行政院國家科學委員會補助專題研究計畫成果報告
計畫名稱: TEL-JAK2 致癌基因在血癌生成機轉的訊息傳遞路
計畫類別: 個別型計畫
計畫編號: NSC
89-2314-B-002-055-執行期間: 88 年 8 月 1 日至 89 年 7 月 31 日
計畫主持人: 周獻堂
共同主持人: 林凱信
田蕙芬
林東燦
盧孟佑
執行單位: 國立台灣大學小兒部
中華民國 89 年 10 月 24 日
中文摘要
JAK/STAT 在血液細胞的訊息傳遞路徑中扮演著非 常重要的角色。藉由 JAK/STAT 使 cytokine 的訊 息能藉由刺激細胞膜上的 receptor 一步步地傳達 到細胞核內以啟動基因的表達。此路徑傳遞血液 細胞絕大部分的生長和分化訊息。目前文獻顯示 所有須與 cytokine receptor superfamily 結合 的 cytokine 都 能 活 化 JAK tyrosine kinase family。 因為 JAK/STAT 路徑在血液細胞訊息傳遞路徑中扮 演著非常重要的角色，所以推論 JAK/STAT 路徑亦 應在血癌的生成機轉中佔重要的角色。愈來愈多 的證據支持此學說，最近 TEL-JAK2 致癌基因在三 個血癌病人細胞中被發現。此致癌基因能使細胞 變性轉型的能力是來自於藉由 TEL 的 PNT domain 使 JAK2 的 kinase domains 彼 此 結 合 (oligomerization)而持續保有 JAK2 kinase 的活 性。 TEL-JAK2 的致癌生成機轉, 主要來自於其 kinase domain 上的酪氨酸基被持續磷酸化。尋找 TEL-JAK2 的下游受脢質蛋白, 對了解癌症的生成機轉 之謎, 是很重要的. 我們已 clone hTEL-mJAK2 , 且證明 TEL-JAK2 的持續保有酪氨酸激脢的特性, 能使 Ba/F3 細胞株由 cytokine-dependent 轉型 成 cytokine-independent. 而 且 我 們 利 用 TEL-JAK2 的持續保有酪氨酸激脢的特性, 發現一個持 續被酪氨酸磷酸化的受脢質蛋白, 只存在於已轉 型的 Ba/F3/ TEL-JAK2 細胞, 但不出現在未轉型 的 Ba/F3/ TEL-JAK2 細胞. 目前正進而研究它的 生物特性, 以了解血癌的生成機轉.Abstract
The JAK/STAT (Janus protein tyrosine kinase/Signal Transducers and Activators of Transcription) pathway has emerged as a major signal transduction mechanism in hematopoietic system, linking cell surface receptors on the membrane to transcriptional events in the nucleus. This pathway plays critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. It was demonstrated that all the cytokines that utilize receptors of the cytokine receptor superfamily were capable of activating members of the JAK tyrosine kinase family.
The importance of the JAK/STAT pathway in cytokine signaling for the hematopoietic cells suggests a potential role in the pathogenesis of hematological malignancies. Some evidences have supported this hypothesis. The TEL-Jak2 fusion oncogene has recently been found in 3 leukemia patients. The
oligomerization by the PNT domain of TEL leads to constitutive activation ( phosphorylation) of the Jak2 tyrosine kinase domain, which is necessary for cellular transformation.
The constitutive tyrosine phosphorylation of the tyrosine kinase domain of TEL-Jak2 plays the critical role for the leukemogenesis. To try to find the down-stream substrate of the TEL-Jak2 is very important for the understanding the signal transduction pathway for the leukemogenesis. We have constructed the hTEL-mJAK2 oncogene including the human TEL-specific oligomerization domain and the catalytic domain of murine JAK2. And our data also showed the TEL-induced oligomerization of TEL-JAK2 resulted in the constitutive activation of its tyrosine kinase activity and conferred cytokine (IL-3)-independent proliferation to the IL-3-dependent Ba/F3 hematopoietic cell line. We have also identified one strongly constitutively tyrosine-phosphorylated protein in the Ba/F3/TEL-JAK2 cells which is not found in the Ba/F3 parent cells by using the character of the constitutively activated catalytic activity of TEL-JAK2. We are studying the biological character of this down-stream substrate of TEL-JAK2 oncogene to elucidate the signal transduction pathway for leukemogenesis in the hematopoietic system.
Backgr ound and Pur poses
The JAK/STAT signal pathway is the main and the most important pathway among the hematopoietic system for signaling almost all cytokines' messages into the nucleus. This pathway was still unknown until early '90, but the whole frame has been established since '961-3.. This pathway links cell surface cytokine
receptors to transcriptional events in nucleus. The JAK kinase family (JAK1, 2, 3 and Tyk2) is cytoplasmic kinases that bind to cytokine receptors that have no kinase domain. The STAT protein family (STAT1-4, 5a, 5b and 6) is latent cytoplasmic transcription factors. Once cytokines bind to specific receptors, they will induce receptors’ dimerization. The associated JAK kinases will respond by phosphorylating each other and also tryosine residues on the receptor cytoplasmic domains, creating phosphotyrosine docking sites that allow SH2-containing signal proteins, including STATs or other down-stream substrate to bind. The recruited STATs will be activated by phosphorylation on conserved tyrosine residues, then dissociate from the receptor and dimerize, forming homo- or heterodimers, and translocation to the nucleus, where the complexes bind to specific DNA response elements and activate transcription of genes.
The importance of the JAK-STAT pathway in cytokine signaling suggests a potential role in the leukemogenesis of hematological malignancies. Some evidences support this hypothesis: A single
amino-acid mutation in the Drosophila melanogaster JAK causes leukemia-like hematopoietic defects 4; 5. The
constitutive activation of the JAK-STAT pathway has been shown in malignant cutaneous T-cell tumor 6; 7.
And, the constitutively activated STATs have been shown in cells from patients with chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia and chronic lymphocytic leukemia 8-10. The leukemic cells from
patients with relapsed ALL have constitutively activated JAK2, and inhibition of JAK2 activity by a specific blocker (AG-490) selectively blocks leukemic cell growth in vitro and in vivo 11. And, the most direct
evidence for involvement of JAK family in leukemogenesis is the chromosomal translocations from leukemic cells involving the JAK2 kinase gene on 9p24 and the TEL gene on 12p13(TEL-Jak2 oncogene) 12.
The TEL is a member of the ETS family of transcription factors. It contains a 60-amino acid oligomerization domain(PNT) at its N-terminus. Three TEL-tyrosine kinase fusion proteins (TEL-PDGFR (platelet-derived growth factor receptor), TEL-ABL and TEL-JAK2) have been found in human leukemia
12-14. The TEL-JAK2 oncogene was characterized to
fuse the C-portion (catalytic kinase domain) of JAK2 to the N-portion (oligomerization domain) of TEL. The oligomerization by the PNT domain of TEL leads to constitutive activation of the PDGFR, ABL and JAK2 tyrosine kinase domains (JH1) activity (Fig.1.), which is critical and necessary for transformation of hematopoietic cells. There are several expected purposes in this study:
1. To clone the TEL-JAK2 oncogene into retroviral vector.
2. To do site-directed mutagenesis on the supposed critical tyrosine residues of the TEL-JAK2.
3. To sequence the cloned genes.
4. To transfect the TEL-JAK2 and mutant oncogene into various cells.
5. To check the protein expression of TEL-JAK2 by GFP marker.
6. To check the TEL-JAK2 protein activity by : In vitro: immunoprecipitation and western blotting
In vivo: the transformation ability of TEL-JAK2 in cells
7. To try to identify the substrate(s) of TEL-JAK2. 8. To try to purify the substrate(s) of TEL-JAK2.
Results and Discussion
Clone TEL-J AK2 oncogene into r etr ovir al vector :
The JAK kinase family share structural domains designated as JH segments 1-7. The critical domain which is involved in the TEL-JAK2 oncogene is the JH1, the kinase domain located at the C-terminus. The JH2 to JH7 domains appear to be involved in the
protein-protein interactions. The sequence of the human JAK2 was 90% identical to that of murine JAK2 at the nucleotide level and 96% identical at the protein level over the JH1 domain. We prepared a chimeric cDNA construct containing the sequence encoding for the human TEL PNT domain (nucleotide 1-1032) fused to the murine JAK2 kinase domain (JH1, nucleotide 2931-3893) by RT-PCR strategy and cloned into PMSCV retrovirus vector with GFP(Green Fluorescence Protein) selective marker. All of the cDNA fragments are checked by sequencing (data not shown).
Pr oduce r etr ovir us and tr ansfer into mur ine hematopoietic cell line (Ba/F3):
We prepared the retroviral stocks by using the 293-T cells as the first virus packaging cell line for transient transfection of the PMSCV retrovirus vector with encoding the TEL-JAK2 construct and the GP+E86 cells as the second virus producer cell line for harvesting the retrovirus. Virus titer was checked by using the 3T3 cells. The virus titer was 10 x 107 /ml.
We used this harvested supernatant which contained the cDNAs encoding TEL-JAK2 retrovirus to transfect the murine Ba/F3 hematopoietic cell line, which is strictly dependent on IL-3 for survival and proliferation. Mock-transfected ( with retrovirus vector only) cells were used as control. The calcium phosphate method is used for this transfection.
Check the pr otein expr ession of TEL-J AK2 by GFP mar ker :
After 48-72 hours of transfection in the presence of IL-3, the infected Ba/F3 cells were sorted by checking the selective marker GFP in the vector by flow cytometry. The sorted GFP positive cells were also frequently checked directly under fluorescent microscopy to assure the stable expression of exogenous TEL-JAK2 protein.
Check the TEL-J AK2 fusion pr otein activity by: In Vitr o: Immunopr ecipitation and Wester n blotting:TEL-J AK2 oncogene r esults in the constitutive activation of J AK2 ’s tyr osine kinase activity:
To check the TEL-induced oligomerization properties and protein kinase activity of TEL-JAK2, the Ba/F3/TEL-JAK2 cells were analyzed with in vivo-translated protein. We have checked the autophosphorylation of TEL-JAK2 which is mediated by the TEL-induced oligomerization by immunoblotting with a phosphotyrosine-specific antibody (anti-PTyr). The Ba/F3/TEL-JAK2 cell lysate was firstly specifically immunoprecipitated by antibodies directed against the C-terminal domain of JAK2 (anti-JAK2), then immunoblotted by anti-Ptyr and anti-JAK2 respectively. A higher level of tyrosine phosphorylation occurred with hTEL-mJAK2 protein than that with endogenous JAK2 protein. The expression and strong tyrosine-phosphorylation of the hTEL-mJAK2 fusion protein shows hTEL-mJAK2 fusion protein is expressed and constitutively tyrosine-phosphorylated in Ba/F3/TEL-JAK2 cells.
In Vivo: To assay the tr ansfor mation ability of TEL-J AK2 pr otein in cells: The constitutive activation of J AK2’s tyr osine kinase activity tr ansfor ms the IL-3-dependent Ba/F3 cells into gr owth factor (cytokine)-independent pr olifer ation:
We deprived the Ba/F3/TEL-JAK2 cells and parent Ba/F3 cells of IL-3 and monitored the cell proliferation by daily cell counting. The growth curve was recorded. In contrast to control cells (Ba/F3, no IL-3), which died with decreased cell number in the absence of IL-3, the similar proliferation curves were traced between the Ba/F3/TEL-JAK2 cells in the absence of IL-3 and Ba/F3 cells in its presence. According to the above data, the TEL-JAK2 has the property of constitutive activation of its protein tyrosine kinase activity. And this constitutively activated tyrosine kinase activity conferred cytokine-independent proliferation property to the IL-3-dependent Ba/F3 hematopoietic cell line.
Compar ison of Ba/F3 and Ba/F3/TEL-J AK2 cells: Identification of one constitutively tyr osine phosphor ylated pr otein(P100) in the Ba/F3/TEL-J AK2 cells which is not phosphor ylated in the Ba/F3 par ent cells:
The total cell lysates of either Ba/F3 or Ba/F3/TEL-JAK2 cells were used in immunoprecipitation with anti-Ptyr antibody, resolved by SDS-PAGE and transferred to PVDF membrane. The blot was probed with the same anti-Ptyr antibody which was initially used for IP. We identify there is one strongly constitutively tyrosine-phosphorylated protein (P100) in the Ba/F3/TEL-JAK2 cells which is not phosphorylated in the Ba/F3 parent cells.
This constitutively tyr osine phosphor ylated pr otein(P100) is not STAT5 or other known pr oteins:
To examine whether or not the P100 was similar to the known molecules, Ba/F3 and Ba/F3/TEL-JAK2 cell lysates were prepared and subjected to immunoprecipitation with anti-Ptyr antibody. Immune complexes were separated by 7.5% SDS-PAGE and transferred to membrane. Many identical blots were prepared and probed with anti-STAT5, anti-PLCγ1,2, SHIP(SH2-containing-inositol phosphatase), anti-CAS(cellular apoptosis susceptibility), HPK-1(hematopoietic progenitor kinase 1),and anti-SP1(sequence-specific transcription factor). All of the above molecules are considered as the potential down-stream substrates of TEL-JAK2 because of their published biological character and/or their similar molecular weights. But, all of these commercialized antibodies were unable to immunoblot the P100 in anti-Ptyr immunoprecipitates from lysates of Ba/F3/TEL-JAK2 cells (some of these data shown in Fig.5.). For further reassurance that the P100 is not STAT5, the Ba/F3 and Ba/F3/TEL-JAK2 cell lysates were firstly specifically immunoprecipitated by anti-STAT5a and anti-STAT5b antibodies, then these lysates were subsequently immunoprecipitated again
by the Ptyr antibody, then immunoblotted by anti-Ptyr, anti-STAT5a and anti-STAT5b respectively. The dada showed the anti-Ptyr antibody still can immunoprecipitate the P100 from the cell lysates which were firstly immunoprecipitated by the anti-STAT5a,b antibodies, and this P100 immune complex still could not be recognized by the anti-STAT5a,b antibodies. These immunological discrepancies suggested that the tyrosine phosphorylated P100 presented in lysates of Ba/F3/TEL-JAK2 cells was different from the above known molecules.
Self-Estimation
We have completed almost all of the expected goals except the cloning this new substrate of of the TEL-JAK2. We are studying the character of this protein. To study its biological character is very important to try to eluciate the mechanism of leukemogenesis by the oncogene.
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