Phosphatidylinositol 4-kinase type IIalpha is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles

Abstract

Phosphorylation of inositol phospholipids plays a key role in cellular regulation via the generation of intracellular second messengers. In addition, it represents a mechanism to regulate interactions of the lipid bilayer with proteins and protein scaffolds involved in vesicle budding, cytoskeletal organization, and signaling. Generation of phosphatidylinositol 4-phosphate [PI(4)P] from phosphatidylinositol (PI) is an important step in this metabolic pathway because PI(4)P is a precursor of other important phosphoinositides and has protein binding properties of its own. We report here that a PI 4-kinase (PI4K) activity previously reported on synaptic vesicles is accounted for by the alpha isoform of the recently characterized type II PI4K (PI4KII) family. PI4KIIalpha, which also accounts for the bulk of PI4K activity in brain extracts, is concentrated at synapses and in the region of the Golgi complex in neuronal perikarya. Our results provide new evidence for the occurrence of a cycle of phosphoinositide synthesis and hydrolysis nested within the exo-endocytic cycle of synaptic vesicles and point to PI4KIIalpha as a critical player in this cycle.

Figure 1

Characterization of anti-PI4KIIα Abs. (A) HEK293 cells were transiently transfected with myc-tagged PI4KIIα and analyzed by Western blotting. Both anti-myc and anti-PI4KIIα Abs recognized a major band at ≈55 kDa and a minor band at ≈66 kDa in transfected cells. (B) Triton X-100 extracts of transfected and mock-transfected cells were immunoprecipitated (IP) with either anti-myc or anti-PI4KIIα rabbit polyclonal Abs. Immunoprecipitates were then subjected to Western blotting (WB) with either mouse monoclonal anti-myc Abs or biotin-labeled anti-PI4KIIα Abs followed by streptavidin. (C) A Triton X-100 extract of rat brain was immunoprecipitated with anti-PI4KIIα or anti-PIPKIγ Abs. The immunoprecipitates (IP) were subjected to a PI kinase assay, and the products were analyzed by TLC and autoradiography. (D) Anti-PI4KIIα immunoprecipitates generated from rat brain Triton X-100 extract were analyzed by the PI kinase assay in the presence of the indicated compounds. (E) TLC analysis of reaction products generated by anti-PI4KIIα immunoprecipitates from Triton X-100 brain extracts in the presence of several synthetic PI substrates. X denotes an unidentified spot.

Figure 2

PI4KIIα accounts for the majority of PI(4)P-synthesizing activity in brain Triton X-100-soluble extracts. (A) Western blot analysis of total homogenates from mouse tissues with Abs directed against the proteins indicated. PI4KIIα is by far the predominant PI4KII expressed in brain. (B) Immunodepletion of PI4PKIIα from rat brain Triton X-100 extracts results in a dramatic reduction of PI 4-kinase activity. (Upper) Western blots for PI4KIIα and for the control protein amphiphysin 1 (Amph) in the starting material (SM), as well as in the corresponding extracts after immunoprecipitation with anti-PI4KIIα Ab, or with control IgGs. (Lower) The immunodepleted extract has lost nearly all PI kinase activity as revealed by TLC. Under our assay conditions, the bulk of PIP generated by the starting extract was found to be PI(4)P.

Figure 3

Subcellular distribution of PI4KIIα and lipid kinase activities. (A) Western blot analysis of subcellular fractions from a rat brain homogenate (H) with Abs directed against PI4KIIα and other neuronal proteins. Fractions were prepared according to Huttner et al. (30). The P2 fraction represents crude synaptosomes, which after lysis yield quickly and slowly sedimenting membrane fractions denoted LP1 and LP2, respectively. The LP2 fraction, which is highly enriched in synaptic vesicles (crude synaptic vesicle fraction), also contains a large pool of PI4KIIα. Note the absence of synaptophysin (a synaptic vesicle membrane marker) and PI4KIIα in the LS2 fraction, which contains only soluble proteins. (B) TLC analysis of ³²P incorporation into PIP and PIP₂ after incubation of the fractions with a brain phosphoinositide mixture, [³²P]ATP, and Triton X-100. X denotes an unidentified spot.

Figure 4

PI4KIIα accounts for the great majority of the PI 4-kinase activity present on synaptic vesicles. (A) CPG leading to a highly purified synaptic vesicle fraction (30). (Upper) Protein content (A₂₈₀ readings) and the PI kinase activity (TLC analysis) of the fractions in arbitrary units. (Lower) Levels of PI4KIIα and synaptophysin in the same fractions (Western blotting). (B) Synaptic vesicle-containing fractions were pooled, solubilized in Triton X-100 (SM, starting material), and subjected to immunoprecipitation with anti-PI4KIIα Abs or control IgGs. (Upper) Depletion of PI4KIIα, but not of the control protein synaptophysin, from the extract as revealed by Western blotting. (Lower) Drastic decrease in PI kinase activity in the PI4KIIα-depleted extract. X denotes an unidentified spot.

Figure 5

Recovery of PI4KIIα and PI 4-kinase activity on synaptobrevin/VAMP2-positive organelles by immunoisolation. Anti-synaptobrevin 2/VAMP2 Abs or control IgGs were conjugated to methacrylate beads and used for organelle immunoisolation from an LP2 fraction. (A) Electron microscopy of the material bound to control beads (Left) and to the anti-synaptobrevin 2/VAMP2 beads (Right). Arrows point to synaptic vesicles, which are not present on the control beads. (B Left) A Western blot analysis of the starting material (LP2) and of the material bound (B) and unbound (U) to the beads. Note that the nearly complete recovery of synaptophysin on the beads correlates with a similar recovery of PI4KIIα. (Right) Similar results were obtained for the PI kinase activity. The partial recovery of synaptic vesicle antigens and PI4KIIα in control IgG bead pellets is due to nonspecific sedimentation of larger membrane fragments present in LP2.

Figure 6

Concentration of PI4KIIα at synapses and in the Golgi complex area. Rat brainstem frozen sections were stained by double immunofluorescence. (A and B) PI4KIIα colocalizes with synaptojanin 1 and synaptobrevin 2/VAMP2 at synapses, which appear as bright fluorescence puncta outlining perikarya and dendrites. (C) Bright perinuclear PI4KIIα immunofluorescence is located in close apposition (see Insets) to elements immunoreactive for the cis-Golgi complex marker GM130. Small arrows point to synapses. In the “merged” images, PI4KIIα immunoreactivity appears in red. (Bar = 30 μm in A and 10 μm in B and C.)

Figure 7

Putative sites of action of PI-metabolizing enzymes in the synaptic vesicle cycle. PI4KIIα, which is tightly membrane-associated and therefore expected to be present on vesicles throughout the cycle, is proposed to function in the resynthesis of PI(4)P from PI after synaptojanin action on endocytic membranes. The figure is modified from ref. .