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PMID:12805633

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Citation

Stevenson-Paulik, J, Love, J and Boss, WF (2003) Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain. Plant Physiol. 132:1053-64

Abstract

Here, we compare the regulation and localization of the Arabidopsis type III phosphatidylinositol (PtdIns) 4-kinases, AtPI4Kalpha1 and AtPI4Kbeta1, in Spodoptera frugiperda (Sf9) insect cells. We also explore the role of the pleckstrin homology (PH) domain in regulating AtPI4Kalpha1. Recombinant kinase activity was found to be differentially sensitive to PtdIns-4-phosphate (PtdIns4P), the product of the reaction. The specific activity of AtPI4Kalpha1 was inhibited 70% by 0.5 mm PtdIns4P. The effect of PtdIns4P was not simply due to charge because AtPI4Kalpha1 activity was stimulated approximately 50% by equal concentrations of the other negatively charged lipids, PtdIns3P, phosphatidic acid, and phosphatidyl-serine. Furthermore, inhibition of AtPI4Kalpha1 by PtdIns4P could be alleviated by adding recombinant AtPI4Kalpha1 PH domain, which selectively binds to PtdIns4P (Stevenson et al., 1998). In contrast, the specific activity of AtPI4Kbeta1, which does not have a PH domain, was stimulated 2-fold by PtdIns4P but not other negatively charged lipids. Visualization of green fluorescent protein fusion proteins in insect cells revealed that AtPI4Kalpha1 was associated primarily with membranes in the perinuclear region, whereas AtPI4Kbeta1 was in the cytosol and associated with small vesicles throughout the cytoplasm. Expression of AtPI4Kalpha1 without the PH domain in the insect cells compromised PtdIns 4-kinase activity and caused mislocalization of the kinase. The green fluorescent protein-PH domain alone was associated with intracellular membranes and the plasma membrane. In vitro, the PH domain appeared to be necessary for association of AtPI4Kalpha1 with fine actin filaments. These studies support the idea that the Arabidopsis type III PtdIns 4-kinases are responsible for distinct phosphoinositide pools.

Links

PubMed PMC167043 Online version:10.1104/pp.103.021758

Keywords

1-Phosphatidylinositol 4-Kinase/chemistry; 1-Phosphatidylinositol 4-Kinase/genetics; 1-Phosphatidylinositol 4-Kinase/metabolism; Amino Acid Sequence; Androstadienes/pharmacology; Animals; Arabidopsis/enzymology; Arabidopsis/genetics; Base Sequence; Cell Line; DNA Primers; Enzyme Inhibitors/pharmacology; Gene Expression Regulation, Enzymologic/genetics; Gene Expression Regulation, Plant/genetics; Genes, Reporter; Green Fluorescent Proteins; Isoenzymes/chemistry; Isoenzymes/genetics; Isoenzymes/metabolism; Kinetics; Luminescent Proteins/genetics; Protein Structure, Tertiary; Recombinant Fusion Proteins/metabolism; Sequence Deletion; Sequence Homology, Amino Acid; Spodoptera; Transfection

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ARATH:P4KB1

enables

GO:0004430: 1-phosphatidylinositol 4-kinase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ARATH:P4KB1

located_in

GO:0030659: cytoplasmic vesicle membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ARATH:P4KA1

located_in

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

ARATH:P4KA1

enables

GO:0004430: 1-phosphatidylinositol 4-kinase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ARATH:P4KA1

enables

GO:0051015: actin filament binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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