Antagonism or synergism. Role of tyrosine phosphatases SHP-1 and SHP-2 in growth factor signaling

Abstract

SHP-1 and SHP-2 are two Src homology 2 domain-containing tyrosine phosphatases with major pathological implications in cell growth regulating signaling. They share significant overall sequence identity, but their biological functions are often opposite. SHP-1 is generally considered as a negative signal transducer and SHP-2 as a positive one. However, the precise role of each enzyme in shared signaling pathways is not well defined. In this study, we investigated the interaction of these two enzymes in a single cell system by knocking down their expressions with small interfering RNAs and analyzing the effects on epidermal growth factor signaling. Interestingly, knockdown of either SHP-1 or SHP-2 caused significant reduction in the activation of ERK1/2 but not Akt. Furthermore, SHP-1, SHP-2, and Gab1 formed a signaling complex, and SHP-1 and SHP-2 interact with each other. The interaction of SHP-1 with Gab1 is mediated by SHP-2 because it was abrogated by knockdown of SHP-2, and SHP-2, but not SHP-1, binds directly to tyrosine-phosphorylated Gab1. Together, the data revealed that both SHP-1 and SHP-2 have a positive role in epidermal growth factor-induced ERK1/2 activation and that they act cooperatively rather than antagonistically. The interaction of SHP-1 and SHP-2 may be responsible for previously unexpected novel regulatory mechanism of cell signaling by tyrosine phosphatases.

Fig. 1

Knockdown of SHP-1 and SHP-2 expressions in Caco2 cells by siRNA. Caco2 cells (30–40% confluency) were transfected with the indicated siRNAs in the presence of lipofectamine 2000 as described in Experimental Procedures. Cell extracts containing equal amounts of total proteins were separated on 10% polyacrylamide gels, transferred to PVDF membranes, and subjected to Western blot analysis with antibodies against SHP-1, SHP-2, ERK1/2, and GAPDH as indicated.

Fig. 2

Effects of SHP-1 and SHP-2 knockdowns on EGF-induced activation of ERK1/2. Caco2 cells were treated as described in Fig. 1 except that immediately before extraction cells were stimulated with 50 ng/ml of EGF for the indicated periods of time. Cell extracts were subjected to Western blot analysis with specific antibodies that detect the indicated proteins. The line graphs in the right panel are quantitative representations of phospho-ERK2 and phospho-Akt levels (normalized with total ERK2 and Akt, respectively) based on gel scanning data from three independent experiments. Data represent relative band intensity with error bars denoting standard deviation.

Fig. 3

Verification of the specific effects of SHP-1 and SHP-2 knockdowns on EGF-induced activation of ERK1/2 by using vector-based siRNAs. Caco2 cells were transfected with empty pSure-neo vector or constructs carrying SHP-1 or SHP-2 siRNAs as described in Experimental Procedures. Cells were serum-starved and then stimulated with 50 ng/ml of EGF for the indicated periods of time. Cell extracts were subjected to Western blot analysis with specific antibodies that detect the indicated proteins.

Fig. 4

Association of SHP-1 and SHP-2 with Gab1. Subconfluenet Caco2 cells were serum-starved for 16 hr and then left untreated or treated with 50 ng/ml of EGF for the indicated periods of time. Cell extracts were immunoprecipitated with anti-Gab1 antibody. The immunoprecipitates were analyzed for the presence of SHP-1 and SHP-2 by Western blotting with antibodies against SHP-1 and SHP-2 as indicated. An irrelevant rabbit IgG was used as control.

Fig. 5

Disruption of Gab1/SHP-1 association by knockdown of SHP-2 expression. Caco2 cells were treated with SHP-1 or SHP-2 siRNA as described in Fig. 1. Cell extracted were immunoprecipitated with anti-Gab1 antibody, and the immunoprecipitates were subjected to Western blotting analysis with anti-SHP-1 or anti-SHP-2 antibodies as indicated.

Fig. 6

Direct binding of SHP-2 but not SHP-1 to tyrosine-phosphorylated Gab1. Non-phosphorylated GST-Gab1CT and phosphorylated GST-pGab1CT were immobilized onto glutathione-Sepharose beads and then incubated with purified SHP-1 and SHP-2. Proteins bound to the beads were analyzed by Western blotting with antibodies against SHP-1, SHP-2, phosphotyrosine, and GST. Purified SHP-1 and SHP-2 were loaded as controls.

Fig. 7

Direct interaction of SHP-1 with SHP-2. A. Subconfluent Caco2 cells were serum-starved for 16 hr and then left untreated or treated with 50 ng/ml of EGF for 3 min. Cell extracts were immunoprecipitated with anti-SHP-1 or ant-SHP-2 antibodies, and the immunoprecipitates were subjected to Western blotting analyses with antibodies against SHP-1 and SHP-2 as indicated. An irrelevant rabbit IgG was used as controls. B. GST and GST-SHP-2 immobilized onto glutathione-Sepharose beads were incubated with purified MBP or MBP-SHP-1. Proteins bound to the beads and remained in supernatants were analyzed by Western blotting with an anti-SHP-1 antibody.