Type I Phosphatidylinositol-4-phosphate 5-Kinases Are Distinct Members of This Novel Lipid Kinase Family

Abstract

Phosphatidylinositol-4-phosphate 5-kinases (PIP5K) synthesize phosphatidylinositol-4,5-bisphosphate, a key precursor in phosphoinositide signaling that also regulates some proteins and cellular processes directly. Two distinct PIP5Ks have been characterized in erythrocytes, the 68-kDa type I (PIP5KI) and 53-kDa type II (PIP5KII) isoforms. Using peptide sequences from the erythroid 68-kDa PIP5KI, we have isolated cDNAs encoding PIP5KI␣ from human brain. Partial cDNAs obtained for a second isoform, PIP5KI␤, established that the human STM7 gene encoded a previously unrecognized PIP5KI. However, the peptide sequences demonstrated that erythroid PIP5KI corresponded to PIP5KI␣. Recombinant, bacterially expressed PIP5KI␣ possessed PIP5K activity and was immunoreactive with erythroid PIP5KI antibodies. By Northern analysis, PIP5KI␣ and PIP5KI␤ had wide tissue distributions, but their expression levels differed greatly. PIP5KIs had homology to the kinase domains of PIP5KII␣, yeast Mss4p and Fab1p, and a new Caenorhabditis elegans Fab1-like protein identified in the data base. These new isoforms have refined the sequence requirements for PIP5K activity and, potentially, regulation of these enzymes. Furthermore, the limited homology between PIP5KIs and PIP5KII␣, which was almost exclusively within the kinase domain core, provided a molecular basis for distinction between type I and II PIP5Ks. Phosphatidylinositol-4-phosphate 5-kinases (PIP5K)1 synthesize phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) by phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P). This conversion is pivotal to the phosphoinositide cycle as PtdIns(4,5)P 2 is hydrolyzed by phosphoinositide-specific phospholipase C to generate the second messengers 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. These second messengers activate several protein kinase C isoforms and effect the release of stored intracellular calcium, respectively. PtdIns(4,5)P 2 is also phosphorylated by agonist-activated phosphatidylinositol 3-kinases, resulting in synthesis of phosphatidylinositol 3,4,5-trisphosphate, a second messenger whose targets are largely unknown, but may include protein kinase C isoforms (1-3). In addition, PtdIns(4,5)P 2 directly modulates the function of proteins, including phospholipase D (4) and many actin-binding proteins (5). It binds pleckstrin homology domains found in some signaling proteins (6, 7) and is required for Ca 2ϩ -regulated secretion of neurotransmitters (8). The diversity of PtdIns(4,5)P 2 actions suggests that PIP5Ks are crucial enzymes for many cellular functions (reviewed in Ref. 9).Multiple PIP5K isoforms have been purified and biochemically characterized (9 -13). The best characterized isoforms are the erythroid enzymes which have been categorized as type I or type II PIP5Ks based on their separation by phosphocellulose ion exchange (10 -12). These PIP5Ks differ in molecular mass and are biochemically and immunologically distinct. Two notable differences are that PIP5KI, but not PIP5KII, can be stimulated by pho...

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

“…Turnover of PI(4,5)P 2 may be needed for the normal growth of a CCP by facilitating dynamic remodeling of the coat. This possibility is consistent with a recent report that type I PIP kinases (PI4P 5-kinases), which are the major PI(4,5)P 2 synthesizing enzymes, interact with AP-2, even though these enzymes are apparently not concentrated at CCPs (42,43).…”

Phosphoinositides in Constitutive Membrane Traffic

“…PIP 5-kinases were originally purified as activities that phosphorylated PtdIns(4)P to PtdIns (4,5)P 2 and subsequently cDNAs for six enzymes have been cloned (25,37,157,159,209). Based on sequence relationships, the PIP5Ks were grouped into two families called types I and II.…”

“…The mammalian PIP5Ks ␣-and ␤-isoforms were cloned independently from human or mouse by two laboratories and named in a reciprocal manner (157,209). A third isoform of PIP5KI, ␥, has also been cloned from both species (158).…”

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

“…Three major PI5KI isoforms (␣, ␤, and ␥) have been identified. [12][13][14][15] Increasing evidences indicate that PI5KIs, providing spatially and functionally distinct PIP2 pools, guarantee the regulation of specific cellular events: PI5KI␥, for instance, is required for focal adhesion and synaptic vesicle trafficking, 16,17 PI5KI␤ for constitutive receptor endocytosis, 18 whereas PI5KI␣ acts in the regulation of processes dependent on local changes of actin dynamics, such as membrane ruffling and phagocytosis. 19,20 In this report, we took advantage of the fusion protein consisting of GFP and the pleckstrin homology (PH) domain of PLC␦1, that is known to bind PIP2 with high affinity and specificity, [21][22][23] to monitor the dynamics and function of PIP2 during the cytotoxic event.…”