IDENTIFICATION OF A 200 KDA POLYPEPTIDE AS TYPE-3 PHOSPHATIDYLINOSITOL 4-KINASE FROM BOVINE BRAIN BY PARTIAL PROTEIN AND CDNA SEQUENCING

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have be en separated on hydroxylapatite after solubilizing bovine brain micros omes with Triton X-114. Employing a newly developed renaturation proce dure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide car ries the enzymic activity of this type 3 isoform. Chromatography on hy droxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE re sults in an approximately 30 000-fold purification. Tryptic peptides g enerated from the 200 kDa polypeptide after SDS-PAGE have been sequenc ed and the obtained data have been used for constructing and synthesiz ing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated fro m 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 align ment of this domain with the mammalian counterpart, the human type 2 p hosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosp hatidylinositol 3-kinases reveals a high degree of identity: 26 of the se approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA b inding properties of the enzyme. Two leucine zipper motifs (amino acid s 358-386, 862-882) are detectable. Furthermore, a helix loop helix mo tif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 iso form in the nucleus.