Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing

摘要

Two phosphatidylinositol 4-kinase iso/.ymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain mierosomes with Triton X-l 14. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide curries the en/ymic activity of this type 3 Lsolbrm. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 anil finally SDS-PACiB results in an approximately 30000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-I'ACIK Iwve been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleo-tides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0,3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids, The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIKI, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.