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

Abstract

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. 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 PIK1, 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.

Phosphoinositide Kinases

“…It encodes an 854-amino-acid protein (p97) that is 50% identical to Stt4 in the catalytic domain and is also more similar to Stt4 than to Pik1 in the noncatalytic region (108). Homologues have now been cloned from rat and bovine brain (109,110). Interestingly, the rat brain protein shares 98% identity with human p97 PtdIns4Kα over the region of overlap but encodes a protein of 2041 amino acids (p220) with a distinct N-terminal half.…”

Subcellular Localization and Structural Function of Endogenous Phosphorylated Phosphatidylinositol 4-Kinase (PI4K92)

“…32 P]ATP into extractable organic solvent material was measured as described previously (24). Produced phospholipids were extracted according to Ref.…”

Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms

“…It has comparatively low K m values for PtdIns and ATP (below 100 m) and is strongly inhibited by adenosine and Ca 2ϩ (Carpenter and Cantley, 1990). The type III enzyme has an apparent molecular mass of 200 kD on gel filtration, and has been renatured from a 200-kD polypeptide after SDS-PAGE (Gehrmann et al, 1996). It has 3-to 7-fold higher K m values for PtdIns and ATP than the type II enzyme and is insensitive to inhibition by adenosine and Ca 2ϩ .…”

“…These represent polypeptides of either 92 kD (Balla et al, 1997;Meyers and Cantley, 1997) or 230 kD (Gehrmann et al, 1996;Nakagawa et al, 1996;Balla et al, 1997), presumably corresponding to the isolated type III enzyme forms. This is in contrast to the only cloned kinase with type II properties (Wong and Cantley, 1994), a 97-kD polypeptide for which no corresponding enzyme has been isolated.…”