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

Abstract

Thrombopoietin (TPO) stimulates a network of intracellular 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 megakaryocytes (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 MEKphosphorylating activity of protein kinase C (PKC) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKC and PI3K 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 PKC in steadystate megakaryopoiesis.Binding of TPO 1 to its receptor, the product of the protooncogene c-mpl, activates a wide variety of signaling molecules and pathways. As for other cytokine systems, it is becoming clear that the response to TPO is characterized by networks of multiple branching and converging signaling pathways, which display extensive cross-talk. As such, blockade of one signaling pathway can be compensated by alternate pathways. This may partially explain relatively mild hematopoietic phenotypes of mice in which supposedly critical signaling pathways are disrupted by homologous recombination (1-3). We demonstrated previously that the ERK/MAPK pathway is activated in response to TPO in both a BaF3 cell line engineered to express full-length Mpl (BaF3/Mpl) and in primary MKs, and plays an important role in MK endomitosis (4). Consistent with our results, MKs from mice engineered to express only a truncated Mpl receptor missing 60 residues from the COOH terminus of the cytoplasmic domain (T61 or ⌬60 mice) display a reduced capacity to activate ERK and have significantly decreased endomitotic capability after TPO administration in vivo (3). The classic pathway of ERK activation is via growth factor-induced Shc phosphorylation followed by its association with Grb2 (5), which then activates Sos, a nucleotide exchange factor for Ras (6). Consequently, ERK can be activated by Ras-GTP through Raf and MEK phosphorylation. Several groups have reported that Shc is strongly activated in response to TPO (7). Hence, Shc-dependent activation of Ras is likely to be an important mechanism...

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