The role of PTEN phosphorylation remodeling in hematopoietic stem cell regulation

描述（由申请人提供）：我们的目标是定义Pten磷酸化重塑在造血干细胞（HSC）调节中的作用。Pten是一种肿瘤抑制因子，具有脂质和蛋白磷酸酶活性，分别抑制PI3K/Akt和fb - mapk信号。它调节许多方面的细胞行为，包括增殖、存活、粘附和迁移。1-4 Pten的磷酸酶活性受其c端尾部磷酸化调控。5,6此外，Pten许多还具有一些与磷酸酶无关的功能。Pten的许多生物学功能依赖于由其pdz基序介导的蛋白-蛋白相互作用敲除小鼠造血组织中Pten导致PI3K/Akt和Src信号异常激活，导致HSC激活（G0到G1过渡）和动员失控，继而导致HSC下降。这些小鼠发展为骨髓增生性疾病（MPD），随后是急性骨髓/T淋巴细胞白血病。尽管Pten突变在包括白血病在内的造血恶性肿瘤中并不常见，但在大多数白血病患者骨髓样本的异常原细胞中，p-Pten （Pten的磷酸化形式）水平升高。Pten的c端尾部（ser380， thr382和thr383）的磷酸化导致构象改变，这可能导致其与其他伴侣蛋白结合的能力受阻，Pten磷酸酶活性降低，和/或Pten蛋白的寿命改变。我们最近的研究表明，Pten的c端尾部磷酸化可能不会影响其脂质磷酸酶活性，但会显著降低其蛋白磷酸酶活性。Pten的非磷酸化形式（non-p-Pten）抑制Src/Fak/p38活性，从而抑制细胞迁移/侵袭性并诱导细胞：细胞接触抑制生长。p-Pten可能具有显性负性功能，诱导细胞接触相关的Src/Fak/p38激活。我们发现非p-Pten在造血干细胞中表达，而p-Pten水平在造血干细胞进入细胞周期后增加；这两种事件都对应于p- Src、p- fak和p-p38水平的升高。转导的非p-Pten过表达以骨髓生态位依赖的方式保存HSC，而转导的p-Pten过表达诱导HSC/祖细胞（HSC/Ps，来自内源性Pten表达的野生型小鼠）分化为骨髓前体细胞。我们提出，非p-Pten通过抑制Src/Fak/p38信号活性，诱导细胞（HSC和小生境细胞）接触诱导的生长抑制，从而维持HSC的静止和自我更新能力，而Pten的c端磷酸化改变了其与其伴侣结合的能力，并损害了其蛋白磷酸酶活性。p-Pten通过诱导细胞接触相关的Src/Fak/p38信号传导来促进与这些相反的功能。这些研究将深入了解静止的造血干细胞是如何被激活和数量增加的，以及我们如何能够诱导活化的造血干细胞恢复到静止状态以增强其植入能力。这将极大地帮助我们在体外扩增造血干细胞的能力，从而改善临床干细胞移植的结果。这也可能有助于我们了解Pten c端磷酸化在白血病发生中的性质。

DESCRIPTION (provided by applicant): Our goal is to define the roles of Pten phosphorylation remodeling in hematopoietic stem cell (HSC) regulation. Pten, a tumor suppressor, has both lipid and protein phosphatase activities that inhibit PI3K/Akt and Fak-MAPK signaling respectively. It regulates many aspects of cell behavior, including proliferation, survival, adhesion and migration.1-4 The phosphatase activities of Pten are regulated by its c-terminal tail phosphorylation.5, 6 In addition, Pten many also have some phosphatase-independent functions. Many of Pten's biological functions are dependent upon protein-protein interactions which are mediated by its PDZ-motif.7 Knockout of Pten in mouse hematopoietic tissues results in abnormal activation of PI3K/Akt and Src signaling, which leads to uncontrolled HSC activation (G0 to G1 transition) and mobilization, followed by HSC decline. These mice develop myeloproliferative disorder (MPD) followed by acute myeloid/T lymphoid leukemia. Although Pten mutations are not commonly found in hematopoietic malignancies, including leukemia, p-Pten (the phosphorylated form of Pten) levels are increased in the abnormal blasts of most leukemic patients' bone marrow samples. Phosphorylation of Pten's c-terminal tail (ser380, thr382, and thr383) leads to a conformation change which may result in the blocking of its ability to bind to other partner proteins, the reduction of Pten phosphatase activity, and/or the alteration of the lifespan of the Pten protein. Our recent studies have suggested that the phosphorylation of Pten's c-terminal tail may not affect its lipid phosphatase activity but significantly compromises its protein phosphatase activity. The non-phosphorylated form of Pten (non-p-Pten) inhibits Src/Fak/p38 activity, thus repressing cell migration/invasiveness and inducing cell:cell contact inhibition of growth. p-Pten might have a dominant-negative function which induces cell-contact-related Src/Fak/p38 activation. We found that non-p-Pten is expressed in HSCs, while p- Pten levels are increased when HSCs enter the cell cycle; both of these events correspond to increased p- Src, p-Fak and p-p38 levels. Transduced over-expression of non-p-Pten preserves HSCs in a bone marrow niche-dependent manner, whereas transduced over-expression of p-Pten induces HSC/progenitors (HSC/Ps, from wild-type mice which have endogenous Pten expression) to differentiate to myeloid precursors. We propose that non-p-Pten maintains HSC quiescence and self-renewal ability through inducing cell:cell (HSCs and niche cells) contact-induced inhibition of growth by inhibiting Src/Fak/p38 signaling activities, whereas Pten's c-terminal phosphorylation alters its ability to bind to its partners and compromises its protein phosphatase activity. p-Pten promotes opposite functions to these through inducing cell:cell contact-related Src/Fak/p38 signaling. These studies will provide insights into how quiescent HSCs become activated and expand in number, and how we might be able to induce activated HSCs to revert back into quiescence in order to enhance their engraftment ability. This should greatly help our ability to expand HSCs in vitro and hence improve the outcome of clinical stem cell transplantation. It might be also help us to understand the nature of Pten c-terminal phosphorylation in leukemogenesis. PUBLIC HEALTH RELEVANCE: This proposed study will provide insights into how quiescent hematopoietic stem cell be induced cell cycle entry and proliferation, and how stem cell function is regulated by PTEN c-terminal phosphorylation. The predicted outcome will greatly help our ability to expand hematopoietic stem cells in vitro and hence improve the outcome of clinical stem cell transplantation.