ISOLATION AND MOLECULAR-CLONING OF WORTMANNIN-SENSITIVE BOVINE TYPE-III PHOSPHATIDYLINOSITOL 4-KINASES

Agonist-sensitive phosphoinositide pools are maintained by recently-id entified 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-sensit ive type III: PI 4-kinases were isolated from bovine adrenal cortex as [H-3]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 r eading frame encoding a 90-kDa protein was isolated from a bovine brai n cDNA library, Expression of this cDNA in COS-7 cells yielded a 110-k Da protein with WT-sensitive PI 4-kinase activity. Northern blot analy sis of a human mRNA panel showed a single similar to 3.8-kb transcript . Peptide sequences obtained from the 210-kDa enzyme corresponded to t hose of a recently described rat 230-kDa PI 4-kinase, A 6.5-kb cDNA co ntaining an open reading frame of 6129 nucleotides that encoded a 230- kDa protein, was isolated from brain cDNA, Northern blot analysis of h uman mRNA revealed a major 7.5-kb transcript, The molecular cloning of these novel WT-sensitive type III PI 4-kinases will allow detailed an alysis of their signaling and other regulatory functions in mammalian cells.