PAK1 Regulates MEC-17 Acetyltransferase Activity and Microtubule Acetylation during Proplatelet Extension.

Juliette van Dijk,Gabriel Rabeharivelo,Nathalie Morin,Julien Cau,Guillaume Bompard,Claude Delsert

doi:10.3390/ijms21207531

Abstract

Mature megakaryocytes extend long processes called proplatelets from which platelets are released in the blood stream. The Rho GTPases Cdc42 and Rac as well as their downstream target, p21-activated kinase 2 (PAK2), have been demonstrated to be important for platelet formation. Here we address the role, during platelet formation, of PAK1, another target of the Rho GTPases. PAK1 decorates the bundled microtubules (MTs) of megakaryocyte proplatelets. Using a validated cell model which recapitulates proplatelet formation, elongation and platelet release, we show that lack of PAK1 activity increases the number of proplatelets but restrains their elongation. Moreover, in the absence of PAK1 activity, cells have hyperacetylated MTs and lose their MT network integrity. Using inhibitors of the tubulin deacetylase HDAC6, we demonstrate that abnormally high levels of MT acetylation are not sufficient to increase the number of proplatelets but cause loss of MT integrity. Taken together with our previous demonstration that MT acetylation is required for proplatelet formation, our data reveal that MT acetylation levels need to be tightly regulated during proplatelet formation. We identify PAK1 as a direct regulator of the MT acetylation levels during this process as we found that PAK1 phosphorylates the MT acetyltransferase MEC-17 and inhibits its activity.

Highlights

Platelets are small anucleate cytoplasts derived from giant precursor cells, the megakaryocytes (MKs), residing in the bone marrow
Using inhibitors of the tubulin deacetylase HDAC6, we demonstrate that abnormally high levels of MT acetylation are not sufficient to increase the number of proplatelets but cause loss of MT integrity
We identify PAK1 as a direct regulator of the MT acetylation levels during this process as we found that PAK1 phosphorylates the MT acetyltransferase MEC-17 and inhibits its activity

Summary

Introduction

Platelets are small anucleate cytoplasts derived from giant precursor cells, the megakaryocytes (MKs), residing in the bone marrow. While F-actin is involved in the branching of PPL shafts, thereby increasing the number of PPL tips available for platelet release [4], MTs provide the driving force for PPL formation. They form bundles which slide up against each other, in a dynein-dependent manner, providing the pushing force to generate initial broad pseudopodia and elongate them to form PPLs [5,6]. At the PPL tips, MT bundles turn around forming loops that will give rise to the MT ring known as the marginal band which forms the skeleton of the platelets This MT ring is mainly composed of β1-tubulin, a hematopoietic-specific beta-tubulin isoform expressed only in mature megakaryocytes. We recently showed that two MT post-translational modifications, acetylation and polyglutamylation, occur in PPLs and are mandatory for PPL formation [12]

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