The protein tyrosine phosphatase-PEST is implicated in the negative regulation of epidermal growth factor on PRL signaling in mammary epithelial cells

Treatment of HC11 mammary epithelial cells with the lactogenic hormone PRIL promotes differentiation and induction of milk protein gene expression via stimulation of the Janus kinase (JAK)/signal transducer and activator of t ranscription pathway. We have previously shown that autocrine activation of epidermal growth factor (EGF) receptor interferes with normal PRL-induced differentiation. Here we show that PRIL activation of JAK2 was dramatically reduced in HC11 cells pretreated with EGF, demonstrating that the target o f EGF receptor activation is JAK2 kinase. Using an in-gel protein tyrosine phosphatase (PTP) assay, we observed that the activity of a 125-kDa PTP was up-regulated in HC11 cells in response to EGF. A specific antiserum was us ed to demonstrate that the 125-kDa PTP was PTP-PEST and to show that EGF tr eatment of HC11 cells led to an increase in the level of PTP-PEST. In intac t HC11 cells, PTP-PEST was constitutively associated with JAK2, and in resp onse to EGF treatment there was an increased level of PTP-PEST in JAK2 comp lexes. An in vitro phosphatase assay, using PRL-activated JAK2 as the subst rate and lysates from HC11 cells as the source of PTP-PEST, revealed that J AK2 could serve as a PTP-PEST substrate. However, in intact cells the regul ation of JAK2 by PTP-PEST was complex, since transient overexpression of PT P-PEST had a negligible effect on PRL-induced JAK2 activation. EGF's negati ve influence on JAK2 activity was blocked by actinomycin D treatment of HC1 1 cells, suggesting that EGF induced a protein that mediated the effects of PTP-PEST on JAK2. In support of this model, PTP-PEST-containing lysates fr om EGF-treated HC11 cells dephosphorylated JAK2 to a greater extent than ly sates prepared from control cells.