Inhibition of Ret oncogene activity by the protein tyrosine phosphatase SHP1

Germline mutations in the Ret protooncogene give rise to the inherited endo crine cancer syndromes MEN types 2A and 2B and familiar medullary thyroid c arcinoma. Although it is well accepted that the constitutive active tyrosin e kinase of Ret oncogenes ultimately leads to malignant transformation, it is not clear whether a decrease in the autophosphorylation of oncogenic Ret forms can affect the mitogenic and transforming activities of Ret. Potenti al modulators of the tyrosine kinase activity of Ret could be tyrosine phos phatases that are expressed in human thyroid tissue. Therefore, we investig ated the impact of the tyrosine phosphatases SHP1 and SHP2 on the intrinsic tyrosine kinase activity and oncogenic potency of Ret with a 9-bp duplicat ion in the cysteine-rich domain (codons 634-636), which was described in a patient with MEN type 2A recently. SHP1 and SHP2 were stably overexpressed in NIH3T3 fibroblasts together with Ret-9bp. Coexpression of SHP1 with Ret- 9bp reduced the autophosphorylation of Ret-9bp by 19 +/- 7% (P = 0.01, n = 4), whereas no effect was seen with SHP2. Furthermore, Ret-9bp could be coi mmunoprecipitated with SHP1 but not with SHP2 antibodies. Suppression of th e Ret-9bp tyrosine kinase activity by SHP1 caused a decrease in activation of Erk2 (extracellular signal-regulated kinase) and abolished PKB/Akt (prot ein kinase B) phosphorylation. In addition, diminished Ret-9bp autophosphor ylation led to reduced phosphorylation of the transcription factor jun-D. F inally, the inhibitory effect on Ret-9bp signaling resulted in a 40-60% red uction of [H-3]thymidine incorporation and in reduced ability of NIH3T3 cel ls to form colonies in soft agar. In conclusion, the data suggest that SHP1 caused a moderate reduction of Ret autophosphorylation, which led to a str ong suppression of the Ret oncogene activity.