GO:0000825 inositol-1,3,4,5-tetrakisphosphate 6-kinase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0000825 describes the enzymatic activity that converts 1D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4) to 1D-myo-inositol 1,3,4,5,6-pentakisphosphate (IP5) using ATP.
• The enzyme belongs to the inositol 1,3,4-trisphosphate 5/6-kinase family, which can phosphorylate at either the 5- or 6-position of the inositol ring.
• This activity is conserved from plants to mammals; the Arabidopsis homolog AtITPK4 is an outlier with an ATP-grasp fold.
• In plants, inositol polyphosphate 6-/3-kinase is nuclear and complements yeast mutants lacking the ArgR-Mcm1 transcription complex.
• The enzyme is inhibited by various inositol polyphosphates, polyanions, and polycations, indicating tight regulation.
• Dysregulation of inositol polyphosphate signaling has been linked to cancer, neurodegeneration, and metabolic disorders, making this enzyme a potential therapeutic target.
Description
Inositol polyphosphates are versatile signaling molecules that regulate diverse cellular processes, including transcription, mRNA export, and calcium signaling. The enzyme inositol-1,3,4,5-tetrakisphosphate 6-kinase (EC 2.7.1.134) catalyzes the phosphorylation of 1D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4) to 1D-myo-inositol 1,3,4,5,6-pentakisphosphate (IP5), a key step in the generation of higher inositol polyphosphates such as IP6. This activity is encoded by the inositol 1,3,4-trisphosphate 5/6-kinase gene family, which was first purified and cloned from rat liver and subsequently characterized in plants. Understanding GO:0000825 is essential for researchers studying phosphoinositide signaling, as it bridges the gap between lipid-derived second messengers and nuclear inositol polyphosphate functions. The enzyme's ability to phosphorylate at both the 5- and 6-positions of the inositol ring underscores its metabolic flexibility and importance in cellular homeostasis.
inositol-1,3,4,5-tetrakisphosphate 6-kinase activity At A Glance
| GO ID | GO:0000825 |
|---|---|
| GO term | inositol-1,3,4,5-tetrakisphosphate 6-kinase activity |
| Ontology | molecular_function |
| Synonym | 1D-myo-inositol-tetrakisphosphate 6-kinase activity; inositol 1,3,4,5-tetrakisphosphate 6-kinase activity; inositol tetrakisphosphate 6-kinase activity |
| Definition | Catalysis of the reaction: 1D-myo-inositol 1,3,4,5-tetrakisphosphate + ATP = 1D-myo-inositol 1,3,4,5,6-pentakisphosphate + ADP + H+. |
| Major function | Phosphorylation of IP4 to IP5, a key step in inositol polyphosphate biosynthesis. |
| EC number | 2.7.1.134 |
| Substrate | 1D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4) |
| Product | 1D-myo-inositol 1,3,4,5,6-pentakisphosphate (IP5) |
What Is GO:0000825?
GO:0000825, inositol-1,3,4,5-tetrakisphosphate 6-kinase activity, is a molecular function defined by the catalytic reaction: 1D-myo-inositol 1,3,4,5-tetrakisphosphate + ATP = 1D-myo-inositol 1,3,4,5,6-pentakisphosphate + ADP + H+. This activity transfers a phosphate group from ATP to the 6-position of the inositol ring, producing IP5. It is synonymous with 1D-myo-inositol-tetrakisphosphate 6-kinase activity, inositol 1,3,4,5-tetrakisphosphate 6-kinase activity, and inositol tetrakisphosphate 6-kinase activity. The enzyme belongs to the inositol 1,3,4-trisphosphate 5/6-kinase family, which can also phosphorylate at the 5-position, but this term specifically refers to the 6-kinase activity.
Why Is inositol-1,3,4,5-tetrakisphosphate 6-kinase activity Important in Cell Biology?
GO:0000825 is critical because it represents a committed step in the synthesis of higher inositol polyphosphates, which act as signaling molecules in processes ranging from mRNA export to chromatin remodeling. The enzyme's product, IP5, is a precursor to inositol hexakisphosphate (IP6), a molecule implicated in diverse cellular functions including vesicle trafficking and apoptosis. In plants, this activity is essential for auxin signaling and stress responses, while in mammals it contributes to nuclear phosphoinositide signaling. Moreover, the enzyme is subject to complex regulation by polyphosphates and ions, highlighting its role in cellular homeostasis. Researchers studying cancer, neurodegeneration, and metabolic diseases are increasingly interested in this pathway as a source of therapeutic targets.
• Provides a key enzymatic step in the biosynthesis of inositol pentakisphosphate and hexakisphosphate.
• Regulates nuclear processes such as transcription and mRNA export through inositol polyphosphate signaling.
• Is conserved across eukaryotes, with homologs in mammals, plants, and yeast.
• Its product IP5 is a precursor to IP6, which has roles in apoptosis and vesicular trafficking.
• Enzyme activity is modulated by inositol polyphosphates, polyanions, and polycations, indicating tight regulation.
• Dysregulation of inositol polyphosphate metabolism is linked to cancer and neurodegenerative disorders.
• Plant homologs are involved in auxin signaling and stress responses.
• The enzyme's ATP-grasp fold in plant isoforms suggests unique structural features for drug design.
• It serves as a model for studying enzyme promiscuity, as the same enzyme can phosphorylate at the 5- or 6-position.
• Understanding this activity aids in interpreting genetic variants in inositol polyphosphate kinases associated with human diseases.
What Happens During inositol-1,3,4,5-tetrakisphosphate 6-kinase activity?
Substrate binding and recognition
In simple terms: The enzyme grabs the IP4 molecule and ATP, positioning them for a phosphate transfer.
The enzyme inositol-1,3,4,5-tetrakisphosphate 6-kinase binds its substrate, 1D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4), and the co-substrate ATP. The binding likely involves conserved residues in the active site that coordinate the inositol ring and the phosphate groups. Studies on the rat liver enzyme showed that it can be purified using an inositol hexakisphosphate affinity column, indicating strong affinity for inositol polyphosphates. The enzyme recognizes the 1,3,4,5-tetrakisphosphate configuration and positions the 6-hydroxyl for phosphorylation.
Phosphoryl transfer
In simple terms: The enzyme transfers a phosphate from ATP to the 6-position of the inositol ring.
The catalytic step involves the transfer of the gamma-phosphate from ATP to the 6-hydroxyl group of IP4, yielding 1D-myo-inositol 1,3,4,5,6-pentakisphosphate (IP5) and ADP. This reaction is magnesium-dependent, as typical for kinases. The enzyme was originally identified as a 5/6-kinase, meaning it can phosphorylate at either the 5- or 6-position, but this GO term specifically refers to the 6-kinase activity. The reaction is essentially irreversible under physiological conditions.
Product release and downstream metabolism
In simple terms: The newly made IP5 is released and can be further converted to other inositol polyphosphates.
After catalysis, IP5 is released from the active site. IP5 can serve as a substrate for further phosphorylation to inositol hexakisphosphate (IP6) or be dephosphorylated. In plants, the enzyme is nuclear and may directly influence transcription by generating IP5 locally. In mammals, IP5 is involved in various signaling pathways, including calcium mobilization and vesicle trafficking.
Regulation by inositol polyphosphates and ions
In simple terms: Other inositol polyphosphates and charged molecules can inhibit the enzyme.
The activity of inositol 1,3,4,5-tetrakisphosphate 6-kinase is inhibited by various inositol polyphosphates, other polyol phosphates, polyanions, and polycations. For example, inositol hexakisphosphate (IP6) and inositol 1,3,4,5,6-pentakisphosphate (IP5) can act as feedback inhibitors. This suggests that the enzyme is part of a tightly regulated network that responds to cellular levels of inositol polyphosphates. Additionally, divalent cations such as calcium may modulate activity.
Key Genes Involved in GO:0000825 inositol-1,3,4,5-tetrakisphosphate 6-kinase activity
The following genes encode enzymes with inositol-1,3,4,5-tetrakisphosphate 6-kinase activity or are closely related to its function across species.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ITPK1 (human) | Inositol-tetrakisphosphate 1-kinase; can phosphorylate IP4 at 5- or 6-position | Implicated in inositol polyphosphate signaling and cancer |
| ITPK1 (rat) | Inositol 1,3,4-trisphosphate 5/6-kinase; first purified and cloned | Model for enzyme kinetics and regulation |
| AtITPK1 (Arabidopsis) | Inositol 1,3,4-trisphosphate 5/6-kinase | Plant homolog involved in stress responses |
| AtITPK4 (Arabidopsis) | Outlier ATP-grasp fold protein | Structural studies on ATP-grasp fold |
| AtIPK2 (Arabidopsis) | Inositol polyphosphate 6-/3-kinase | Nuclear protein complementing yeast mutants |
| IPMK (human) | Inositol polyphosphate multikinase | Regulates IP4 and IP5 levels |
| IPK1 (yeast) | Inositol pentakisphosphate 2-kinase | Model for inositol polyphosphate synthesis |
| PLC (various) | Phospholipase C; produces IP3 from PIP2 | Upstream of IP4 production |
| IP3K (various) | Inositol 1,4,5-trisphosphate 3-kinase | Generates IP4 from IP3 |
| IP5P (various) | Inositol polyphosphate 5-phosphatase | Degrades IP5 and IP6 |
| IP6K (various) | Inositol hexakisphosphate kinase | Produces IP7 from IP6 |
| PPIP5K (various) | Diphosphoinositol pentakisphosphate kinase | Synthesizes IP7 and IP8 |
| ITPK1 (zebrafish) | Inositol-tetrakisphosphate 1-kinase | Developmental studies |
| ITPK1 (Drosophila) | Inositol 1,3,4-trisphosphate 5/6-kinase | Genetic studies on inositol signaling |
| ITPK1 (C. elegans) | Inositol 1,3,4-trisphosphate 5/6-kinase | Model for neuronal function |
| ITPK1 (Dictyostelium) | Inositol 1,3,4-trisphosphate 5/6-kinase | Chemotaxis studies |
| ITPK1 (plant) | Inositol 1,3,4-trisphosphate 5/6-kinase | Auxin signaling and development |
How Is inositol-1,3,4,5-tetrakisphosphate 6-kinase activity Regulated?
The activity of inositol-1,3,4,5-tetrakisphosphate 6-kinase is regulated by the availability of its substrates, ATP and IP4, as well as by feedback inhibition from downstream products such as IP5 and IP6. In plants, the enzyme is nuclear and may be regulated by developmental cues and stress signals. In mammals, the enzyme's activity can be influenced by calcium ions and other polycations, which may modulate its interaction with membranes or substrates. Additionally, the enzyme's expression levels may be controlled transcriptionally, although specific transcription factors have not been fully elucidated.
inositol-1,3,4,5-tetrakisphosphate 6-kinase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ITPK1 | Cancer (e.g., colorectal, breast) | Knockout in cancer cell lines; xenograft models |
| ITPK1 | Neurodegeneration (Alzheimer's) | Neuronal KO models; iPSC-derived neurons |
| IPMK | Metabolic disorders (diabetes) | Liver-specific KO mice; insulin resistance models |
| AtITPK1 | Plant stress responses | Arabidopsis knockout lines |
| ITPK1 | Developmental disorders | Zebrafish knockdown; mouse KO |
Cancer
Inositol polyphosphate signaling is frequently dysregulated in cancer. The enzyme inositol-1,3,4,5-tetrakisphosphate 6-kinase contributes to the production of IP5 and IP6, which have been implicated in cell growth, apoptosis, and migration. Altered expression of inositol polyphosphate kinases has been observed in various cancers, suggesting that this activity could be a therapeutic target.
Neurodegeneration
Inositol polyphosphates play roles in neuronal signaling and calcium homeostasis. Dysregulation of inositol metabolism has been linked to neurodegenerative diseases such as Alzheimer's and Parkinson's. The enzyme's product IP5 is involved in synaptic vesicle trafficking, and its perturbation may contribute to neuronal dysfunction.
Metabolic disorders
Inositol polyphosphates are involved in insulin signaling and glucose metabolism. Changes in inositol polyphosphate levels have been associated with diabetes and metabolic syndrome. The enzyme's activity may influence these pathways through the generation of IP5 and IP6.
From inositol-1,3,4,5-tetrakisphosphate 6-kinase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Enzyme kinetics and substrate specificity | Recombinant protein overexpression in E. coli or HEK293 cells |
| Role in inositol polyphosphate levels | CRISPR knockout of ITPK1 in cell lines followed by HPLC analysis |
| Nuclear function in transcription | Knockout in Arabidopsis or yeast complementation |
| Structural studies of ATP-grasp fold | Point mutations in AtITPK4 followed by crystallography |
| Disease relevance in cancer | Knockout in cancer cell lines; proliferation and apoptosis assays |
| Regulation by inhibitors | In vitro kinase assays with varying polyphosphate concentrations |
How to Study the inositol-1,3,4,5-tetrakisphosphate 6-kinase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Kinase assay with radiolabeled ATP | Enzymatic activity | In vitro characterization of recombinant enzyme |
| HPLC separation of inositol phosphates | Substrate and product levels | Purification and kinetic studies |
| LC-MS/MS | Quantification of inositol polyphosphates | In vivo metabolic profiling |
| CRISPR knockout | Loss-of-function phenotypes | Cell line and animal models |
| Complementation assay | Functional rescue | Yeast and plant genetics |
| X-ray crystallography | Three-dimensional structure | Structural studies of ATP-grasp fold |
| Site-directed mutagenesis | Residue function | Active site mapping |
| Affinity chromatography | Protein purification | Enzyme isolation using IP6 column |
Enzymatic assays
In vitro kinase assays using recombinant enzyme and radiolabeled ATP are standard for measuring inositol-1,3,4,5-tetrakisphosphate 6-kinase activity. Substrate IP4 can be generated enzymatically or chemically, and products are separated by HPLC or thin-layer chromatography.
Genetic knockout and complementation
CRISPR/Cas9 knockout of ITPK1 in cell lines or model organisms allows assessment of the enzyme's role in inositol polyphosphate metabolism. Complementation with wild-type or mutant cDNA can rescue phenotypes, as shown in yeast and plants.
Mass spectrometry
LC-MS/MS can quantify inositol polyphosphates in cell extracts, providing a direct readout of enzyme activity in vivo. This method is highly sensitive and can detect changes in IP5 and IP6 levels upon genetic manipulation.
Structural biology
X-ray crystallography and cryo-EM can elucidate the structure of the enzyme, particularly the ATP-grasp fold in plant isoforms. Site-directed mutagenesis of active-site residues can confirm their roles in catalysis.
How CRISPR Can Be Used to Study GO:0000825 inositol-1,3,4,5-tetrakisphosphate 6-kinase activity
Knockout
CRISPR/Cas9-mediated knockout of ITPK1 or related genes can abolish inositol-1,3,4,5-tetrakisphosphate 6-kinase activity, leading to reduced IP5 and IP6 levels. This approach is used to study the enzyme's role in cell proliferation, signaling, and development.
Point Mutation
Introducing point mutations in the catalytic domain of ITPK1 can dissect the contribution of specific residues to substrate binding and catalysis. For example, mutating the ATP-binding lysine can eliminate kinase activity, providing a negative control.
Knock-in
Knock-in of tagged versions of ITPK1 (e.g., GFP or FLAG) allows visualization and immunoprecipitation of the enzyme. This can reveal its subcellular localization and interaction partners, as shown for the nuclear plant homolog.
Overexpression
Overexpression of ITPK1 in cell lines or model organisms can increase IP5 and IP6 levels, enabling gain-of-function studies. This is useful for assessing the impact of elevated inositol polyphosphate signaling on cellular phenotypes.
How EDITGENE Supports inositol-1,3,4,5-tetrakisphosphate 6-kinase activity Research
Researchers studying inositol-1,3,4,5-tetrakisphosphate 6-kinase activity-related genes often need to determine whether a candidate gene is causally involved in a specific pathway or disease. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models, enabling functional validation of genes encoding this enzymatic activity.
Contact EDITGENE today to design your custom CRISPR model for inositol-1,3,4,5-tetrakisphosphate 6-kinase activity research.
Frequently Asked Questions About inositol-1,3,4,5-tetrakisphosphate 6-kinase activity
What is inositol-1,3,4,5-tetrakisphosphate 6-kinase activity?
It is the enzymatic activity that converts 1D-myo-inositol 1,3,4,5-tetrakisphosphate (IP4) to 1D-myo-inositol 1,3,4,5,6-pentakisphosphate (IP5) using ATP, as defined by GO:0000825.
What genes are involved in inositol-1,3,4,5-tetrakisphosphate 6-kinase activity?
The primary genes include ITPK1 in mammals and AtITPK1/AtITPK4 in Arabidopsis, which encode inositol 1,3,4-trisphosphate 5/6-kinases.
What is the reaction catalyzed by GO:0000825?
The reaction is: 1D-myo-inositol 1,3,4,5-tetrakisphosphate + ATP = 1D-myo-inositol 1,3,4,5,6-pentakisphosphate + ADP + H+.
How is inositol-1,3,4,5-tetrakisphosphate 6-kinase activity regulated?
It is regulated by substrate availability, feedback inhibition by inositol polyphosphates, and interactions with polyanions and polycations.
What diseases are associated with inositol-1,3,4,5-tetrakisphosphate 6-kinase activity?
Dysregulation has been linked to cancer, neurodegeneration, and metabolic disorders through altered inositol polyphosphate signaling.
What is the difference between 5-kinase and 6-kinase activity?
The same enzyme can phosphorylate IP4 at either the 5- or 6-position, producing different IP5 isomers; GO:0000825 specifically refers to the 6-kinase activity.
Which model organisms are used to study this activity?
Common models include rat liver, Arabidopsis thaliana, Saccharomyces cerevisiae, and various mammalian cell lines.
What methods measure inositol-1,3,4,5-tetrakisphosphate 6-kinase activity?
In vitro kinase assays with radiolabeled ATP, HPLC, and LC-MS/MS are standard methods.
Is inositol-1,3,4,5-tetrakisphosphate 6-kinase activity conserved in plants?
Yes, plant homologs such as AtITPK1 and AtITPK4 exist, and AtIPK2 complements yeast mutants, indicating functional conservation.
How can CRISPR be used to study this activity?
CRISPR knockout, point mutation, knock-in, and overexpression of ITPK1 and related genes allow functional dissection of the enzyme's role in cells and organisms.
Conclusion
GO:0000825, inositol-1,3,4,5-tetrakisphosphate 6-kinase activity, represents a critical enzymatic step in the biosynthesis of higher inositol polyphosphates. Its products, IP5 and IP6, are involved in diverse cellular processes, from nuclear signaling to stress responses. The enzyme is conserved across eukaryotes and is tightly regulated by its substrates and downstream metabolites. Dysregulation of this activity has been implicated in cancer, neurodegeneration, and metabolic disorders, making it a promising target for therapeutic intervention. Researchers can leverage CRISPR-based models to dissect its function and identify novel regulatory mechanisms.
References
- 1. Wilson MP et al.. 1996. Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme.. J Biol Chem 271(20):11904-10 PMID: 8662638
- 2. Abdullah M et al.. 1992. Purification and characterization of inositol-1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.. J Biol Chem 267(31):22340-5 PMID: 1331051
- 3. Chang SC et al.. 2006. Inositol polyphosphate multikinase regulates inositol 1,4,5,6-tetrakisphosphate.. Biochem Biophys Res Commun 339(1):209-16 PMID: 16293229
- 4. Shears SB. 1989. The pathway of myo-inositol 1,3,4-trisphosphate phosphorylation in liver. Identification of myo-inositol 1,3,4-trisphosphate 6-kinase, myo-inositol 1,3,4-trisphosphate 5-kinase, and myo-inositol 1,3,4,6-tetrakisphosphate 5-kinase.. J Biol Chem 264(33):19879-86 PMID: 2584198
- 5. Wilson MP et al.. 1997. Characterization of a cDNA encoding Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase.. Biochem Biophys Res Commun 232(3):678-81 PMID: 9126335
- 6. Sweetman D et al.. 2007. Arabidopsis thaliana inositol 1,3,4-trisphosphate 5/6-kinase 4 (AtITPK4) is an outlier to a family of ATP-grasp fold proteins from Arabidopsis.. FEBS Lett 581(22):4165-71 PMID: 17698066
- 7. Xia HJ et al.. 2003. Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex.. Plant Cell 15(2):449-63 PMID: 12566584
- 8. Hughes PJ et al.. 1994. Inhibition of porcine brain inositol 1,3,4-trisphosphate kinase by inositol polyphosphates, other polyol phosphates, polyanions and polycations.. Biochim Biophys Acta 1223(1):57-70 PMID: 8061054
