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Literature summary for 2.7.1.127 extracted from

  • Windhorst, S.; Fliegert, R.; Blechner, C.; Mollmann, K.; Hosseini, Z.; Gunther, T.; Eiben, M.; Chang, L.; Lin, H.Y.; Fanick, W.; Schumacher, U.; Brandt, B.; Mayr, G.W.
    Inositol-1,4,5-trisphosphate-3-kinase-A is a new cell motility-promoting protein that increases the metastatic potential of tumour cells by two functional activities (2009), J. Biol. Chem., 285, 5541-5554.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
Ca2+/calmodulin for enzyme activation, Ca2+/calmodulin, 2.15 microg/ml calmodulin and 10-20 microM free CaCl2 is added to the assay mixture Homo sapiens

Cloned(Commentary)

Cloned (Comment) Organism
establishment of A549 or H1299 cells overexpressing the enzyme: the cDNA fragment encoding for ITPKAis cloned into the retroviral vector MigRI, for virus production this construct is co-transfected with SVgp (M57) and pHCMV-VSV-G (M75) into HEK293 phoenix ampho cells, after 48 h of incubation the supernatant containing the viruses is filtered and transferred to A549 or H1299 cells Homo sapiens
expression of full length enzyme in Escherichia coli: a cDNA fragment encoding the full length form of ITPKA is inserted into the vector pGEX-4T-3 encoding a N-terminal Glutathione S-transferase-tag Homo sapiens
for stable knock-down of enzyme expression in MDA231 and H1299 cells a lentiviral transduction approach is employed Homo sapiens

Protein Variants

Protein Variants Comment Organism
L34P a point mutant that blocks actin-binding is overexpressed in H1299 cells, the morphology of cells expressing the mutant and those of cells expressing green fluorescent protein do not show visible differences Homo sapiens

Inhibitors

Inhibitors Comment Organism Structure
F-actin
-
Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + 1D-myo-inositol 1,4,5-trisphosphate Homo sapiens
-
ADP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate
-
?

Organism

Organism UniProt Comment Textmining
Homo sapiens P23677
-
-
Homo sapiens P23677 isoform inositol-1,4,5-trisphosphate 3-kinase A
-

Purification (Commentary)

Purification (Comment) Organism
purification of GST-inositol 1,4,5-trisphosphate-3-kinase-A protein Homo sapiens

Source Tissue

Source Tissue Comment Organism Textmining
A-549 cell
-
Homo sapiens
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Hep-G2 cell
-
Homo sapiens
-
HMEpC cell
-
Homo sapiens
-
IMR-90 cell
-
Homo sapiens
-
MCF-7 cell
-
Homo sapiens
-
MDA-MB-231 cell
-
Homo sapiens
-
MeVo cell
-
Homo sapiens
-
NCI-H1299 cell
-
Homo sapiens
-
neuron
-
Homo sapiens
-
SK-BR-3 cell
-
Homo sapiens
-
skin fibroblast cell line
-
Homo sapiens
-
T-47D cell
-
Homo sapiens
-
testis
-
Homo sapiens
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
activity of ITPKA is reduced by 94-95% in H1299 knock-down cells as compared to controls Homo sapiens
additional information
-
high ITPKA activity can significantly reduce the concentration of the 1D-myo-inositol 1,4,5-trisphosphate signal by rapidly removing the Ca2+ releasing second messenger Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 1D-myo-inositol 1,4,5-trisphosphate
-
Homo sapiens ADP + 1D-myo-inositol 1,3,4,5-tetrakisphosphate
-
?

Synonyms

Synonyms Comment Organism
inositol 1,4,5-trisphosphate-3-kinase-A
-
Homo sapiens
Ins(1,4,5)P3 3-kinase
-
Homo sapiens
ITPKA
-
Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Homo sapiens

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
additional information
-
the full length form of inositol 1,4,5-trisphosphate-3-kinase-A bundles F-actin in a concentration dependent manner, inhibition of the initial rate of gelsolin induces F-actin severing and the degree of bundling are half maximal at an similar concentration of about 300 nM, whereas inhibition of the rate of actin depolymerization after dilution to 100 nM actin is half maximal at about 30 nM, the much lower EC 50 for the latter effect may reflect the high affinity of enzyme for binding to F actin Homo sapiens F-actin

Expression

Organism Comment Expression
Homo sapiens cancer cell lines show large differences in their enzyme levels: whereas SKBR-3 and T47D cells expressed very low enzyme levels, very high expression levels are found in the highly metastatic cell lines H1299, MDA231, and HepG2, most of the transformed cell lines (H1299, MDA231, Mevo, Jurkat T-cells, HEK293, HepG2) examined express increased levels of ITPKA compared to the low levels in non-transformed cells (IMR-90, HmEpc, HSF-14, leucocytes) additional information
Homo sapiens phenotypic consequences of enzyme expression in tumour cells: enzyme is stably knocked-downed in 2 cell lines with high enzyme expression levels (MDA231, H1299) and is stably overexpressed in low enzyme expressing A549 cells. MDA231, H1299 and A549-ITPKA cells show dramatically changed phenotypes compared with their respective control cells: highly metastatic MDA231 control cells migrate completely through the matrix and grow in an evenly scattered pattern, knock down variants grow as cell clusters inside the gelatin layer or adhered to the bottom of the culture dish in tight aggregates. Also H1299 control cells completely migrate through the gelatin. H1299 kd cells by contrast remain inside the gelatin matrix, forming tight aggregates of round cells. In cells with up-regulated enzyme expression, the opposite behaviour as in cells with low enzyme expression is observed. Most of the A549-ITPKA cells migrate through the gelatin layer and adhered to the bottom of the culture dish. Fewer A549 control cells migrated through the gelatin. Non-migrating cells remain in the matrix and cell clusters have a rounded morphology. To analyze the influence of enzyme on invasive migration, transwell migration assays are performed. Transwell migration of MDA231 kd cells is reduced by 73%, and that of H1299 kd cells by 58%, as compared to the respective control cells exhibiting high levels of enzyme. Approximately twice as many A549-ITPKA cells penetrate as A549 control cells (101%). A549-ITPKA cells show 2.5fold higher migration compared to control A549 cells, while migration of H1299 kd cells is reduced 3.4fold relative to H1299 control cells additional information
Homo sapiens Western blot analysis reveals that nontransformed cells express low (IMR-90, HSF-14) to nearly undetectable (HmEpc, leucocytes) levels of ITPKA additional information
Homo sapiens H1299 control and H1299 cells overexpressing inositol 1,4,5-trisphosphate-3-kinase-A (H1299-ITPKA) cells are injected subcutaneously in BALB/c severe combined immunodeficient SCID mice, high expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model, high expression of ITPKA during invasion of tumour cells from the primary tumour to the neighbouring tissue up

General Information

General Information Comment Organism
physiological function inositol-1,4,5-trisphosphate-3-kinase-A is a cell motility-promoting protein that increases the metastatic potential of tumour cells by 2 functional activities: it promotes migration of tumour cells by 2 different mechanisms: growth factor independently, high levels of inositol-1,4,5-trisphosphate-3-kinase-A induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton, the F-actin binding activity of inositol-1,4,5-trisphosphate-3-kinase-A stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin, in growth factor stimulated cells, the catalytically active domain enhances basal inositol-1,4,5-trisphosphate-3-kinase-A induced migration by activating store-operated calcium entry through production of inositol-1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol-phosphate-5-phosphatase Homo sapiens
physiological function isoform inositol 1,4,5-trisphosphate 3-kinase A is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of isoform 1,4,5-trisphosphate 3-kinase A increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. 1,4,5-Trisphosphate 3-kinase A promotes migration of tumor cells by two different mechanisms: growth factor independently high levels of enzyme induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of 1,4,5-trisphosphate 3-kinase A stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal 1,4,5-trisphosphate 3-kinase A-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase Homo sapiens