PSTPIP2 dysregulation contributes to aberrant terminal differentiation in GATA-1-deficient megakaryocytes by activating LYN

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.

PSTPIP2, a Protein Associated with Autoinflammatory Disease, Interacts with Inhibitory Enzymes SHIP1 and Csk

“…However, it is possible that PESTPTPs together with Csk specifically inhibit the pool of SFKs associated with PSTPIP2. The interaction between PSTPIP2 and SFK Lyn was described previously in megakaryocytes (38), and we detected a similar interaction in macrophages (data not shown). The effect of Csk binding could not be fully analyzed because we were unable to identify its binding site in PSTPIP2.…”

“…The C-terminal tyrosines of PSTPIP2 were identified as major phosphorylation sites in PSTPIP2, and in some experiments they also appeared to be among the functionally most important residues in the PSTPIP2 protein (32,38). In our experiments, Csk binding did not depend on the presence of these tyrosines.…”

“…Moreover, additional evidence supports the importance of PEST-PTPs for the functionality of PSTPIP2. The W232A mutation in PSTPIP2, which prevented its binding to PEST-family phosphatases, diminished the ability of PSTPIP2 to inhibit the differentiation of osteoclasts and megakaryocytes (32,38). In addition, we show in this article that this tryptophan is also important for the suppression of IL-1b processing in neutrophils.…”

“…Their mutation completely abolished the ability of PSTPIP2 to suppress the differentiation of osteoclasts and reduced its ability to inhibit megakaryocyte differentiation (32,38). The most likely explanation was that these tyrosines recruit a negative regulatory molecule responsible for these effects.…”

“…The effects of PSTPIP2 binding to PEST-family phosphatases have been tested recently in osteoclasts and megakaryocytes, where it had an effect on their differentiation. However, in these experiments, a profound effect was also seen after the mutation of C-terminal tyrosines, which rendered PSTPIP2 especially in the case of osteoclasts essentially nonfunctional without affecting the interaction with PEST-family phosphatases (32,38). This suggested that the binding partners of these tyrosines are critical for PSTPIP2 function.…”