A PHOSPHATIDYLINOSITOL 4-KINASE PLECKSTRIN HOMOLOGY DOMAIN THAT BINDSPHOSPHATIDYLINOSITOL 4-MONOPHOSPHATE

Pleckstrin homology (PH) domains are found in many proteins involved i n signal transduction, including the family of large molecular mass ph osphatidylinositol (PI) 4-kinases. Although the exact function of thes e newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this stud y, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs o f the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4K alph a) and Arabidopsis (AtPI4K alpha). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contai ned the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, P H, and catalytic domains identified a polypeptide of 205 kDa in Arabid opsis microsomes and an F-actin-enriched fraction from carrot cells. T he 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 pho sphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-b isphosphate (PI-4,5-P-2), and phosphatidic acid and did not bind to th e S-phosphoinositides. The PH domain had the highest affinity for PI-4 -P, the product of the reaction. Consideration is given to the potenti al impact that this has on cytoskeletal organization and the PI signal ing pathway in cells that have a high PI-4-P/PI-4,5-P-2, ratio.