Cloned (Comment) | Organism |
---|---|
gene ipmk, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli | Homo sapiens |
Crystallization (Comment) | Organism |
---|---|
purified recombinant core catalytic domain of HsIMPK, that contains residues 50 to 416, from which an internal domain comprising residues 263 to 377 is deleted and replaced with a simple Gly-Gly-Ser-Gly-Gly linker, apostructure and catalytic core domain with bound ADP-Ins (1,4,5)P3-Mg and ADP-DiC4-PtdInsP2-Mg, hanging drop vapor diffusion, mixing of 0.002 ml of 38 mg/ml protein solution with 0.002 ml of well solution containing 35% w/v PEG 400, 0.1 M Li2SO4, 100 mM MES-imidazole, pH 6.0, and 50 mM 2-mercaptoethanol, 25°C, to obtain complex structures, apoenzyme crystals are further soaked for 1 day in 35% w/v PEG 400, 100 mM Li2SO4, 100 mM HEPES, pH 7.5, at 25°C, in the presence of 20 mM Ins(1,4,5)P3 or a soluble diC4-analogue of PtdIns(4,5)P2, 10 mM MgCl2, and 5 mM of either Na2ATP or Li2AMP-PNP, X-ray diffraction structure determination and analysis at 1.63-1.93 A resolution. The structure of the IPMK apoenzyme is determined by a molecular replacement approach using a model constructed from the template of yeast ScIPMK (PDB ID 2IF8) | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
H388A | site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
K160A | site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
K167A | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
additional information | generation of the core catalytic domain of the enzyme that contains residues 50 to 416, from which an internal domain comprising residues 263 to 377 is deleted. The deletion is necessary to obtain crystals, it is replaced with a simple Gly-Gly-Ser-Gly-Gly linker. This deletion does not compromise catalytic activity, it is a non-catalytic region of the protein. It contains a nuclear localization sequence, flanked by residues that host protein kinase phosphorylation sites that regulate nuclear localization sequence functionality. Gln residues at positions 163, 164, and 196 are mutated each to Arg and Lys, both of which have side chains that are larger and also positively charged at physiological pH. The results are quite dramatic: in each case, the rate of Ins(1,4,5)P3 3-kinase activity declines, but in contrast, the rate of Ins(1,3,4,5)P4 6-kinase activity is not impaired, three of these mutants show increased 6-kinase activity, analysis of the structural basis, overview | Homo sapiens |
Q163A | site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q163K | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q163R | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q164A | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q164K | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q164R | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q196A | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q196K | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q196R | site-directed mutagenesis, the mutant shows reduced Ins(1,4,5)P3 3-kinase activity and increased Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Q78A | site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
R82A | site-directed mutagenesis, the mutant shows no Ins(1,4,5)P3 3-kinase activity and reduced Ins(1,3,4,5)P4 6-kinase activity compared to wild-type | Homo sapiens |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
nucleus | the internal domain comprising residues 263 to 377 contains the nuclear localization sequence | Homo sapiens | 5634 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate | Homo sapiens | - |
ADP + 1D-myo-inositol 1,3,4,5,6-pentakisphosphate | - |
? | |
ATP + 1D-myo-inositol 1,4,5-trisphosphate | Homo sapiens | - |
ADP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate | - |
? | |
ATP + 1D-myo-inositol 4,5-bisphosphate | Homo sapiens | - |
ADP + 1D-myo-inositol 3,4,5-trisphosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | Q8NFU5 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity and heparin affinity chromatography, followed by tag cleavage through TEV protease, another step of heparin affinity chromatography, and gel filtration | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate | - |
Homo sapiens | ADP + 1D-myo-inositol 1,3,4,5,6-pentakisphosphate | - |
? | |
ATP + 1D-myo-inositol 1,4,5-trisphosphate | - |
Homo sapiens | ADP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate | - |
? | |
ATP + 1D-myo-inositol 4,5-bisphosphate | - |
Homo sapiens | ADP + 1D-myo-inositol 3,4,5-trisphosphate | - |
? | |
additional information | recombinant HsIMPK uses 3-kinase activity to phosphorylate Ins(1,4,5)P3 to Ins(1,3,4,5)P4, and then phosphorylates the latter with a 6-kinase activity that yields Ins(1,3,4,5,6)P5. It also shows PtdIns(4,5)P2 3-kinase activity. Substrate binding structures, overview | Homo sapiens | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
HsIPMK | - |
Homo sapiens |
inositol phosphate multikinase | - |
Homo sapiens |
IP3K | - |
Homo sapiens |
IPMK | - |
Homo sapiens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.2 | - |
assay at | Homo sapiens |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Homo sapiens |
General Information | Comment | Organism |
---|---|---|
evolution | IPMK is a member of the so-called IP-kinase family that includes IP3Ks and IP6Ks, evolutionary relationships and structure comparisons, overview. There has been co-evolution of Ins(1,4,5)P3 and PtdIns(4,5)P2 3-kinase activities | Homo sapiens |
additional information | HsIPMK owns a catalytic pocket that is more constrained than those of the plant and yeast orthologues. Also unique to mammalian IPMK is a catalytically important proline-loop, and a preponderance of Gln residues in the active site. Description of two versions of Ins(1,4,5)P3 within the active site, first as a free inositol phosphate, and second as the headgroup of a soluble analogue of PtdIns(4,5)P2. The structure of the IPMK apoenzyme is determined by a molecular replacement approach using a model constructed from the template of yeast ScIPMK (PDB accession code 2IF8), and this apo-structure is used for further elucidation of the structures of crystal complexes with ADP plus either Ins(1,4,5)P3 or diC4-PtdIns(4,5)P2. Domains that are similar to the so-called N- and C-lobes that comprise the ATP-binding site. The C-terminal lobe comprising residues 136-149 and 175-416, which is an alphabeta-fold with five, central antiparallel beta-strands including beta4-6, beta8, and beta9, a pair of small antiparallel beta-strands (beta7 and beta10), and three alpha-helices (alpha5-alpha7). Also in the C-lobe of HsIPMK, a 310 helix is observed between the beta6 strand and alpha5 helix. His388 is at the catalytic center. Structure comparisons, overview | Homo sapiens |
physiological function | human inositol phosphate multikinase (HsIPMK) critically contributes to intracellular signaling through its inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) 3-kinase activities. HsIPMK is both an inositol phosphate kinase and a PtdIns(4,5)P2 kinase | Homo sapiens |