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Literature summary for 2.7.1.149 extracted from

  • Richardson, J.P.; Wang, M.; Clarke, J.H.; Patel, K.J.; Irvine, R.F.
    Genomic tagging of endogenous type IIbeta phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localization (2007), Cell. Signal., 19, 1309-1314.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
The last 2.6 kbp of the human PIPkin IIbeta gene and the following 3.6 kbp are cloned and inserted into a plasmid which placed a FLAG-(His6)2 double-tag, followed by a puromycin selection cassette between them. Transfection of this into DT40 cells followed by puromycin selection produces cells which have the tag attached to the C-terminus of the protein when expressed by one of the two alleles. Experiments are conducted on one clonal line derived from this procedure calling JPR3. Successful integration of the tag at the correct genomic location was confirmed by PCR analysis. Gallus gallus
The last 2.6 kbp of the human PIPkin IIbeta gene and the following 3.6 kbp are cloned and inserted into a plasmid which placed a FLAG-(His6)2 double-tag, followed by a puromycin selection cassette between them. Transfection of this into DT40 cells followed by puromycin selection produces cells which have the tag attached to the C-terminus of the protein when expressed by one of the two alleles. Experiments are conducted on one clonal line derived from this procedure calling JPR3. Successful integration of the tag at the correct genomic location was confirmed by PCR analysis. Homo sapiens

Protein Variants

Protein Variants Comment Organism
E300D Site-directed mutagenesis is performed on the human Type IIbeta PIPkin cloned into pEGFP-C1 using the QuikChange mutagenesis method, to introduce an Glu300Asp amino acid substitution into the human nuclear localisation sequence producing the equivalent chicken sequence. Gallus gallus
E300D Site-directed mutagenesis is performed on the human Type IIbeta PIPkin cloned into pEGFP-C1 using the QuikChange mutagenesis method, to introduce an Glu300Asp amino acid substitution into the human nuclear localisation sequence producing the equivalent chicken sequence. Homo sapiens

Localization

Localization Comment Organism GeneOntology No. Textmining
cytoplasm small amount, it is unclear how much of PIPkin II in the cytoplasmic fraction can be accounted for by nuclear disintegration or by leakage of PIPkinIIbeta during the fractionation. Homo sapiens 5737
-
cytoplasm small amount, it is unclear how much of PIPkin IIbeta in the cytoplasmic fraction can be accounted for by nuclear disintegration or by leakage of PIPkinIIbeta during the fractionation. Gallus gallus 5737
-
nucleus
-
Gallus gallus 5634
-
nucleus
-
Homo sapiens 5634
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
53000
-
FLAG-(His6)2 tagged PIPkin IIbeta, confirmed by western blotting Gallus gallus
53000
-
FLAG-(His6)2 tagged PIPkin IIbeta, confirmed by western blotting Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + 1-phosphatidyl-1D-myo-inositol 4-phosphate Gallus gallus 5' phosphorylation ADP + 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate
-
r
ATP + 1-phosphatidyl-1D-myo-inositol 4-phosphate Homo sapiens 5' phosphorylation ADP + 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate
-
r

Organism

Organism UniProt Comment Textmining
Gallus gallus
-
Site-directed mutagenesis is performed on the human type IIbeta PIPkin cloned into pEGFP-C1 using QuikChange mutagenesis method, to introduce an Glu300Asp amino acid substitution into the human nuclear localisation sequence producing the equivalent chicken sequence 289-DRAEQEEMEVE(E300D)RAEDEE-306
-
Homo sapiens P78356 Site-directed mutagenesis is performed on the human type IIbeta PIPkin cloned into pEGFP-C1 using QuikChange mutagenesis method, to introduce an Glu300Asp amino acid substitution into the human nuclear localisation sequence producing the equivalent chicken sequence 289-DRAEQEEMEVE(E300D)RAEDEE-306
-

Purification (Commentary)

Purification (Comment) Organism
affinity purification of FLAG-His6 tagged proteins using TALON beads Gallus gallus
affinity purification of FLAG-His6 tagged proteins using TALON beads Homo sapiens

Source Tissue

Source Tissue Comment Organism Textmining
cell culture DT40 cell from chicken, that expresses active PIPkin IIbetA tagged at the C-terminus of Hef, a protein component of the Fanconi anemia-related tumor-suppressor complex. Gallus gallus
-
cell culture DT40 cell from chicken, that expresses active PIPkin IIbetA tagged at the C-terminus of Hef, a protein component of the Fanconi anemia-related tumor-suppressor complex. Homo sapiens
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
In three independent experiments, the ratio (tagged cells to wild-type) of immunoprecipitated enzyme activity, quantified by densitometry or by scintillation counting, is 2.37 +/-0.27 Gallus gallus
additional information
-
In three independent experiments, the ratio (tagged cells to wild-type) of immunoprecipitated enzyme activity, quantified by densitometry or by scintillation counting, is 2.37 +/-0.27 Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 1-phosphatidyl-1D-myo-inositol 4-phosphate 5' phosphorylation Gallus gallus ADP + 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate
-
r
ATP + 1-phosphatidyl-1D-myo-inositol 4-phosphate 5' phosphorylation Homo sapiens ADP + 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate
-
r

Synonyms

Synonyms Comment Organism
PIPkinIIbeta
-
Gallus gallus
PIPkinIIbeta
-
Homo sapiens
type IIbeta phosphatidylinositol 5-phosphate 4-kinase
-
Gallus gallus
type IIbeta phosphatidylinositol 5-phosphate 4-kinase
-
Homo sapiens
type IIbeta PIPkin
-
Gallus gallus
type IIbeta PIPkin
-
Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Gallus gallus
30
-
assay at Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
assay at Gallus gallus
7.4
-
assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
ATP
-
Gallus gallus
ATP
-
Homo sapiens