A phosphatidylinositol 4-kinase pleckstrin homology domain that binds phosphatidylinositol 4-monophosphate.
Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kalpha) and Arabidopsis (AtPI4Kalpha). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio.
Botany Department, North Carolina State University, Raleigh, North Carolina 27695, USA.
J M
JM
Stevenson
I Y
IY
Perera
W F
WF
Boss
D019870:1-Phosphatidylinositol 4-Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018552:Daucus carota / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010750:Phosphoproteins; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001798:Blood Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D011994:Recombinant Proteins; C037178:phosphatidylinositol 4-phosphate; C039077:platelet protein P47; D019870:1-Phosphatidylinositol 4-Kinase