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 1

Mild thrombocytopenia in Mastl(Δ/Δ) and Mastl(ED/ED) mice.

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Mild thrombocytopenia in Mastl(Δ/Δ) and Mastl(ED/ED) mice. (A) Represent...
(A) Representation of the Mastl(lox) and Mastl(Δ) alleles used in this study. A Pf4-Cre transgene was used to generate megakaryocyte-specific Mastl-null mice. (B) Generation of the Mastl(E166D) [Mastl(ED)] allele. The frt-neo_resistance-frt cassette was removed after crossing with Flp-expressing mice. In A and B, White and orange triangles represent lox or frt sequences, respectively. (C) Expression of Mastl in CD41+ bone marrow cells from mice of the indicated Mastl genotypes. Lysates from NIH3T3 cells were used as a control. Representative image from 2 independent experiments. See complete unedited blots in the supplemental material. (D) Platelet levels in 12-week-old male (blue) or female (pink) mice with the indicated genotypes. Blue line indicates a concentration of 800 × 106 platelets/ml as a reference. (E) Percentage of mice with fewer than 800 × 106 platelets/ml (using mice from panel D). Male and female mice are represented by separate bars (right). (F) Levels of TPO in peripheral blood from the indicated mice. Cdc20-deficient mice, which display severe thrombocytopenia (33), were used as a control (n ≥4 mice per genotype). (G) Representative micrographs of bone marrow from mice of the indicated genotypes. Images represent more than 3 mice per genotype analyzed. Scale bars: 50 μm. Plot shows quantification of the VWF signal in more than 100 megakaryocytes per genotype. Data are shown as the mean. (H) Quantification of CD41+ and CD42+ cells in bone marrow cells from mice of the indicated genotypes. (I) Quantification of the ploidy in CD41+CD42+ double-positive bone marrow cells from mice of the indicated genotypes. No significant differences were observed among the different genotypes (n = 3 per genotype; data are shown as the mean). *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t test with Welch’s correction (D, F, G, and I) and 1-way ANOVA (E).