoblotted with 4G10 anti-phosphotyrosine antibodies (pTyr). The arrows to the right indicate either specific proteins or a molecular mass range in which total tyrosyl-phosphorylation changes are observed during differentiation. The molecular mass markers (Gibco-BRL) are depicted on the left. (Bottom) Representative photomicrographs of C2C12 myoblasts, fixed in methanol and visualized with Wright Giemsa Stain, at each stage of differentiation corresponding to the total tyrosyl-phosphorylation analysis in top panel. (B) C2C12 cell lysates (50 µg) prepared from A were immunoblotted with the indicated antibodies. The positions of molecular mass markers are shown.

View larger version (20K): [in a new window]   Fig. 2. SHP-2 forms a complex with a p120 tyrosyl-phosphorylated protein during C2C12 myogenesis. C2C12 myoblasts cultured at the indicated times were subjected to (A) immunoprecipitation (IP) with either preimmune (PI) antisera or anti-SHP-2 antibodies. Immune complexes were resolved by SDS-PAGE, transferred to Immobilon and immunoblotted with anti-phosphotyrosine (pTyr) antibodies. The arrows to the right indicate the positions of the p120 and p180 tyrosyl-phosphorylated proteins that associate with SHP-2 during differentiation. The immunoblot was reprobed with anti-SHP-2 antibodies (bottom). (B) Cell lysates from C2C12 myoblasts cultured as indicated were immunoprecipitated with anti-SHP-2 antibodies and immunoblotted with pTyr antibodies. As a control, this immunoblot was reprobed with anti-SHP-2 antibodies (bottom). The positions of molecular mass markers are shown.

View larger version (37K): [in a new window]   Fig. 3. The SHP-2-interacting p120 tyrosyl-phosphorylated protein is SHPS-1. (A) (Left) Proliferating C2C12 myoblasts cultured in GM were lysed and immunoprecipitated with either pre-immune (PI) or anti-SHP-2 antibodies. Immune complexes were resolved by SDS-PAGE and immunoblotted with anti-SHPS-1 (epitope directed to the conserved cytoplasmic domain of SIRP family members). (Right) C2C12 myoblast lysates prepared as described above were subjected to immunoprecipitation with anti-SHPS-1/MFR (10C4) antibodies, which recognize the V1 hypervariable extracellular region of SHPS-1. Immune complexes were immunoblotted with anti-SHPS-1 antibodies. (Bottom) The above immunoblots were reprobed with anti-SHP-2 antibodies. (B) Lysates were prepared from C2C12 myoblasts cultured in either GM or DM for the indicated times and were immunoblotted with anti-SHPS-1 antibodies. (C,D) Tyrosyl phosphorylation of SHPS-1 and association with SHP-2 during C2C12 myogenesis. Lysates prepared from C2C12 myoblasts cultured in either GM or DM for the indicated times were subjected to immunoprecipitation with either anti-SHPS-1 antibodies, followed by pTyr immunoblotting (C) or immunoprecipitation with anti-SHP-2 antibodies followed by anti-SHPS-1 immunoblotting (D). The lower panel in C represents an equal fraction of the lysates from the experiment above, which was immunoprecipitated for SHPS-1 and immunoblotted with anti-SHPS-1 antibodies as a control. The lower panel in D is a reprobe of the above experiment with anti-SHP-2 antibodies. The positions of molecular mass markers are shown.

View larger version (40K): [in a new window]   Fig. 4. SHP-2 associates with Gab-1 in C2C12 myoblasts. (A) Lysates prepared from C2C12 myoblasts cultured in GM were immunoprecipitated with either preimmune (PI) antisera or anti-SHP-2 antibodies. Immune complexes were immunoblotted with anti-Gab-1 antibodies. The bottom panel is a reprobe with anti-SHP-2 antibodies from the above experiment. (B,C,D) Tyrosyl dephosphorylation of Gab-1 and constitutive association with SHP-2 during C2C12 myogenesis. Lysates prepared from C2C12 myoblasts were cultured in either GM or DM for the indicated times. (B) Total cell lysates were immunoblotted with anti-Gab-1 antibodies. Lysates prepared from C2C12 myoblasts under the indicated conditions were also subjected to immunoprecipitation with either (C) anti-Gab-1 antibodies followed by pTyr immunoblotting or (D) immunoprecipitation with anti-SHP-2 antibodies followed by anti-Gab-1 immunoblotting. The bottom panel in C represents an equal fraction of the lysates from the experiment above that was immunoprecipitated for Gab-1 and immunoblotted with anti-Gab-1 antibodies as a control. Bottom panel in D is a reprobe with anti-SHP-2 antibodies of the above experiment. The positions of molecular mass markers are shown.

View larger version (43K): [in a new window]   Fig. 5. Induction of SHPS-1 tyrosyl phosphorylation by constitutive expression of MyoD in 10T fibroblasts. (A) 10T and 10T-MyoD fibroblasts cultured in GM were subjected to immunoprecipitation with either pre-immune (PI) antisera or anti-SHP-2 specific antibodies. Immune complexes and one-tenth of the total cell lysates (TCL) were resolved and immunoblotted with pTyr antibodies. The bottom panel represents a reprobe of the above immunoblot with anti-SHP-2 antibodies. (B) Equal amounts (50 µg) of total cell lysates from 10T and 10T-MyoD cells were immunoblotted with anti-SHPS-1 antibodies. (C) 10T and 10T-MyoD cells were immunoprecipitated with either pre-immune (PI) or anti-SHP-2 antibodies and immunoblotted for SHPS-1. The bottom panel shows the immunoblot reprobed for SHP-2. (D) Lysates prepared from 10T and 10T-MyoD cells were immunoprecipitated with anti-SHPS-1 antibodies and immune complexes were immunoblotted with pTyr antibodies. (Bottom) SHPS-1 was also immunoprecipitated from a fraction of these same cell lysates and immune complexes were immunoblotted with anti-SHPS-1 antibodies as a control. The positions of molecular mass markers are shown.

View larger version (27K): [in a new window]   Fig. 6. Inducible activation of MyoD in 10T MyoD-ER fibroblasts promotes SHPS-1 tyrosyl phosphorylation and association with SHP-2. 10T MyoD-ER cells were cultured in GM or DM in the absence (-E) or presence (+E) of estradiol (0.1 µM) for 18 hours. Cell lysates prepared from these cultures were (A) immunoblotted with anti-MHC antibodies or (B) immunoprecipitated with anti-SHPS-1 antibodies followed by pTyr immunoblotting (top). As a control an equal fraction of these lysates were immunoprecipitated for SHPS-1 and immunoblotted with anti-SHPS-1 antibodies. Gab-1 was also immunoprecipitated under the indicated conditions and subjected to anti-pTyr immunoblotting (bottom). (C) SHP-2 was immunoprecipitated under the conditions indicated; tyrosyl phosphorylation of the p120/SHPS-1 complexed with SHP-2 was assessed by immunoblotting with anti-pTyr antibodies (top) and the amount of SHPS-1 in the complex was determined by immunoblotting for SHPS-1 (middle). The bottom panel shows the SHP-2 reprobe as a control.

View larger version (71K): [in a new window]   Fig. 7. p38 MAPK promotes MyoD induction, SHPS-1 tyrosyl phosphorylation and association with SHP-2 during myogenesis. (A) Photomicrographs of C2C12 myoblasts treated either with DMSO (0.2% (v/v)) or with 30 µM SB203580 for 72 hours in differentiation medium (DM). (B) C2C12 myoblasts were induced to differentiate in the presence of DMSO as control or SB203580 (30 µM). Cell lysates were prepared from these cultures at the indicated times. These lysates were subjected to immunoblotting with (B) SHPS-1, SHP-2 or MyoD antibodies, (C) SHPS-1 immunoprecipitation followed by pTyr immunoblotting or (D) SHP-2 immunoprecipitation followed by SHPS-1 immunoblotting. The bottom panel in C shows a fraction of the same cell lysates from the above experiments immunoprecipitated with anti-SHPS-1 antibodies and immunoblotted with SHPS-1 antibodies. The bottom panel in D shows the reprobe with anti-SHP-2 antibodies.

View larger version (74K): [in a new window]   Fig. 8. Insulin, IGF-I and IGF-II fail to induce SHPS-1 tyrosyl phosphorylation. C2C12 myoblasts were either left unstimulated, or were stimulated with insulin, IGF-I or IGF-II following serum deprivation for 6 hours. Cell lysates prepared from C2C12 myoblasts following stimulation with the indicated growth factor were immunoblotted with anti-phospho MAPK and reprobed with anti-MAPK antibodies (top). These cell lysates were also immunoblotted with anti-phospho-Akt antibodies and reprobed with anti-Akt antibodies (middle). These immunoblots were also reprobed with anti-Akt antibodies. SHPS-1 immunoprecipitated from the cell lysates shown in the top panels were immunoblotted with either anti-pTyr or anti-SHPS-1 antibodies (bottom).

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