Importance of tubulin detyrosination in platelet biogenesis

Abstract

Background: The functional diversity of microtubules is regulated through the expression of distinct x-and p-tubulin isotypes together with several posttranslational modifications, a concept known as tubulin code. Tubulin detyrosination is a reversible posttranslational modification that consists of the removal of the genetically encoded C-terminal tyrosine residue of most x-tubulins. While this modification has been observed in the megakaryocyte lineage, its importance remains poorly understood in platelet biogenesis. Objectives: To assess the role of x-tubulin detyrosination in platelet biogenesis. Methods: The responsible enzymes and the relative abundance of detyrosinated x-tubulins were monitored by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively, in human cultured megakaryocytes and platelets differentiated from CD34+ hematopoietic stem and progenitor cells. The function of x-tubulin detyrosination was assessed in human cultured megakaryocytes treated with the VASH-SVBP inhibitor EpoY, and in mice constitutively inactivated for Svbp (which encodes the cofactor of the VASH detyrosinases). Results: Transcriptional analysis identified VASH1-SVBP and MATCAP as the predominant detyrosinases in the megakaryocyte lineage. During megakaryocyte maturation, their transcript levels progressively increased and correlated with the accumulation of detyrosinated alpha-tubulins. Remarkably, inhibition of VASH1-SVBP by EpoY abolished tubulin detyrosination, establishing VASH1-SVBP as the main functional detyrosinase in megakaryocytes. More importantly, EpoY enhanced proplatelet formation and platelet production in vitro. These in vitro data were confirmed in vivo in SVBP-deficient mice, which exhibited an increase in platelet counts. Conclusion: These findings reveal, for the first time, a role for tubulin detyrosination in proplatelet formation, thereby expanding our understanding of the megakaryocyte tubulin code beyond tubulin isotypes.