We have previously mapped critical TPO-induced proliferation to the proximal Box I and anti-apoptosis effects of TPO to Box 11 regions of c-mpl while TPO-induced activation signals such as CD61 expression, c-fos expression and SHIP/SHC tyrosine phosphorylation to the regions of c-mpl distal to Box II (Blood 92:251, 1998). In order to further define specific regions of c-mpl responsible for TPO-induced SHIP/SHC tyrosine phosphorylation, S/T phosphorylation and activation of AKT kinase, and tyrosine phosphorylation and expression of a novel phosphoprotein, pi 10, we over expressed wild type, deletion mutants and F to Y site-directed cytoplasmic mutants of c-mpl in the murine IL-3 -dependent Baf-3 cells. Deletion mutants downstream of Box II (AA#585) demonstrated normal TPO-induced proliferation and inhibition of apoptosis but demonstrated no TPO-induced SHIP/SHC tyrosine phosphorylation, AKT activation and S/T phosphorylation and tyrosine phosphorylation of a novel protein, pi 10, which could be co-immunoprecipitated using monoclonal antibodies to pBAD ser 112. Site directed mutagenesis of all cytoplasmic tyrosine residues of c-mpl demonstrated that TPO-induced S/T phosphorylation of AKT, tyrosine phosphorylation of pi 10 and tyrosine phosphorylation of SHC/SHIP mapped to the penultimate Y626 of c-mpl. AKT activation was blocked by Wortmanin and LY294002 (PI3 kinase inhibitors) but was unaffected by all other Y and S/T kinase inhibitors tested, pi 10 phosphorylation were blocked by all tyrosine kinase inhibitors tested but co-IP with pBAD was not affected. TPO-induced pi 10 expression was induced after 5 minutes and peaked in 30 minutes while TPO-induced S/T phosphorylation of AKT was maximal after only 15 seconds. Mutation of Y626 from Y to F resulted in inhibition of TPO-induced c-fos and CD61 expression as well as two early response genes, osteopontin and murine b-globin. Mutation of Y626 to F626 had no effect on TPO-induced proliferation or TPO-induced inhibition of apoptosis. Conclusion: The penultimate Y626 of c-mpl is essential for a variety of signaling events including CD61, pi 10 expression and tyrosine phosphorylation, SHIP/ SHC tyrosine phosphorylation, activation and S/T phosphorylation of AKT but not for TPO-induced proliferation and survival signals both of which map to regions c-mpl proximal to Y626 and Box II and distal to Box I. AKT activation is not critical for TPOinduced viability.
|Issue number||11 PART I|
|State||Published - Dec 1 2000|