Unconventional interaction of the zebrafish thrombopoietin receptor with Mapk14a determines thrombocyte development and function.

Background: Thrombopoietin receptor signalling permits the thrombocyte lineage development in human, mice as well as zebrafish. While the sequence, structure, signalling, and function of TpoR is well-conserved in humans and mice, the zebrafish thrombopoietin receptor (zTpoR) remains less understood.

Objectives: In this article, we identified the interactome of the cytoplasmic domain of zTpoR.

Methods: Using recombinant zTpoR cytoplasmic domain, we designed a pull down assay combined with mass spectrometry to identify possible interactors of the cytoplasmic domain. We further used biochemistry, flow cytometry and zebrafish bleeding time assay to demonstrate the effects of these interactions.

Results: We observed a specific interaction of the zTpoR with Mitogen-activated protein kinase14a (Mapk14a), a homolog of the human and mouse p38 Mitogen-activated protein kinaseα (p38-MAPKα). Interestingly, this interaction was not observed with human TpoR, highlighting a unique aspect of zebrafish biology. To understand the physiological consequence of zTpoR-Mapk14a interaction, we used inhibitors of Janus kinase (Jak) (ruxolitinib) and Mapk14a (BIRB 796) in zebrafish. Both ruxolitinib and BIRB 796 reduced the thrombocyte, myeloid and progenitor cell count while increasing the bleeding time. Moreover, combinatorial treatment with BIRB 796 and ruxolitinib showed an additive effect on bleeding time without any further decrease in the myeloid and progenitor cell counts.

Conclusion: We thus identified and characterized a specific interaction of zTpoR with Mapk14a that regulates zebrafish thrombopoiesis in both embryonic and adult stages.