Regulated fusion of secretory granules with the plasma membrane in secretory cells requires ATP, Ca2+ and cytosolic as well as membrane proteins. ATP-dependent steps in Ca(2+)-activated secretion from PC12 cells require three cytosolic PEP proteins (priming in exocytosis proteins, PEP1-3), the identity of which will provide insights into the required ATP-using reactions. PEP3 was recently identified as phosphatidylinositol transfer protein (PtdInsTP), and here we report that PEP1 consists of the type I phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K). The roles of PEP3/PtdInsTP and PEP1/PtdInsP5K in sequential phosphoinositide recruitment and phosphorylation explains their synergistic activity in ATP-dependent priming. Moreover, inhibition of Ca(2+)-activated secretion by PtdIns(4,5)P2-specific antibodies and phospholipase C implies that 5-phosphorylated inositides play a novel, necessary role in the regulated secretory pathway. The results indicate that lipid kinase-mediated phosphorylation is an important basis for ATP use in the exocytotic pathway.

Abstract. In interphase cells, a-casein kinase I (et-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which a-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to a-CKI. In nonneuronal cells, a-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, o~-CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to o~-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with ot-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membranecontaining fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion. CELLS transport proteins and lipids throughout their cytoplasm using small vesicles containing specific integral membrane proteins (2-5, 13, 24, 35, 38, 52, 58, 62, 72-74). This transport system is microtubule dependent and requires motor proteins (16,19,31). In this process, vesicles bud from donor membranes and are shunted to specific acceptor membranes where the vesicles fuse and release their contents. These general events can be divided into constituitive and regulated pathways (13,17,24,35,41,65,(72)(73)(74). Although distinct, the two pathways appear to have similar components in common. A typical constitutive pathway would be the shuttling of vesicles within and between the endoplasmic reticulum, the Golgi organelle, and the plasma membrane. The movement and CaE+-dependent fusion of synaptic vesicles provides an example of a regulated secretory pathway.Stidhof and Jahn (74) physin and synaptobrevin, and two proteins integral to the plasma membrane, syntaxin and neurexin are involved in docking (4,5,10,11,26,62,68,75). Synaptotagmin, another vesicle integral membrane protein, is thought to act in both the positioning of vesicles at the plasma membrane and the Ca2+-dependent step of the fusion process (12,22,34,37,55,60,61). Finally, the SNAPs (N-ethylmaleimidesensitive factor-associated proteins) have been linked to vesicle targeting and fusion (11,15,35,71,77).Within each of these stages, the myriad of processes involved are extensively regulated. Evidence exists showing that G-proteins (16-18, 23, 41, 42), phosphoinositide kinases (23, 45-48, 69),...

No abstract