Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets
Begoña Hurtado, … , Pablo García de Frutos, Marcos Malumbres
Begoña Hurtado, … , Pablo García de Frutos, Marcos Malumbres
Published September 25, 2018
Citation Information: J Clin Invest. 2018;128(12):5351-5367. https://doi.org/10.1172/JCI121876.
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Research Article Cell biology Hematology

Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets

Abstract

MASTL, a Ser/Thr kinase that inhibits PP2A-B55 complexes during mitosis, is mutated in autosomal dominant thrombocytopenia. However, the connections between the cell-cycle machinery and this human disease remain unexplored. We report here that, whereas Mastl ablation in megakaryocytes prevented proper maturation of these cells, mice carrying the thrombocytopenia-associated mutation developed thrombocytopenia as a consequence of aberrant activation and survival of platelets. Activation of mutant platelets was characterized by hyperstabilized pseudopods mimicking the effect of PP2A inhibition and actin polymerization defects. These aberrations were accompanied by abnormal hyperphosphorylation of multiple components of the actin cytoskeleton and were rescued both in vitro and in vivo by inhibiting upstream kinases such as PKA, PKC, or AMPK. These data reveal an unexpected role of Mastl in actin cytoskeletal dynamics in postmitotic cells and suggest that the thrombocytopenia-associated mutation in MASTL is a pathogenic dominant mutation that mimics decreased PP2A activity resulting in altered phosphorylation of cytoskeletal regulatory pathways.

Authors

Begoña Hurtado, Marianna Trakala, Pilar Ximénez-Embún, Aicha El Bakkali, David Partida, Belén Sanz-Castillo, Mónica Álvarez-Fernández, María Maroto, Ruth Sánchez-Martínez, Lola Martínez, Javier Muñoz, Pablo García de Frutos, Marcos Malumbres

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Figure 7

Platelet defects in Mastl(ED/ED)-mutant mice are mimicked with actin polymerization inhibitors and rescued by inhibiting inside-out kinases.

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Platelet defects in Mastl(ED/ED)-mutant mice are mimicked with actin pol...
(A) Representative images of WT platelets treated with cytochalasin D (CytD) or jasplakinolide (Jasplak) 15 minutes after activation with thrombin. Untreated WT or mutant platelets are shown as controls. (B) Percentage of annexin V+ cells, as quantified by flow cytometry with specific antibodies, in platelets (as labeled with CD41-specific antibodies) from mice of the indicated genotypes in the absence or presence of cytochalasin D, jasplakinolide, or the Bcl2-family inhibitor ABT-263 (used as a control for the induction of cell death), 1, 3, or 6 hours after activation with thrombin. Data are shown as the mean ± SEM. (C) Representative images of Mastl(ED/ED) platelets activated with 0.05 IU/ml thrombin and spread on fibrinogen for 30 minutes in the presence of vehicle (DMSO 0.01%) or the kinase inhibitors compound C (10 nM), H89 (10 nM), or Gö6983 (500 nM). In A and C, platelets were stained with α-tubulin (red) and phalloidin (green), and plots show the percentage (mean ± SEM) of cells in the categories described in Figure 4E. n > 150 cells per experiment from 3 independent experiments. Scale bars: 5 μm. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t test with Welch’s correction (B) and Mann-Whitney U test (A and C).