TY - JOUR
T1 - Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe synaptojanin and SHIP2
AU - Chi, Yuling
AU - Zhou, Bo
AU - Wang, Wei Qing
AU - Chung, Sung Kee
AU - Kwon, Yong Uk
AU - Ahn, Young Hoon
AU - Chang, Young Tae
AU - Tsujishita, Yosuke
AU - Hurley, James H.
AU - Zhang, Zhong Yin
PY - 2004/10/22
Y1 - 2004/10/22
N2 - 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+, Co 2+, 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) ≈ Ins(4,5)P2 ≈ Ins(1,4,5)P 3 ≈ 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. The rank order for SHIP2 is Ins(1,2,3,4,5)P 5 > 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)P 4. 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.
AB - 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+, Co 2+, 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) ≈ Ins(4,5)P2 ≈ Ins(1,4,5)P 3 ≈ 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. The rank order for SHIP2 is Ins(1,2,3,4,5)P 5 > 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)P 4. 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.
UR - http://www.scopus.com/inward/record.url?scp=7244226331&partnerID=8YFLogxK
U2 - 10.1074/jbc.M406416200
DO - 10.1074/jbc.M406416200
M3 - Article
C2 - 15316017
AN - SCOPUS:7244226331
SN - 0021-9258
VL - 279
SP - 44987
EP - 44995
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -