Abstract
GATA1 mutations are tightly associated with transient myeloproliferative disorder (TMD) and acute megakaryoblstic leukemia (AMKL) in children with Down syndrome. Numerous genes are altered in GATA-1-deficient megakaryocytes, which may contribute to the hyperproliferation and abnormal terminal differentiation of these malignant cells. In this study, we demonstrate that Pstpip2 is a GATA-1-repressed gene that controls megakaryopoiesis. Ectopic expression of PSTPIP2 impaired megakaryocytic differentiation as evidenced by a decrease of CD41 expression and reduced DNA content in K562 cells. PSTPIP2 overexpression also caused enhanced activation of Src family kinases and subsequently reduced ERK phosphorylation. Consistently, PSTPIP2 knockdown showed the opposite effect on differentiation and signaling. Moreover, the W232A mutant of PSTPIP2, defective in its interaction with PEST family phosphatases that recruit c-Src terminal kinase (CSK) to suppress Src family kinases, failed to inhibit differentiation and lost its ability to enhance Src family kinases or reduce ERK phosphorylation. In fact, the W232A mutant of PSTPIP2 promoted megakaryocyte differentiation. These observations suggest that PSTPIP2 recruiting PEST phosphatases somehow blocked CSK activity and led to enhanced activation of Src family kinases and reduced ERK phosphorylation, which ultimately repressed megakaryocyte differentiation. Supporting this idea, PSTPIP2 interacted with LYN and the expression of a dominant negative LYN (LYN DN) overwhelmed the inhibitory effect of PSTPIP2 on differentiation and ERK signaling. In addition, a constitutively active LYN (LYN CA) normalized the enhanced megakaryocyte differentiation and repressed ERK signaling in PSTPIP2 knockdown cells. Finally, we found that PSTPIP2 repressed ERK signaling, differentiation, and proliferation and verified that PSTPIP2 upregulation repressed megakaryocyte development in primary mouse bone marrow cells. Our study thus reveals a novel mechanism by which dysregulation of PSTPIP2 due to GATA-1 deficiency may contribute to abnormal megakaryocyte proliferation and differentiation in pathogenesis of related diseases.
Highlights
Several cytokine signaling components have been shown to be GATA-1 target genes
Our study demonstrates that Pstpip[2] is a GATA-1 target gene and that it inhibits megakaryocyte differentiation by repressing ERK activating through recruiting PEST phosphatases and activating LYN
GATA-1 significantly inhibited P2-pro/G1BS activity (Figure 1a). These results suggest that the putative GATA-1-binding site in the intron 1 region of Pstpip[2] gene may mediate inhibitory effect on its promoter
Summary
Several cytokine signaling components have been shown to be GATA-1 target genes. For instance, JAK2 has been found to be significantly downregulated in GATA-1low megakaryocytes that display reduced TPO signaling with. Compared with PSTPIP1, PSTPIP2 lacks the SH3 domain that is necessary for interaction with the Wiskott–Aldrich syndrome protein (WASP) Instead, it binds to the CTH (carboxyl-terminal homology) region of PEST family phosphatases.[17] PSTPIP2 is tyrosine-phosphorylated on colony-stimulating factor-1 (CSF-1) treatment.[17] It is efficiently phosphorylated after v-Src transfection.[18] In mouse models, PSTPIP2 deficiency causes autoinflammatory disease involving extramedullary hematopoiesis, as evidenced by expansion of macrophage progenitors. Dysregulation of PSTPIP2 due to GATA-1 deficiency may contribute to abnormal megakaryocyte terminal differentiation in the pathogenesis of the related diseases
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