Identification and analysis of tyrosine phosphatase CD45 interacting proteins

酪氨酸磷酸酶 CD45 相互作用蛋白的鉴定和分析

• 批准号: 09680653
• 负责人: FURUKAWA Tatsuhiko
• 金额: $ 2.11万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680653/
• 关键词: tyrosine phosphatase; CD45; T cell; CD3zeta; LAR; insulin receptor; CD35; CD3; LCK; ZAP70

CD45 and leukocyte common antigen-related protein(LAR) have two tandem-repeated PTPase domains in the cytoplasmic segment like most of other transmembrane protein tyrosine phosphatases (RTPases). PTPase activity is associated only with the first of the two domains, PTPase domain 1(D1), and the membrane-distal PTPase domain 2(D2), which has no catalytic activity, would regulate substrate specificity.From the lysate of co-expression of CD3zeta, lck and CD45D1CS, whose cystein at 828 has replaced with serine, phosphorylated CD3zeta was coprecipitated with CD45D1CS using anti-CD45 antibody. This finding indicates CD3zeta is a good substrate of CD45 also in vivo. Three kinds mutants MC1, MC2, MC3 of CD3zeta, which are carrying only 2 tyrosine residues, 2nd and 3rd, 1st and 3rd, 1st and 2nd tyrosine from C terminus, were made. All 3 mutants can be poorly phosphorylated with Ick and has almost same affinty with CD45D1CS, but SHC-SH2 can not bind with phosphorylated MC2 mutant.CD45D1D2CS, whose cystein at 1144 was replaced with serine, has lower affinity with CD3zeta, and phsophorylated CD3zeta than CD45D1CS.Cystein of D2 has an important role of recognition of CD3zeta, independently of phosphorylation.We studied on LAR and insuline receptor (IR) interaction also. LAR was associated with and preferentially dephosphorylated the insulin receptor which was tyrosine-phosphorylated after insulin stimulation. LARD1CS can bind phosphorylated IR stably. IR can be a phsiological substrate of LAR.LAR is phsophorylated with IRkinase after insuline stimulation. LARD1D2CS , whose cystein 1813 is changed to serine in the domain 2, has lower affinity with IR and higher phosphrylation than LARDICS.These findings suggests LARD2 has auto-dephosphorylation activity. These data indicate that D1PTP of CD45 and LAR has main phosphatase activity and D2 PTP of them can recognize the substrates independently of phosphorylation.

CD45 和白细胞共同抗原相关蛋白 (LAR) 与大多数其他跨膜蛋白酪氨酸磷酸酶 (RTPases) 一样，在细胞质片段中具有两个串联重复的 PTPase 结构域。 PTPase 活性仅与两个结构域中的第一个 PTPase 结构域 1(D1) 相关，膜远端 PTPase 结构域 2(D2) 无催化活性，可调节底物特异性。来自 CD3zeta、lck 和 CD45D1CS 共表达的裂解物，其 828 处的半胱氨酸已被丝氨酸取代，磷酸化 使用抗 CD45 抗体将 CD3zeta 与 CD45D1CS 共沉淀。这一发现表明 CD3zeta 在体内也是 CD45 的良好底物。制备了CD3zeta的三种突变体MC1、MC2、MC3，它们仅携带2个酪氨酸残基，即来自C末端的第2和第3、第1和第3、第1和第2酪氨酸。 3个突变体均可被Ick弱磷酸化，与CD45D1CS的亲和力几乎相同，但SHC-SH2不能与磷酸化的MC2突变体结合。CD45D1D2CS的1144位半胱氨酸被丝氨酸取代，与CD3zeta的亲和力较低，磷酸化的CD3zeta比CD45D1CS更重要。D2的半胱氨酸具有重要作用 CD3zeta 的识别，与磷酸化无关。我们还研究了 LAR 和胰岛素受体 (IR) 的相互作用。 LAR 与胰岛素受体相关并优先去磷酸化，胰岛素受体在胰岛素刺激后被酪氨酸磷酸化。 LARD1CS可以稳定结合磷酸化IR。 IR可以是LAR的生理底物。LAR在胰岛素刺激后被IRkinase磷酸化。 LARD1D2CS 的半胱氨酸 1813 在结构域 2 中变为丝氨酸，与 LARDICS 相比，其与 IR 的亲和力较低，磷酸化程度较高。这些发现表明 LARD2 具有自动去磷酸化活性。这些数据表明CD45和LAR的D1PTP具有主要的磷酸酶活性，而它们的D2PTP可以独立于磷酸化来识别底物。

项目成果

Tomoyuki Sumizawa: "Reversal of MRP-associated drug resistance by the pyridine analog, PAK-104P" Mol.Pharmacol.51. 399-405 (1997)

Tomoyuki Sumizawa：“吡啶类似物 PAK-104P 逆转 MRP 相关耐药性”Mol.Pharmacol.51。

Yuji Takebayashi, Shin-ichi Akiyama, Shoji Natsugoe, Shuichi Hokita, Kiyoshi Niwa, Masaki Kitazono, Tomoyuki Sumizawa, Misako Haraguchi, Tatsuhiko Furukawa, Shuichi Nagayama and Takeshi Aikou: "The expression of multidrug resistance protein (MRP) in human

Yuji Takebayashi、Shin-ichi Akiyama、Shoji Natsugoe、Shuichi Hokita、Kiyoshi Niwa、Masaki Kitazono、Tomoyuki Sumizawa、Misako Haraguchi、Tatsuhiko Furukawa、Shuichi Nagayama 和 Takeshi Aikou：“人类多药耐药蛋白 (MRP) 的表达

Tomoyuki Sumizawa: "Reversal of MRP-associated drug resistance by the pyridine analog,PAK-104P" Molecular Pharmacology. 51. 399-405 (1997)

Tomoyuki Sumizawa：“吡啶类似物 PAK-104P 逆转 MRP 相关耐药性”分子药理学。

Zhe-Sheng Chen: "An active efflux system for heavy metals in cisplatin-resistant human KB carcinoma cells" Experimental Cell Research. (発表予定). (1998)

Zhe-Sheng Chen：“抗顺铂人类 KB 癌细胞中重金属的主动外排系统”实验细胞研究（即将发表）。

Yutaka Chuman: "Reversal of MRP-mediated vincristine resistance in KB cells by buthionine sulfoximide in combination with PAK-104P" Cancer Lett.129. 69-76 (1998)

Yutaka Chuman：“通过丁硫氨酸亚磺酰亚胺与 PAK-104P 组合逆转 KB 细胞中 MRP 介导的长春新碱耐药性”Cancer Lett.129。