Evidence for a novel thrombopoietin signalling event: Activation of protein kinase a in human megakaryoblastic CMK cells

Thrombopoietin (TPO) plays a crucial role in megakaryocyte development. TPO signalling, which is mediated by its receptor Mpl, includes Janus kinase, (JAK) signal transducer and activator of transcription (STAT) and Shc/Ras/m itogen-activated protein kinase (MAPK) pathways. The precise nature of thes e signalling routes has not been clarified in detail up until now. We inves tigated the effect of TPO on activation of cAMP-dependent protein kinase (P KA) and its involvement in MAPK signalling in human megakaryoblastic leukae mia CMK cells. For estimation of PKA activity, phosphorylation of a PKA-spe cific peptide substrate was assayed in CMK cell lysates. Since activation o f PKA is associated with translocation of its catalytic subunit alpha (C-PK A) into the cell nucleus, Western blot analysis of nuclear fractions with a n anti-C-PKA antibody was additionally performed. The activation of TPO-ind uced MAPK activation and the effect of the PKA inhibitor H-89 was measured using immunoblotting with a monoclonal anti-pERK antibody. TPO enhanced cAM P and induced activation of PKA in CMK cells. In addition, H-89 partly bloc ked TPO-induced MAPK activation in CMK cells. Our results indicate a novel TPO-triggered signalling event, activation of the cAMP/PKA pathway in human megakaryoblastic CMK cells. This signal transduction route seems to be inv olved in TPO-induced MAPK signaling. (C) 2001 Academic Press.