Chi Yuling, Zhou Bo, Wang Wei-Qing, Chung Sung-Kee, Kwon Yong-Uk, Ahn Young-Hoon, Chang Young-Tae, Tsujishita Yosuke, Hurley James H, Zhang Zhong-Yin
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 2004 Oct 22;279(43):44987-95. doi: 10.1074/jbc.M406416200. Epub 2004 Aug 16.
Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.
肌醇-5-磷酸酶是一类重要的酶，参与从突触小泡循环到胰岛素信号传导等多种细胞过程的调控。我们描述了对两种代表性肌醇-5-磷酸酶——粟酒裂殖酵母突触素（SP突触素）和人含SH2结构域的肌醇-5-磷酸酶SHIP2的比较研究。我们发现，除了Mg2+外，过渡金属如Mn2+、Co2+和Ni2+也是SP突触素的有效激活剂。相比之下，Ca2+和Cu2+具有抑制作用。我们提供的证据表明，Mg2+结合的位点与在与肌醇1,4-二磷酸（Ins(1,4)P2）复合的SP突触素晶体结构中观察到的Ca2+所占据的位点相同。确定了对SP突触素催化的Ins(1,4,5)P3反应中底物结合和催化重要的离子化作用。对四种带有5-磷酸的磷脂酰肌醇脂质和54种水溶性肌醇磷酸进行的动力学分析表明，SP-突触素和SHIP2具有比以前认识到的更广泛的底物特异性。SP突触素的底物特异性排序为：Ins(2,4,5)P3 > 磷脂酰肌醇-4,5-二磷酸（PtdIns(4,5)P2）≈ Ins(4,5)P2 ≈ Ins(1,4,5)P3 ≈ Ins(4,5,6)P3 > PtdIns(3,5)P2 ≈ PtdIns(3,4,5)P3 ≈ Ins(1,2,4,5)P4 ≈ Ins(1,3,4,5)P4 ≈ Ins-(2,4,5,6)P4 ≈ Ins(1,2,4,5,6)P5。SHIP2的底物特异性排序为：Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 ≈ PtdIns(3,5)P2 ≈ Ins(1,4,5,6)P4 ≈ Ins(2,4,5,6)P4。由于肌醇磷酸异构体引发不同的生物学活性，SP突触素和SHIP2扩展的底物特异性表明这些酶可能在细胞信号传导中具有多种作用，并可能调节不同的途径。独特的底物特异性谱以及2-位磷酸在结合中的重要性对于设计用于药理学研究的强效和选择性SP突触素及SHIP2抑制剂也具有重要意义。
