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. 2006 Oct 9;27(1):220–228. doi: 10.1128/MCB.00899-06

FIG. 2.

FIG. 2.

Src kinase mediates hyperphosphorylation of Tyr877 in ErbB2-5M. (a) Src kinase-specific inhibitor PP1 inhibits ErbB2 Tyr877 phosphorylation and reduces Tyr1248 phosphorylation. COS7 cells expressing ErbB2-5M were treated with 10 μM PP1 for the indicated times or with the same amount of vehicle (dimethyl sulfoxide) for 1 h. ErbB2 protein and phosphorylation were determined as described in Fig. 1c. (b) Dominant-negative (DN) Src inhibits ErbB2-5M phosphorylation on Tyr877. COS7 cells transiently expressing ErbB2-5M together with dominant-negative or wild-type (WT) Src were lysed with modified RIPA buffer. Total cell lysates were used to detect ErbB2 protein and Tyr877 phosphorylation levels by Western blotting. The expression level in cell lysate of DN and wild-type Src protein is shown. The ratio of intensities of the bands corresponding to phospho-Y877 and total ErbB2, as determined by densitometry, are displayed below the blots. (c) Wild-type Src increases ErbB2 phosphorylation on Tyr877. COS7 cells were transfected with wild-type or kinase-deficient ErbB2, together with wild-type Src or empty vector (v). ErbB2 protein and phosphorylation were determined as described in the legend to Fig. 1c. (d) Src phosphorylates ErbB2 on Tyr877 in an in vitro assay. Wild-type ErbB2 protein expressed in COS7 cells was immunoprecipitated (IP) from total cell lysate, aliquoted into four tubes, and mixed with purified Src proteins as indicated. The in vitro kinase assay was carried out at 30°C for 30 min in the presence or absence of Src inhibitor PP1 and ErbB inhibitor CI-1033. Samples were examined by Western blotting. Duplicate blots were used to detect ErbB2 phosphorylation on Tyr877 and the protein levels of ErbB2 and Src. (e) Src associates to a higher level with ErbB2-5M than with wild-type ErbB2. COS7 cells were transfected with wild-type Src together with wild-type ErbB2 or ErbB2-5M. Src was immunoprecipitated from total cell lysate, and coprecipitation of ErbB2 was examined by Western blotting. Expression levels of Src and ErbB2 were also examined by Western blotting.