The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3-kinase/Akt signaling pathway

We have recently demonstrated that the gene encoding the osteopontin (OPN) protein is activated both by interleukin-3 and granulocyte-macrophage colon y-stimulating factor signaling pathways and that, through binding to the ce ll surface receptor CD44, OPN contributes to the survival activities of int erleukin (IL)-3 and GM-CSF (Lin, Y.-H., Huang, C.-J., Chao, J.-R., Chen, S. -T., Lee, S.-F., Yen, J. J.-Y., and Yang-Yen, H.-F. (2000) Mot. Cell. Biol. 20, 2734 -2742). In this report, we demonstrate that the CD44-binding doma in of OPN involves a region containing amino acid residues from 121 to 140 and that both threonine and serine at positions 137 and 147, respectively, are essential for the survival stimulatory effect of OPN. Substitution of e ither residue with alanine results into a dominant negative mutant that ove rrides the survival effect of IL-3. Upon binding to the CD44 receptor, the wild-type OPN but not the inactive mutant induces activation of phosphatidy linositol 3-kinase and Akt. Last, we demonstrate that two waves of Akt acti vation are detected in IL-3-treated cells and that the survival promoting e ffect of OPN is mediated predominantly through the phosphatidylinositol 3-k inase/Akt signaling pathway. Together, our results suggest that a positive autoregulatory loop is involved in the survival pathway of IL-3.