SH2-containing protein tyrosine phosphatase-1 (SHP-1) association with Jak2 in UT-7/Epo cells

We have investigated the interaction of the SH2-containing protein tyrosine phosphatase-1 (SHP-1) and Jak2 in an erythropoietin (Epo)-dependent human leukemia cell line, UT-7/Epo, using reciprocal immunoprecipitation and immu noblotting. The Epo-induced kinetics and dose response on phosphorylated Ja k2 in anti-SHP-1 precipitates of UT-7/Epo cell lysates were similar to thos e in direct anti-Jak2 precipitates, suggesting that Jak2 coprecipitated wit h SHP-1. Furthermore, immunoblotting with anti-Jak2 and anti-SHP-1 antibodi es indicated that SHP-1 appeared to be constitutively associated with non-t yrosine-phosphorylated Jak2 in UT-7/Epo cells in the absence of Epo and wit hout phosphorylation of the Epo receptor (EpoR). Competition studies with C -terminal SHP-1 and Jak2 peptides decreased the amounts of SHP-1 and Jak2 d etected in immunoprecipitates supporting the specific coprecipitation of SH P-1 and Jak2. In the presence of a recombinant GST-fusion protein containin g both the N-terminal and C-terminal SH2 domains of SHP-1, anti-GST precipi tated the fusion protein but not cellular Jak2. These studies suggest that SHP-1 and Jak2 are constitutively associated in UT-7/EPO cells. The associa tion is not dependent upon Epo and is not mediated via SHP-1 SH2 binding. S equential double immunoprecipitation demonstrated that only a small portion of intracellular Jak2 and SHP-1 molecules are constitutively associated. T his partial association pattern may allow a more flexible and diverse regul ation of Jak2 and SHP-1 activities. Whether Jak2 and SHP-1 are directly ass ociated with each other or are part of a larger complex needs further inves tigation. (C) 2000 Academic Press.