PKA-LNK-14-3-3信号通路在造血干细胞及其前体细胞中的功能研究

The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis. The adaptor protein LNK is a key negative regulator of hematopoietic stem cell (HSC) self-renewal by limiting JAK2 activity. LNK-deficient （Lnk-/-） mice have a >10-fold expansion in the HSC pool. To our knowledge, Lnk-/- mouse is one out of very few select mouse models that show increased phenotypic and functional HSCs with superior self-renewal and competitive multi-lineage reconstitution, yet without premature exhaustion during aging or in serial transplants. To explore mechanisms of LNK functions, we recently identified 14-3-3 proteins as novel LNK binding partners. We found that 14-3-3 interferes with the LNK-JAK2 interaction thereby alleviating LNK inhibition of JAK2 signaling and cell proliferation. Binding of 14-3-3 requires previously unappreciated serine (S) phosphorylation site in LNK, which is phosphorylated by Protein Kinase A (PKA). Importantly, bone marrow transplantation studies revealed an essential role for 14-3-3 in HSPC reconstitution that can be partially mitigated by LNK deficiency. Together, our recent work generously supported by the AHA fellowship implicates 14-3-3 proteins as novel and positive HSPC regulators by impinging on the LNK/JAK2 pathway (JCI, 2012). We will further investigate the role of 14-3-3 and LNK phosphorylation in JAK2-mediated signaling and HSPC expansion, and to exploit this pathway to expand HSCs ex vivo by targeting LNK. .Aim 1: Investigate the signals that phosphorylate/inactivate LNK and promote murine HSPC expansion..Aim 2: Ex vivo expansion of human cord blood HSCs by inactivating LNK..Our results implicate that this novel PKA- 14-3-3 – LNK signaling pathway inactivates LNK in HSCs thereby promoting HSC expansion and engraftment. If successful, the proposed studies will likely reveal LNK as a signaling nodal point that integrates multiple pathways that control HSC homeostasis. Defining the mechanisms by which LNK coordinates distinct signaling pathways might lead to strategies to expand HSCs more efficiently in culture and novel therapeutic approaches in treating hematologic disorders.

酪氨酸激酶JAK2在造血过程细胞因子信号通路中具有非常重要的作用。调控蛋白LNK通过抑制JAK2的活性负调节造血干细胞(HSC)的自我更新能力。LNK敲除小鼠骨髓中HSCs增加10倍之多，LNK缺失能够促进HSC的自我更新以及骨髓移植后HSC的重建。前期研究发现14-3-3蛋白是LNK新的结合元件，通过干扰LNK-JAK2的相互作用从而削弱LNK对JAK2信号通路以及对细胞增殖的抑制作用。而预实验发现14-3-3与鼠或人LNK的结合需要PKA磷酸化LNK。本项目将继续研究14-3-3和LNK的磷酸化在JAK2介导的信号通路和骨髓前体细胞增值领域的研究，有两个方面：(1)研究通过LNK磷酸化和去磷酸化促进小鼠骨髓前体细胞增殖的信号通路；(2) 发展通过抑制人源LNK获得人脐带血HSC增殖的方法。研究PKA-LNK-14-3-3信号通路能够为HSCs体外培养和临床治疗血液学失调寻找新的治疗策略

在造血过程的细胞因子信号通路中酪氨酸激酶JAK2扮演着非常重要的角色。淋巴细胞调控蛋白LNK通过抑制JAK2的活性负调节造血干细胞的自我更新能力。LNK基因缺失小鼠骨髓中造血干细胞增加10倍之多。本人之前研究发现14-3-3蛋白质是LNK新的结合元件，14-3-3蛋白通过干扰LNK-JAK2的相互作用从而削弱LNK对JAK2信号通路以及LNK对细胞增殖的抑制作用。.本项目在前期工作的基础上，继续研究14-3-3和LNK的磷酸化作用在JAK2介导的信号通路和骨髓前体细胞增殖领域的研究。该研究进一步证实了PKA是內源性LNK磷酸化所必需的激酶，PKA磷酸化LNK后能促进LNK结合更多的14-3-3蛋白。高表达LNK的白血病细胞系，经PKA抑制剂处理后其LNK的磷酸化水平明显降低，并且LNK结合的14-3-3蛋白也明显减少。而PKA激活剂会促进LNK的磷酸化水平以及结合14-3-3的量。同样用特异性慢病毒（shRNA）敲除高表达内源性LNK的白血病细胞中的PKA后，免疫共沉淀结果显示LNK S150位点的磷酸化水平降低，并且LNK结合的14-3-3蛋白也明显减少。细胞生长实验表明敲除PKA后会促进高表达内源性LNK的白血病细胞的生长。集落刺激实验也表明PKA激活剂能明显促进骨髓造血干细胞前体细胞的生长。说明在人白血病细胞中，PKA激活剂能通过提高LNK的磷酸化水平从而结合更多的14-3-3来消弱LNK的抑制作用。.该项研究围绕之前发现的LNK-JAK2通路的新调节元件，在调控HSC/HSPC增殖和发育领域提出新的机制。此外，本研究也为干细胞治疗提供新的参考，并为恶性和非恶性血细胞失调症开发新的治疗策略。