Isolation and molecular cloning of wortmannin-sensitive bovine type III phosphatidylinositol 4-kinases.
Agonist-sensitive phosphoinositide pools are maintained by recently-identified wortmannin (WT)-sensitive phosphatidylinositol (PI) 4-kinase(s) (Nakanishi, S., Catt, K. J., and Balla, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5317-5321). Two loosely membrane-associated WT-sensitive type III PI 4-kinases were isolated from bovine adrenal cortex as [3H]WT-labeled 110- and 210-kDa proteins. Based on peptide sequences from the smaller enzyme, a 3. 9-kilobase pair (kb) cDNA with an open reading frame encoding a 90-kDa protein was isolated from a bovine brain cDNA library. Expression of this cDNA in COS-7 cells yielded a 110-kDa protein with WT-sensitive PI 4-kinase activity. Northern blot analysis of a human mRNA panel showed a single approximately 3.8-kb transcript. Peptide sequences obtained from the 210-kDa enzyme corresponded to those of a recently described rat 230-kDa PI 4-kinase. A 6.5-kb cDNA containing an open reading frame of 6129 nucleotides that encoded a 230-kDa protein, was isolated from brain cDNA. Northern blot analysis of human mRNA revealed a major 7.5-kb transcript. The molecular cloning of these novel WT-sensitive type III PI 4-kinases will allow detailed analysis of their signaling and other regulatory functions in mammalian cells.
Balla
T
T
Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510, USA. tambal@box-t.nih.gov
Downing
G J
GJ
Jaffe
H
H
Kim
S
S
Zólyomi
A
A
Catt
K J
KJ
D019870:1-Phosphatidylinositol 4-Kinase; D000302:Adrenal Cortex / Q000201:enzymology*; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D019556:COS Cells; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004330:Drosophila; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; D000077191:Wortmannin