The formation of erythroid progenitor cells depends sharply upon erythropoietin (EPO), its cell surface receptor (erythropoietin receptor, EPOR), and Janus kinase 2 (JAK2). Clinically, recombinant human EPO (rhEPO) additionally is an important anti-anemia agent for chronic kidney disease (CKD), myelodysplastic syndrome (MDS) and chemotherapy, but induces hypertension, and can exert certain pro-tumorigenic effects. Cellular signals transduced by EPOR/JAK2 complexes, and the nature of EPO-modulated signal transduction factors, therefore are of significant interest. By employing phospho-tyrosine post-translational modification (p-Y PTM) proteomics and human EPO- dependent UT7epo cells, we have identified 22 novel kinases and phosphatases as novel EPO targets, together with their specific sites of p-Y modification. New kinases modified due to EPO include membrane palmitoylated protein 1 (MPP1) and guanylate kinase 1 (GUK1) guanylate kinases, together with the cytoskeleton remodeling kinases, pseudopodium enriched atypical kinase 1 (PEAK1) and AP2 associated kinase 1 (AAK1). Novel EPO- modified phosphatases include protein tyrosine phosphatase receptor type A (PTPRA), phosphohistidine phosphatase 1 (PHPT1), tensin 2 (TENC1), ubiquitin associated and SH3 domain containing B (UBASH3B) and protein tyrosine phosphatase non-receptor type 18 (PTPN18). Based on PTPN18's high expression in hematopoietic progenitors, its novel connection to JAK kinase signaling, and a unique EPO- regulated PTPN18-pY389 motif which is modulated by JAK2 inhibitors, PTPN18's actions in UT7epo cells were investigated. Upon ectopic expression, wt-PTPN18 promoted EPO dose-dependent cell proliferation, and survival. Mechanistically, PTPN18 sustained the EPO- induced activation of not only mitogen-activated protein kinases 1 and 3 (ERK1/2), AKT serine/threonine kinase 1-3 (AKT), and signal transducer and activator of transcription 5A and 5B (STAT5), but also JAK2. Each effect further proved to depend upon PTPN18's EPO- modulated (p)Y389 site. In analyses of the EPOR and the associated adaptor protein RHEX (regulator of hemoglobinization and erythroid cell expansion), wt-PTPN18 increased high molecular weight EPOR forms, while sharply inhibiting the EPO-induced phosphorylation of RHEX-pY141. Each effect likewise depended upon PTPN18-Y389. PTPN18 thus promotes signals for EPO-dependent hematopoietic cell growth, and may represent a new druggable target for myeloproliferative neoplasms.

中文翻译：

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Phospho-PTM蛋白质组学发现了新型EPO调节的激酶和磷酸酶，包括PTPN18作为EPOR / JAK2 Signaling的正向调节剂。

红细胞祖细胞的形成严重依赖于促红细胞生成素（EPO），其细胞表面受体（促红细胞生成素受体，EPOR）和Janus激酶2（JAK2）。在临床上，重组人EPO（rhEPO）另外是用于慢性肾脏疾病（CKD），骨髓增生异常综合症（MDS）和化学疗法的重要抗贫血药，但会诱发高血压，并能发挥一定的促肿瘤作用。因此，由EPOR / JAK2复合物转导的细胞信号以及EPO调节的信号转导因子的性质引起了人们的极大兴趣。通过采用磷酸酪氨酸翻译后修饰（pY PTM）蛋白质组学和人类EPO依赖性UT7epo细胞，我们已经鉴定出22种新型激酶和磷酸酶作为新型EPO靶标，以及它们的pY修饰特异性位点。由EPO修饰的新激酶包括膜棕榈酰化蛋白1（MPP1）和鸟苷酸激酶1（GUK1）鸟苷酸激酶，以及细胞骨架重塑激酶，富含伪足蛋白的非典型激酶1（PEAK1）和AP2相关激酶1（AAK1）。新型EPO修饰的磷酸酶包括蛋白酪氨酸磷酸酶受体A型（PTPRA），磷酸组氨酸磷酸酶1（PHPT1），肌腱蛋白2（TENC1），与泛素相关且含SH3结构域的B（UBASH3B）和蛋白酪氨酸磷酸酶非受体18型（PTPN18） ）。基于PTPN18在造血祖细胞中的高表达，其与JAK激酶信号转导的新颖联系以及受JAK2抑制剂调节的独特的EPO调节的PTPN18-pY389基序，研究了PTPN18在UT7epo细胞中的作用。异位表达后 wt-PTPN18促进EPO剂量依赖性细胞增殖和存活。从机制上讲，PTPN18不仅维持EPO诱导的丝裂原活化蛋白激酶1和3（ERK1 / 2），AKT丝氨酸/苏氨酸激酶1-3（AKT）以及信号转导和转录激活子5A和5B（STAT5）的激活。 ），也可以使用JAK2。每种效应进一步证明取决于PTPN18的EPO调节（p）Y389位点。在对EPOR和相关的衔接蛋白RHEX（血红蛋白化和红系细胞扩张的调节剂）的分析中，wt-PTPN18增加了高分子量EPOR的形式，同时急剧抑制了EPO诱导的RHEX-pY141的磷酸化。每个效果同样取决于PTPN18-Y389。因此，PTPN18促进依赖EPO的造血细胞生长的信号，并可能代表骨髓增生性肿瘤的新药物靶标。