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Type I phosphatidylinositol 4-phosphate (PI(4)P) 5-kinases (PIP5Ks) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic. The protein localizes to the plasma membrane where its activity has been shown to be regulated by small GTPase ARFs and/or phosphatidic acid. Deletion analysis of amino- or carboxy-terminal sequences of PIP5Kgamma fused with EGFP demonstrated that the presence of central kinase homology domain (KHD), a 380 amino acid-long region highly conserved among PIP5K family, was necessary and sufficient for the plasma membrane localization of PIP5Kgamma. Particularly, the dibasic Arg-Lys sequence located at the carboxy-terminal end of KHD was shown to be crucial for the plasma membrane targeting of PIP5Kgamma, since the deletion or charge-reversal mutation of this dibasic sequence resulted in the mislocalization of the protein to the cytoplasm. Mislocalized mutants also failed to complement the temperature-sensitive growth of Saccharomyces cerevisiae mss4-1 mutant defective in PIP5K function. The presence of dibasic residues at the C-terminal end of KHD was conserved among mammalian as well as invertebrate PIP5K family members, but not in the type II PIPKs that are not targeted to the plasma membrane, suggesting that the conserved dibasic motif provides a mechanism essential for the proper localization and cellular function of PIP5Ks.

Toll-like receptor 4 (TLR4) is essential for recognizing a Gram-negative bacterial component, lipopolysaccharide (LPS). A single amino acid mutation at position 712 of murine TLR4 leads to hyporesponsiveness to LPS. In this study we determined that an amino acid, a leucine at position 815 of human TLR4, is also pivotal for LPS responsiveness and sub- cellular distribution. By replacing the leucine with alanine, the mutant TLR4 lost responsiveness to LPS and did not localize on the plasma membrane. In addition, it does not coprecipitate with myeloid differentiation-2, an accessory protein that is necessary for TLR4 to recognize LPS. These results suggest that the leucine at position 815 is required for the normal maturation of TLR4 and for formation of the TLR4∙MD-2 complex. [ABSTRACT FROM AUTHOR]