The Roles of Phosphatidylinositol 3-Kinase and Protein Kinase Cζ for Thrombopoietin-induced Mitogen-activated Protein Kinase Activation in Primary Murine Megakaryocytes

Thrombopoietin (TPO) stimulates a network of intra-cellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryo-cytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Cζ (PKCOζ) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCζ and PI3K P13K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCζ in steady-state megakaryopoiesis.