Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling

Abstract

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation and differentiation through binding to and stimulation of the cell surface receptor c-MPL. Activating mutations in c-MPL constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-MPL that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study we focused on c-MPL tyrosine 591 (Y591). We found Y591 of wild-type c-MPL to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using an SH2/PTB domain binding microarray, we identified novel c-MPL binding partners for phosphorylated Y591, including Src homology phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through siRNA treatment of Ba/F3-MPL cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by SYK. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development.

Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients

“…46 Furthermore, this tyrosine is also phosphorylated after TPO binding, and negatively regulates MPL signaling by promoting its internalization degradation as well as ERK signaling. [46][47][48] Asparagine is a polar residue in contrast to the negative charge represented by tyrosine phosphorylation and, thus, the Y591N mutation resembles a Y591 lossof-function, like Y591F, but possibly weaker because unlike phenylalanine, asparagine is polar. When the Y591N mutant was expressed in Ba/F3 cells it induced a slight TPO hypersensitivity, which was associated with constitutive activation of STAT3 and ERK.…”

Secondary Pulmonary Alveolar Proteinosis Following Treatment with Azacitidine for Myelodysplastic Syndrome

“…In terms of the clinical course of genetic and cytogenetic abnormalities during azacitidine treatment, the present case showed 3 significant points: (i) an increased proportion of the normal karyotype from 75% to 100%, (ii) a slightly increased VAF of the SH2B3 P344 frameshift insertion, and (iii) stable VAFs of the MPL Y591N and R592Q mutations before and after azacitidine treatment. Although these gene alterations have been reported in patients with myeloproliferative neoplasm (26)(27)(28), the clinical significance of these gene alterations in MDS, particularly in cases with a low VAF at both the diagnosis and the onset of sPAP, remains unclear. However, these mutations might contribute to the abnormal function of alveolar macrophages, which may be related to the development of sPAP.…”

Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia

“…1C). While Y591F has been shown to be a negative regulator of TPO signaling and dispensable for proliferation (32), Y625F is essential for it. All three mutant cell lines showed high levels of phosphorylation with AK119 treatment (equal or higher than TPO).…”

“…The MPL intracellular domain point mutations (Y591F, Y625F and Y630F) were introduced using the QuikChange mutagenesis kit (Stratagene). Ba/F3-MPL cells lines were generated by retroviral transduction as described previously(32). Prior to stimulation, Ba/F3-MPL cells were cytokine starved for 16hrs.…”