Starved PEPs have been mock stimulated or pretreated with 0, 25, 50 or one hundred nM wortmannin for 30 min or with 30 or one hundred M LY294002 for 1 h then stimu lated with 0. three U ml Epo where indicated. For comparison, PEPs starved and pretreated with one hundred nM WM or one hundred M LY where indicated have been stimulated with 25 ng ml stem cell fac tor for ten min to activate c Kit signaling. 100g total cell protein have been immunoblotted with P STAT5, P Akt or P Erk1 2 antibodies as indicated. GTP loaded Ras was precipi tated with GST c Raf1 RBD from 500g total cell protein and immunoblotted with anti Ras. indicates non starved and non treated PEPs. PEPs pretreated with one hundred nM WM for 30 min where indicated had been mock stimulated or treated with 0. 3 U ml Epo or 25 ng ml SCF for ten min.
inhibitor MK-0752 100g total cell protein were immunoblotted with P MEK1 2, Erk1 two, P Erk1 2 or P GSK3 antibodies as indicated. Phosphorylated EpoR was immunoprecipitated with anti phosphotyrosine mAb from 500g cell protein and immunoblotted with anti EpoR. indicates non starved and non treated PEPs. its PI3Ks, which was not expected to influence Erk activation, was also tested. Surprisingly, not simply the phosphoryla tion with the kinase Akt, a target of PI3Ks, on Ser 473 was inhibited, but in addition a block of Erk activation was seen with LY, albeit at greater concentrations. Dose dependent Erk inhibition was additional observed with the structurally and mechanistically distinct PI3K inhibitor wortmannin, once more at concentrations somewhat higher than these expected to suppress Epo effects on Akt Ser473 phosphorylation.
Phospho Erk inhibi tion by LY and WM was also not found when PEPs have been stimulated with low concentrations of your c Kit ligand SCF, suggesting that that is an Epo specific signal. WM also inhibited the phosphorylation of the Akt targets GSK3 and GSK3 as well as the activation of MEKs. In contrast, effects on tyrosine phosphoryla tions of STAT5 or EpoR Rhein by WM were not detected, and autophosphorylation of tyrosines 1007 and 1008 within the EpoR connected kinase Jak2 was not inhibited. Ras is activated upon Epo therapy of PEPs and generally upstream of MEKs. Consequently, the effects LY and WM have on GTP loading of Ras were also investigated. Both inhibitors totally blocked Ras activa tion by Epo but not by SCF, indicating that Ras is down stream of a PI3K activity in Epo stimulated PEPs. This mode of signal transmission from PI3K to Ras is distinct from signaling routes described for many other cell sorts or stimuli but not unprecedented, since the PI3Ks can, by way of example, induce the release of intracellular calcium, that is recognized to regulate Ras by way of Pyk2 and Ras GRFs.