Abstract
Rasa3 is a GTPase activating protein of the GAP1 family which targets Ras and Rap1. Ubiquitous Rasa3 catalytic inactivation in mouse results in early embryonic lethality. Here, we show that Rasa3 catalytic inactivation in mouse hematopoietic cells results in a lethal syndrome characterized by severe defects during megakaryopoiesis, thrombocytopenia and a predisposition to develop preleukemia. The main objective of this study was to define the cellular and the molecular mechanisms of terminal megakaryopoiesis alterations. We found that Rasa3 catalytic inactivation altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet forming megakaryocytes. These megakaryocyte alterations were associated with an increased active Rap1 level and a constitutive integrin activation. Thus, these mice presented a severe thrombocytopenia, bleeding and anemia associated with an increased percentage of megakaryocytes in the bone marrow, bone marrow fibrosis, extramedular hematopoiesis, splenomegaly and premature death. Altogether, our results indicate that Rasa3 catalytic activity controls Rap1 activation and integrin signaling during megakaryocyte differentiation in mouse.
Highlights
Ras families GTPase-activating proteins (GAP), like Ras GAPs, Rho GAPs and Arf GAPs, are tumor suppressors as the loss of their GAP activity allows uncontrolled Ras, Rho and Arf activities and promotes cancer
Interactions of megakaryocytes with extracellular matrix proteins are essential in this process since constitutive megakaryocyte integrin activity caused by specific mutations in ITGA2B or ITGB3 genes encoding for extracellular matrix protein receptors may result in abnormal adherent megakaryocytes, defect in proplatelet formation and thrombocytopenia
We found that Rasa3 catalytic inactivation in mice altered megakaryocyte development, adherence, migration, actin cytoskeleton organization and differentiation into proplatelet
Summary
Ras families GTPase-activating proteins (GAP), like Ras GAPs, Rho GAPs and Arf GAPs, are tumor suppressors as the loss of their GAP activity allows uncontrolled Ras, Rho and Arf activities and promotes cancer. Rasa protein structure is characterized by a conserved basic domain structure comprising two N-terminal tandem C2 domains, a central GAP domain and a C-terminal pleckstrin homology (PH) domain that is associated with a Bruton’s tyrosine kinase (Btk) motif [8] Binding of the latter domain to phosphoinositides determines Rasa targeting to the cytosolic leaflet of the plasma membrane where it inactivates Ras and Rap1 [9,10,11]. We found that catalytic inactivation of Rasa in the hematopoietic system results in a lethal syndrome characterized by major alterations during megakaryopoiesis These alterations were associated with increased active Rap level and constitutive integrin activation in megakaryocytes, a phenotype quite different clinically, biologically and mechanistically from that of recently published mice with a spontaneous missense mutation between the two N-terminal tandem C2 domains of Rasa3 [18]
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