Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2.
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.
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Yuling
Y
Chi
Bo
B
Zhou
Wei-Qing
WQ
Wang
Sung-Kee
SK
Chung
Yong-Uk
YU
Kwon
Young-Hoon
YH
Ahn
Young-Tae
YT
Chang
Yosuke
Y
Tsujishita
James H
JH
Hurley
Zhong-Yin
ZY
Zhang
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D000072181:Inositol Polyphosphate 5-Phosphatases; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008982:Molybdenum / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D012568:Schizosaccharomyces / Q000378:metabolism*; D015398:Signal Transduction; D013053:Spectrophotometry; D013379:Substrate Specificity
D007477:Ions; D008055:Lipids; D009419:Nerve Tissue Proteins; D003035:Cobalt; D003300:Copper; D009532:Nickel; D008982:Molybdenum; D004492:Edetic Acid; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D008274:Magnesium; C022175:ammonium molybdate; D002118:Calcium