Báo cáo khoa học: WD-repeat-propeller-FYVE protein, ProF, binds VAMP2 and protein kinase Cf pptx

WD-repeat-propeller-FYVE protein, ProF, binds VAMP2 and protein kinase Cf Thorsten Fritzius, Alexander D Frey*, Marc Schweneker , Daniel Mayerà and Karin Moelling Institute of Medical Virology, University of Zurich, Switzerland Keywords protein interaction; protein kinase Cf; VAMP2; vesicle transport; WD repeats Correspondence K Moelling, Institute of Medical Virology, University of Zurich, Gloriastrasse 30, Zurich CH-8006, Switzerland Fax: +41 44 6344967 Tel: +41 44 6342652 E-mail: moelling@immv.unizh.ch Website: http://www.imv.unizh.ch/ Present address *Institute of Microbiology, ETH Zurich, Switzerland  Gladstone Institute of Virology and Immunology,San Francisco,CA,USA àDepartment of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Germany We have recently identified a protein, consisting of seven WD repeats, presumably forming a b-propeller, and a domain identified in Fab1p, YOTB, VAC1p, and EEA1 (FYVE) domain, ProF The FYVE domain targets the protein to vesicular membranes, while the WD repeats allow binding of the activated kinases Akt and protein kinase (PK)Cf Here, we describe the vesicle-associated membrane protein (VAMP2) as interaction partner of ProF The interaction is demonstrated with overexpressed and endogenous proteins in mammalian cells ProF and VAMP2 partially colocalize on vesicular structures with PKCf and the proteins form a ternary complex VAMP2 can be phosphorylated by activated PKCf in vitro and the presence of ProF increases the PKCf-dependent phosphorylation of VAMP2 in vitro ProF is an adaptor protein that brings together a kinase with its substrate VAMP2 is known to regulate docking and fusion of vesicles and to play a role in targeting vesicles to the plasma membrane The complex may be involved in vesicle cycling in various secretory pathways (Received 20 December 2006, accepted 16 January 2007) doi:10.1111/j.1742-4658.2007.05702.x We have recently identified the propeller-FYVE (domain identified in Fab1p, YOTB, VAC1p, and EEA1) protein (ProF) as a binding partner for Akt and protein kinase (PK)Cf [1] ProF contains seven WD repeats, which form a b-propeller-like structure, providing a protein-binding platform [2] Furthermore, ProF harbors a FYVE domain that specifically interacts with phosphatidylinositol-3-phosphate [3] and targets ProF to internal vesicles Deletion of the FYVE domain or inhibition of phosphatidylinositol-3-phosphate forma- tion by a phosphoinositide-3-kinase inhibitor resulted in loss of vesicular localization ProF preferentially bound to the kinases Akt and PKCf upon hormonal stimulation of the cells with insulin-like growth factor (IGF-1) [1] Because of this stimulation-dependent binding to kinases and due to its vesicular localization and its broad tissue distribution, we suggested that ProF plays a role in a number of secretory pathways [1] In order to better understand the role of ProF in inducible vesicle trafficking, we searched for substrates Abbreviations EGF-1, epidermal growth factor 1; FYVE, domain identified in Fab1p, YOTB, VAC1p, and EEA1; GLUT4, glucose transporter type 4; GST, glutathione S-transferase; HA, hemagglutinin; Hrs, hepatocyte growth factor-related tyrosine kinase substrate; IGF-1, insulin-like growth factor 1; MBP, myelin basic protein; PK, protein kinase; ProF, propeller FYVE protein; P-VAMP2, phosphorylated VAMP2; SNAP, synaptosomal-associated protein; SNARE, soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor; t-SNARE, targetSNARE; VAMP2, vesicle-associated membrane protein 2; Vps4, vacuolar protein sorting-associating protein 4; v-SNAREs, vesicular-SNARE 1552 FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS T Fritzius et al of Akt and PKCf on vesicles While this work was in progress, the Akt substrate of 160 kDa has been found to be located in adipocytes on vesicles containing the glucose transporter (GLUT4) [4] The Akt substrate of 160 kDa affects the trafficking of GLUT4-containing vesicles to the plasma membrane upon Akt phosphorylation [5–7] Several PKCf substrates on vesicles have been described previously The vesicle-associated membrane protein (VAMP2) may be one of them [8] VAMP2 belongs to the vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (v-SNARE) This protein family comprises eight members involved in secretory pathways [9] VAMP2 is widely expressed in a large variety of tissues, such as brain, kidney, adrenal gland, liver and pancreas [10] VAMP2 is crucial for stimulus-dependent secretion in various cell-types including insulinstimulated GLUT4 translocation in adipocytes and muscle cells [11], fusion of early and sorting endosomes [12,13], and synaptic vesicle fusion with the plasma membrane in neurons [14,15] The fusion of VAMP2containing vesicles with the plasma membrane is mediated by complex formation of the v-SNARE with the target (t)-SNARE synaptosome-associated protein (SNAP) and syntaxin [16] VAMP2 has previously been reported to be phosphorylated in myotubes overexpressing PKCf, which correlated with increased GLUT4 translocation and glucose uptake [8] Previous analyses have shown that ProF binds specifically to the atypical PKC isoform PKCf [1] In the present study, we show that ProF also interacts with VAMP2 both in vitro and in vivo We demonstrate that all three proteins can form a complex and that ProF can mediate the binding of PKCf to VAMP2 in a concentration-dependent manner Furthermore, we show that VAMP2 is directly phosphorylated by activated PKCf in vitro and that ProF leads to increased phosphorylation of VAMP2 by activated PKCf in vitro Thus, ProF can integrate the kinase PKCf, and its substrate VAMP2, which, upon phosphorylation, may contribute to vesicle cycling in secretory pathways Results VAMP2 is a binding partner of ProF We have recently identified a protein, consisting of seven WD repeats, presumably folding into a b-propeller-type structure, and a FYVE domain (Fig 1A), designated as ProF ProF interacted via its WD repeats with the serine ⁄ threonine kinases Akt and PKCf, and was located on internal vesicles via its FYVE domain These two kinases preferentially bound to ProF after WD-FYVE protein interacts with VAMP2 and PKCf hormonal stimulation of the cell [1] Therefore, the question arose whether ProF can bring together the kinase with putative kinase substrates In order to identify such candidate substrates, we performed a yeast two-hybrid screen using a human B-cell-specific embryonic cDNA library and full-length ProF as bait [17] Out of this screen, two positive clones were obtained One of them was identified as VAMP2, the other as an as yet not described protein VAMP2 is a v-SNARE protein, associated with vesicular membranes via its C-terminal transmembrane domain Its central SNARE domain of 60 amino acids allows the interaction of VAMP2 with its cognate t-SNARE proteins (Fig 1A) [9] The VAMP2 fragment, which interacted with the full-length ProF in the yeast two-hybrid assay, contained the amino acids 1–111 Because the complete human VAMP2 protein consists of only 116 amino acids, little information about the ProF interaction domain of VAMP2 could be deduced from the yeast two-hybrid screen All serine (Ser) residues of VAMP2 are indicated in Fig 1(A) Four of them were mutated to alanine (Ser to Ala) in order to generate a VAMP2 mutant mt(1–4) To verify the results obtained by the yeast twohybrid screen, we first analyzed the interaction of VAMP2 with ProF by coimmunoprecipitation of overexpressed proteins For this purpose, Myc-tagged ProF- and Flag-tagged VAMP2-expression constructs were cotransfected in COS-7 cells Cell lysates were treated with an anti-Flag IgG (Fig 1B, left) or an anti-Myc IgG (Fig 1B, right) and the precipitates were analyzed by immunoblotting for the presence of coprecipitating proteins As can be seen, Myc-tagged ProF indeed coprecipitated with Flag-tagged VAMP2 (Fig 1B, left, lane 3) and Flag-tagged VAMP2 coprecipitated with Myc-tagged ProF (Fig 1B, right, lane 3) VAMP2 did not interact with the hepatocyte growth factor-regulated tyrosine kinase substrate, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) (Fig 1B, left, lane 4), another FYVEdomain containing protein that is also localized to intracellular vesicles [18,19], or with the vacuolar protein sorting-associating protein 4, Vps4 (Fig 1B, left, lane 7), a protein involved in intracellular vesicle formation and protein trafficking [20] ProF interacted weakly with Hrs (Fig 1B, right, lane 5), possibly via heterodimerization of the FYVE domains of ProF and hepatocyte growth factor-regulated tyrosine kinase substrate, as ProF can form oligomers via its FYVE domain [1] Furthermore, ProF did not interact with Vps4 (Fig 1B, right, lane 6), demonstrating the specificity of the interaction of ProF with VAMP2 FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS 1553 WD-FYVE protein interacts with VAMP2 and PKCf T Fritzius et al Fig VAMP2, a new interaction partner of ProF (A) Domain structure of ProF and VAMP2 ProF consists of seven WD repeats (WD1–7), binding to proteins, and a FYVE domain, binding to phosphatidylinositol-3-phosphate on vesicular membranes (top, left) A model of the three-dimensional structure of ProF without the FYVE domain is shown, with the seven WD repeats indicated (top, right) VAMP2 is anchored to vesicular membranes through its C-terminal transmembrane domain A central SNARE motif is essential for the interaction with its target SNARE proteins Serine (S) residues, which are potential PKCf-phosphorylation sites are indicated below as wild-type (wt) and mutant [mt(1–4)] (bottom) (B) Coimmunoprecipitation assay of overexpressed Flag-tagged VAMP2 (lanes 1, 3–4, and 7) in the presence or absence of Myc-tagged ProF (lanes 2–3 and 5–6), HA-tagged Hrs (lanes 4–5), and GFP-tagged Vps4 (lanes 6–7) in COS-7 cells Immunoprecipitation was performed with an antibody against the Flag-tag (left) or the Myc-tag (right) and immunoprecipitates were analyzed by immunoblotting with antibodies against Flag, Myc, HA and GFP epitopes Direct lysates (DL) are shown as expression control (bottom) (C) MycProF wild-type (wt), ProF lacking the FYVE domain (Myc-ProFDFYVE), ProF lacking the FYVE domain and containing only blades 1–3 or 4–7 (Myc-ProF 1–3 or Myc-ProF 4–7) were overexpressed together with Flag-VAMP2 in COS-7 cells Immunoprecipitation and subsequent immunoblot show the interactions We further characterized the interaction between ProF and VAMP2 with deletion mutants of ProF We analyzed a Myc-tagged mutant of ProF, lacking the FYVE domain (ProFDFYVE) and two mutants lacking the FYVE domain and containing only blades 1–3 (ProF 1–3) or blades 4–7 (ProF 4–7) of the 1554 seven-bladed b-propeller (Fig 1A) Interaction of truncated WD-repeat proteins, containing only one or two b-propeller blades, with its binding partners has been shown earlier ([21,22]), indicating that the remaining blades are still able to fold properly We coexpressed these proteins together with Flag-tagged VAMP2 in FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS T Fritzius et al COS-7 cells and tested their interaction by coimmunoprecipitation assays As can be seen, all ProF mutants interacted equally well with VAMP2 (Fig 1C) In summary, this result suggested that multiple binding sites on ProF are involved in the binding of VAMP2 VAMP2, ProF, and PKCf colocalize on vesicular structures Further indications for the interaction of both proteins were obtained by confocal immunofluorescence analysis showing their subcellular distribution For that, Flag-tagged VAMP2 and Myc-tagged ProF were coexpressed in COS-7 cells and analyzed by confocal microscopy As can be seen, a partial colocalization of WD-FYVE protein interacts with VAMP2 and PKCf VAMP2 (green signal) and ProF (red signal) on vesicular structures was detectable (Fig 2A) Colocalization of the two proteins is indicated by the orange color, detectable in the merged picture, showing the superposition of the two signals We have previously reported that ProF binds PKCf [1] and show here the interaction between ProF and VAMP2 This raised the question whether ProF could interact with both proteins, VAMP2 and PKCf First, this was tested by colocalization analysis using confocal microscopy Flag-tagged VAMP2, hemagglutinin (HA)-tagged PKCf and Myc-tagged ProF were transiently expressed either together (Fig 2B) or alone (Fig 2C) in COS-7 cells and analyzed by confocal microscopy When PKCf, VAMP2, and ProF were Fig Colocalization of VAMP2, ProF, and PKCf (A) Flag-VAMP2 and Myc-ProF were overexpressed in COS-7 cells Confocal microscopy analysis with antibodies against Flag- and Myc-tag revealed areas of colocalization as visualized in yellow on the merged picture (right) (B) COS-7 cells were transiently transfected with HA-PKCf (green), Flag-VAMP2 (red), and Myc-ProF (blue) and analyzed by confocal microscopy using antibodies against HA, Flag, and Myc epitopes The lower part shows detail A partial colocalization on cytoplasmic punctuate structures (white) is observed (merged) (C) As a control, COS-7 cells were transfected with either HA-PKCf (green), Flag-VAMP2 (red), or Myc-ProF (blue) and were analyzed by confocal microscopy Flag-VAMP2 (red) and Myc-ProF (blue), signals are confined to specific structures, while HA-PKCf (green) and is distributed throughout the cell (D) Confocal microscopy analysis of COS-7 cells cotransfected with HA-PKCf (red) and Myc-ProF (green) revealing colocalization of both proteins on punctuate structures (top) Confocal microscopy analysis of COS-7 cells cotransfected with HA-PKCf (green) and Flag-VAMP2 (red) show colocalization of both proteins on punctuate structures (bottom) (E) For confocal immunofluorescence analysis of 3T3-L1 pre-adipocytes, cells were serum-starved for h prior to staining for endogenous PKCf (green), endogenous VAMP2 (red), and stably expressed Myc-tagged ProF (magenta) A partial colocalization on cytoplasmic punctuate structures (white) is observed (merged) Lower part shows detail FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS 1555 WD-FYVE protein interacts with VAMP2 and PKCf T Fritzius et al expressed together, a partial colocalization of the three proteins on intracellular vesicles was detected (Fig 2B, right) Colocalization of the three proteins is indicated by the white color in the merged picture, showing the superposition of the three signals When expressed alone, overexpressed VAMP2 (red) and ProF (blue) were found to be located on distinct intracellular vesicles, while PKCf (green) was more evenly distributed in the cytoplasm (Fig 2C) These data indicate that VAMP2 and ProF can alter the subcellular localization of PKCf In order to find out whether ProF and VAMP2 alone were also able to target PKCf to vesicles, HA-PKCf and Myc-ProF (Fig 2D, top) or HA-PKCf and Flag-VAMP2 (Fig 2D, bottom) were transiently coexpressed in COS-7 cells and analyzed by confocal microscopy As can be seen, expression of HA-PKCf together with Myc-ProF (Fig 2D, top) and Flag-VAMP2 (Fig 2D, bottom), led to a partial localization of the kinase on punctuate structures We further substantiated these data by confocal microscopy studies with 3T3-L1 pre-adipocyte cells, stably expressing Myc-tagged ProF As can be seen, Myc-ProF, endogenous VAMP2, and endogenous PKCf colocalized on perinuclear vesicular structures (Fig 2E) These results are in agreement with the previous findings that ProF is located on internal vesicles in various cell lines, e.g in 3T3-L1 pre-adipocyte cells [1] VAMP2, ProF, and PKCf form a complex We have shown earlier that ProF interacts specifically with the atypical PKC isoform PKCf, but not with novel PKC isoforms, and binds weakly to the classical PKC isoform PKCa [1] To investigate whether there was also a specific interaction between PKCf and VAMP2, human embryonic kidney 293T cells were transiently transfected with constructs expressing FlagVAMP2 and VAMP2 was immunoprecipitated with an antibody against Flag Overexpressed Flag-VAMP2 coprecipitated endogenous aptyical PKCf ⁄ k but not the novel isoforms PKCd and PKCe or the classical isoform PKCa (Fig 3A), suggesting a specificity of VAMP2 for atypical PKC isoforms Next, we analyzed whether these three proteins physically interacted by performing a sequential precipitation procedure We overexpressed the epitope-tagged forms of all three proteins in human embryonic kidney 293T cells Then we immunoprecipitated Myc-ProF and showed coprecipitation of FlagVAMP2 and HA-PKCf by western blot analysis of an aliquot of the immunoprecipitate (Fig 3B, lane 2) The precipitated complex was thereafter eluted with a Myc-peptide and half of the eluate was used for 1556 immunoprecipitation of HA-PKCf As can be seen, the coimmunoprecipitation of Myc-ProF and FlagVAMP2 was demonstrated by western blotting (Fig 3B, lane 3) Furthermore, immunoprecipitation of Flag-VAMP2 using the other half of the lysate led to the coimmunoprecipitation of HA-PKCf and MycProF (Fig 3B, lane 4) Additionally, reciprocal immunoprecipitation was performed to verify the existence of a complex Immunoprecipitation of HA-PKCf allowed the coprecipitation of Flag-VAMP2 and Myc-ProF, as evidenced by western blotting analysis (Fig 3C, lane 2) The precipitated complex was thereafter eluted with a PKCf-peptide and Myc-ProF (Fig 3C, lane 3) or Flag-VAMP2 was immunoprecipitated (Fig 3C, lane 4) A strong coimmunoprecipitation of HA-PKCf and a weak coimmunoprecipitation of Flag-VAMP2 in the case of Myc-ProF (Fig 3C, lane 3) can be demonstrated by western blotting Furthermore, we found coimmunoprecipitation of HAPKCf and Myc-ProF in the case of Flag-VAMP2 (Fig 3C, lane 4) As only a weak VAMP2 signal was detected after immunoprecipitation of Myc-ProF, this could indicate that ProF might bind more strongly to PKCf than to VAMP2, or that the VAMP2 binding to the protein complex is more susceptible to the mechanical disruptions performed during the elution of the complex Nevertheless, we have shown that Myc-ProF forms a complex with both proteins, FlagVAMP2 and HA-PKCf These findings raised the question how ProF would affect the interaction between VAMP2 and PKCf In order to test this, we expressed Flag-VAMP2 and HA-PKCf in the absence or presence of increasing amounts of Myc-ProF in COS-7 cells (Fig 3D) As can be seen, in the absence of Myc-ProF, only small amounts of HA-PKCf were coprecipitated (Fig 3D, lane 5) Coexpression of small amounts of Myc-ProF led to the coprecipitation of large amounts of HAPKCf by Flag-VAMP2 (Fig 3D, lane 6) Further increasing concentrations of ProF caused the opposite effect, a decreased coprecipitation of HA-PKCf by Flag-VAMP2 (Fig 3D, lanes and 8) This is further corroborated by a quantification of three individual experiments, performed by densitometric scanning of western blots (Fig 3D, bottom), demonstrating that low concentrations of ProF lead to a strongly (3.5fold) and significantly increased binding of VAMP2 to PKCf In summary, these results indicate that ProF can regulate the binding of VAMP2 and PKCf in an adaptor protein-like fashion It increases the binding of PKCf to VAMP2 under optimized conditions, which is a characteristic of adaptor proteins FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS T Fritzius et al WD-FYVE protein interacts with VAMP2 and PKCf Fig Interactions of VAMP2 with ProF and PKCf (A) Flag-VAMP2 was transiently expressed in human embryonic kidney 293T cells The complex comprising Flag-VAMP2 and endogenous PKC isoforms was subjected to immunoprecipitation using an antibody against Flag Interaction of VAMP2 with the PKC isoforms was analyzed as shown by immunoblot against PKCa, PKCd, PCKe, and PKCf (from left to right, top) and VAMP2 (bottom) Direct lysates show expression controls (B) HA-PKCf, Flag-VAMP2, and Myc-ProF were transiently coexpressed in human embryonic kidney 293T cells The complex comprising HA-PKCf, Myc-ProF, and Flag-VAMP2 was subjected to immunoprecipitation with an anti-Myc IgG (lane 2) The complex was eluted by addition of an excess of a competing Myc-peptide followed by immunoprecipitation using an antibody directed against PKCf (lane 3) or the Flag-epitope (lane 4) Immunoprecipitations of the different steps were analyzed by immunoblot against the indicated proteins Samples were loaded onto one gel, separating lines were included later for clarity (C) The complex comprising HA-PKCf, Myc-ProF, and Flag-VAMP2 was subjected to immunoprecipitation with an anti-PKCf IgG (lane 2) The complex was eluted by addition of excess of competing PKCf-peptide followed by immunoprecipitation against the Myc- (lane 3) or the Flagepitope (lane 4) Immunoprecipitations of the different steps were analyzed by immunoblot against the indicated proteins Samples were loaded onto one gel; separating lines were included for clarity (D) HA-PKCf, Myc-ProF, and Flag-VAMP2 were transiently overexpressed in COS-7 cells The interactions of VAMP2 with ProF alone (lane 4), PKCf alone (lane 5), or PKCf in the presence of increasing amounts (0.25 lg, lg, and lg) of Myc-ProF (lanes 6–9) were analyzed as shown by immunoprecipitation and subsequent immunoblot (top) DL (direct lysates) show expression controls (bottom) To the right, a quantification of PKCf binding to VAMP2 in the absence or presence of ProF is shown Results were obtained by densitometric scanning of immunoblot bands from three independent experiments The interaction of PKCf with VAMP2 was normalized to binding in the absence of ProF ¼ Values represent mean±SD of three separate experiments (*P < 0.05, **P < 0.01) FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS 1557 WD-FYVE protein interacts with VAMP2 and PKCf T Fritzius et al Endogenous VAMP2 interacts with ProF and PKCf Fig Interactions of endogenous VAMP2 with ProF and PKCf in brain (A) Immunoprecipitation of murine brain lysates, performed with an anti-ProF IgG in the absence or presence of an excess of competing peptide, used before as an antigen to raise the anti-ProF IgG, and subsequent immunoblot (B) Immunoprecipitation of murine brain lysates, performed with an irrelevant antibody (antiMyc, lane 1), and anti-VAMP2 IgG (lane and 3) The subsequent immunoblot was performed with anti-ProF IgG in the absence (lane 2) or presence (lane 3) of an excess of competing peptide Direct lysates (right) show expression of the endogenous proteins in mouse brain lysate (C) Immunoprecipitation of murine brain lysates with an anti-ProF IgG and subsequent immunoblot (left) Direct lysates (right) show expression of the endogenous proteins in mouse brain lysate So far we have analyzed overexpressed proteins; we now need to confirm these results with endogenous proteins ProF and VAMP2 have been reported to be expressed in the brain [1,23] Therefore, we used mouse brain lysates to test the interaction between endogenous ProF and VAMP2 For this, brain lysates were treated with an anti-ProF IgG with and without peptide competition to demonstrate the specificity of the reaction The precipitates were analyzed by western blotting As can be seen, coimmunoprecipitation of VAMP2 with ProF was detectable, while the presence of a competing peptide inhibited ProF precipitation and VAMP2 coprecipitation (Fig 4A) To verify the interaction of ProF and VAMP2, we performed a reciprocal immunoprecipitation by treatment of mouse brain lysates with an anti-VAMP2 IgG (Fig 4B, lane 2) As can be seen, coprecipitation of ProF with VAMP2 was detectable Peptide competition during western blotting (Fig 4B, lane 3) and immunoprepitation performed with an irrelevant antiMyc IgG (Fig 4B, lane 1) demonstrated the specificity of the reaction Thus we also confirmed the interaction of ProF and VAMP2 for endogenous proteins in brain tissue In order to show the interaction of all three endogenous proteins, we immunoprecipitated ProF from mouse brain lysates and analyzed the precipitates by western blotting for the presence of ProF, VAMP2, and PKCf As can be seen, after precipitation with Fig VAMP2 is phosphorylated by PKCf in vitro (A) Recombinant GST-VAMP2 wild-type (wt) and VAMP2 serine to alanine mutant [mt(1– 4)] were expressed in bacteria, purified and subjected to an in vitro kinase assay with c-32P-ATP in the presence of lg of recombinant active Akt (lane 2), 100 ng recombinant active PKCf (lanes and 5) and 200 ng recombinant active PKCf (lanes and 6) GST-VAMP2 wt and mt(1–4) phosphorylation and Akt ⁄ PKCf autophosphorylation were analyzed using a PhosphoImager Expression of GST-VAMP2 wt and mt(1–4) was analyzed by immunoblot as indicated One representative of three independent experiments is shown (top) A quantification of VAMP2 phosphorylation (P-VAMP2) in the presence of PKCf is shown (bottom, left) Results were obtained by densitometric scanning of PhosphoImager signal bands from three independent experiments The PhosphoImager signal of P-VAMP2 was normalized to GST-VAMP2 substrate phosphorylation in the presence of 100 ng recombinant active PKCf ¼ PKCf activity was verified by addition of the PKCf substrate MBP (bottom, right) (B) Flag-VAMP2 was overexpressed in COS-7 cells and subjected to immunoprecipitation with an antibody against the Flag-epitope Immunoprecipitations were subjected to in vitro kinase assay with c-32P-ATP without (lane 1) or with (lane 2–7) increasing amounts of active PKCf (10 ng, 50 ng and 200 ng) PKCf inhibitor (100 lM) was added in lanes 5–7 as indicated VAMP2 phosphorylation and PKCf autophosphorylation were analyzed by PhosphoImager Immunoprecipitation and immunoblot were performed as indicated A quantification of VAMP2 phosphorylation in the presence of PKCf is shown at the bottom Results were obtained by densitometric scanning of PhosphoImager signal bands from three independent experiments The PhosphoImager signal of P-VAMP2 was normalized to Flag-VAMP2 substrate phosphorylation in the presence of 200 ng recombinant active PKCf ¼ (C) COS-7 cells, transiently overexpressing HA-PKCf, were left either unstimulated or stimulated for 10 with 100 ngỈmL)1 EGF-1, 100 nM insulin or 100 ngỈmL)1 IGF-1, as indicated before lysis Lysates were subjected to immunoprecipitation with an antibody against PKCf Immunoprecipitations were subjected to in vitro kinase assay with c-32P-ATP in the presence of GST-VAMP2 VAMP2 phosphorylation and PKCf autophosphorylation were analyzed by PhosphoImager (top) Immunoprecipitation and immunoblot were performed as indicated A quantification of VAMP2 phosphorylation in the presence of PKCf is shown (bottom) The PhosphoImager signal of P-VAMP2 was normalized to GST-VAMP2 substrate phosphorylation in the presence of unstimulated HA-PKCf ¼ Results were obtained by densitometric scanning of PhosphoImager signal bands from three independent experiments (**P < 0.01) PKCf activity was verified by addition of MBP to immunoprecipitated PKCf (right) 1558 FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS T Fritzius et al anti-ProF IgG all three proteins were present in the immunoprecipitates (Fig 4C) The specificities of the three antibodies used in this study were confirmed by western blotting, which did not lead to any significant unspecific detection of unrelated proteins (data not shown) This result also supports the role of ProF as interaction partner for VAMP2 and PKCf for the endogenous proteins in brain tissue VAMP2 is a substrate of PKCf Next, we wanted to find out whether VAMP2 is a substrate of activated PKCf To investigate whether WD-FYVE protein interacts with VAMP2 and PKCf VAMP2 is directly phosphorylated by active PKCf, we generated glutathione S-transferase (GST)-tagged VAMP2 for expression in bacteria Furthermore, we generated a mutant of GST-VAMP2, in which several serine residues were mutated to alanine Out of the six serine residues conserved in mouse and rat VAMP2 (Ser2, Ser28, Ser61, Ser75, Ser80, Ser115) (Fig 1A), we excluded Ser2 from mutation because of its position at the very N-terminus and Ser115, because of its C-terminal position and its location inside the vesicle, which seemed to be an unlikely target for phosphorylation The four remaining serine residues were mutated together to alanine [VAMP2 mt(1–4); Fig 1A] Three FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS 1559 WD-FYVE protein interacts with VAMP2 and PKCf T Fritzius et al of these sites are located within the SNARE motif (Ser61, Ser75 and Ser80) The fourth one is located in the N-terminal sequence (Ser28) and has previously been reported to represent a PKC phosphorylation site in vitro [24] Purified recombinant wild-type GSTVAMP2 and the mutant GST-VAMP2 mt(1–4) were subjected to in vitro kinase assay using recombinant active PKCf, expressed in bacteria, and were subsequently analyzed for the presence of 32P-phosphorylation (Fig 5A) We found that wild-type GST-VAMP2 was specifically phosphorylated by PKCf in a concentration-dependent manner (Fig 5A, lanes and 4), but not by Akt (lane 2), while phosphorylation of GSTVAMP2 mt(1–4) was strongly decreased (70%) when compared with Flag-VAMP2 wild-type (lanes and 7) The activity of PKCf was verified by addition of the PKC substrate myelin basic protein (MBP) This result indicates that VAMP2 is directly phosphorylated by PKCf We further investigated the specificity of the PKCfdependent VAMP2 phosphorylation by means of a PCKf inhibitory peptide For this, we transiently overexpressed Flag-VAMP2 in COS-7 cells After lysis VAMP2 was immunoprecipitated using an anti-Flag IgG and the precipitates were subjected to an in vitro kinase assay using increasing amounts of recombinant active PKCf in the absence (Fig 5B, lane 2–4) or presence (Fig 5B, lane 5–7) of a PKCf inhibitory peptide The precipitates were subjected to SDS ⁄ PAGE and analyzed for radioactive signal by using a PhosphoImager (Molecular Dynamics, Sunnyvale, CA, USA) As can be seen, addition of recombinant active PKCf led to a concentration-dependent substrate phosphorylation of the immunoprecipitated Flag-VAMP2 Furthermore, addition of a PKCf inhibitory peptide decreased PKCf autophosphorylation and abolished the substrate phosphorylation of VAMP2 These data confirm that VAMP2 is specifically phosphorylated by active PKCf in vitro So far we have shown that VAMP2 is a substrate of PKCf Next, we wanted to find out whether VAMP2 could also be phosphorylated by PKCf that is activated by hormonal stimulation of the cells We addressed this question by transient overexpression of HA-PKCf in COS-7 cells These cells were either left unstimulated or were stimulated with 100 ngỈmL)1 epidermal growth factor (EGF)-1, 100 nm insulin, or 100 ngỈmL)1 IGF-1 as indicated in order to activate PKCf Ten minutes later, cells were lysed and HA-PKCf was immunoprecipitated using an anti-PKCf IgG GST-VAMP2 was added to the immunoprecipitates and subjected to an in vitro kinase assay Subsequently the proteins were separated by SDS ⁄ PAGE and analyzed for radioactive 1560 signals using PhosphoImager As can be seen, hormonal stimulation of the cells by epidermal growth factor (EGF-1), insulin and IGF-1 led to strongly increased substrate phosphorylation of recombinant VAMP2 (3.5- to 4.5-fold) and to phosphorylation of the immunoprecipitated PKCf (Fig 5C) The activity of PKCf was verified by addition of a MBP These results further support the idea that VAMP2 phosphorylation depends on activated PKCf ProF increases the PKCf -dependent VAMP2 phosphorylation in vitro In a final experiment, we tested the effect of ProF on the phosphorylation of VAMP2 by PKCf In order to test this, Flag-VAMP2 wild-type (wt) and FlagVAMP2 mt(1–4), were transiently expressed with and without Myc-ProF in COS-7 cells Flag-VAMP2 was immunoprecipitated with an anti-Flag IgG The precipitates were subjected to an in vitro kinase assay using recombinant active PKCf and subsequently analyzed for the presence of 32P-phosphorylation We found that phosphorylation of Flag-VAMP2 wt by active PKCf was slightly (30%) but significantly (P < 0.05) increased in the presence of Myc-ProF (Fig 6, lane and 2) A strongly decreased in vitro 32P-phosphorylation (90%) was found when Flag-VAMP2 mt(1–4) was used as substrate (Fig 6, lane 3), proving the specificity of the substrate phosphorylation The activity of PKCf was verified by addition of MBP In summary, these data indicate that Myc-ProF increases the in vitro phosphorylation of Flag-VAMP2 by activated PKCf Discussion We have previously identified ProF as a molecule that is located on internal vesicles and which preferentially binds to the activated kinases Akt and PKCf upon hormonal stimulation of the cells [1] This raised the question of putative kinase substrates, which might also interact with ProF To address this question, we performed a yeast two-hybrid screen, which indicated VAMP2 as binding partner of ProF We confirmed the physical interaction of VAMP2 and ProF by coimmunoprecipitation of overexpressed and endogenous proteins in both directions VAMP2 is known to be anchored via its transmembrane domain to secretory vesicles in numerous cell lines, where it represents the v-SNARE protein responsible for mediating fusion of vesicles Many vesicle cycling events rely on the interaction of v-SNARE and t-SNARE proteins, which allow docking of vesicles to their target membranes SNARE complex formation is thought to bring the FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS T Fritzius et al WD-FYVE protein interacts with VAMP2 and PKCf Fig Phosphorylation of VAMP2 by PKCf is increased in vitro by ProF Flag-VAMP2 wt and the VAMP2 mutant mt(1–4) were overexpressed either with or without Myc-ProF in COS-7 cells Flag–VAMP2 and Flag–VAMP2–Myc-ProF complexes were obtained by immunoprecipitation with an antibody against the Flag epitope Immunoprecipitations were phosphorylated by addition of 200 ng active recombinant PKCf and c-32P-ATP VAMP2 phosphorylation and PKCf autophosphorylation were analyzed by PhosphoImager, immunoprecipitation and immunoblot were performed as indicated (top, left) Direct lysate shows expression controls (bottom, left) A quantification of PKCf- mediated phosphorylation of VAMP2 in the absence or presence of ProF is shown (top, right) Results were obtained by densitometric scanning of immunoblot bands from three independent experiments The PhosphoImager signal of P-VAMP2 was normalized to Flag-VAMP2 substrate phosphorylation in the absence of ProF ¼ Values represent mean±SD of three separate experiments (*P < 0.05, **P < 0.01) PKCf activity was verified by addition of MBP (bottom, right) opposing membranes close enough for fusion [25] These SNARE-dependent fusion events include a number of secretory processes, such as insulin release from pancreatic b-cells [26–28], synaptic vesicle exocytosis [29], granule release in hematopoetic cells [30], and aquaporin- [31], or GLUT4 translocation to the plasma membrane [11,32] In general, secretory events are regulated by a variety of mechanisms including phosphorylation of SNARE and accessory proteins [29] In adipocytes and skeletal muscle cells, VAMP2 has been described to bind to the t-SNARE proteins syntaxin-4 and SNAP-23, found at the plasma membrane [33,34], whereas in neurons VAMP2 interacts with syntaxin-1 and SNAP-25 at the plasma membrane for neurotransmitter release [14,15] These findings highlight the importance of VAMP2 in a number of secretory systems In this study, we showed that ProF can act in an adaptor protein-like fashion to mediate the interaction between PKCf and VAMP2 ProF, VAMP2, and PKCf partially colocalized on vesicular structures and formed a complex The contribution of additional proteins to the formation of this complex cannot be excluded at the moment Furthermore, because ProF is able to form oligomers [1], it is possible that one ProF molecule is not simultaneously interacting with VAMP2 and PKCf, but instead that different ProFs may individually bind to VAMP2 and PKCf Further studies using mutants of ProF will investigate this question Finally, we hypothesized that ProF may be important for the phosphorylation of VAMP2 We found that VAMP2 can be phosphorylated by activated PKCf in vitro and that the presence of ProF increased the PKCf- dependent VAMP2 phosphorylation These data support and expand earlier studies, which showed that insulin-stimulated or overexpressed PKCf induced serine phosphorylation of GLUT4 vesicle-associated VAMP2 in vivo in rat myotubes, while expression of dominant-negative PKCf completely abolished VAMP2 phosphorylation [8] Furthermore, it has been shown that PKCf specifically associated with a GLUT4- and VAMP2-positive cellular compartment, and that overexpression of PKCf led to GLUT4 translocation to the plasma membrane and increased glucose uptake even in the absence of insulin stimulation [8] Based on these studies, it is conceivable that the PKCf-mediated VAMP2 phosphorylation affects the fusion of vesicle with the plasma membrane It is currently unknown if the PKCf-dependent phosphorylation of VAMP2 influences the interaction of the v-SNARE protein with its cognate t-SNAREs or with accessory proteins Whether the PKCf-dependent phosphorylation of VAMP2 decreases or increases, the interaction between the v-SNARE and t-SNARE proteins and how this phosphorylation might regulate vesicle cycling should be investigated in future studies We specified four serine residues within the VAMP2 molecule as potential phosphorylation sites and FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS 1561 WD-FYVE protein interacts with VAMP2 and PKCf T Fritzius et al mutated them to alanine Three of the four mutated serine residues were found within the highly conserved SNARE motif of VAMP2 This motif fits to the SNARE motifs of syntaxin-4 and SNAP-23 and would allow a twisted, parallel 4-helical bundle [35] As the driving forces for the generation of the helical bundle are mostly hydrophobic interactions, one or several highly polar phosphorylated serine residues could disturb the formation of the bundle However, several reports showed that decreased binding of SNARE proteins to each other could lead to an increased fusion of vesicles For example, SNAP-25 is phosphorylated by PKC at Ser187, which lies within the C-terminal SNARE motif [36–38] Activation of PKC by various agents resulted in phosphorylation of SNAP-25 [37] This phosphorylation decreased binding of SNAP-25 to syntaxin-1 and increased neurotransmitter release, possibly by accelerating the SNARE complex dissociation and thus enhancing the rate of vesicle cycling [37] Thus, PKCf-mediated phosphorylation of VAMP2 in response to stimulation of the cells, as shown here, may affect the interaction of the v-SNARE protein VAMP2, thereby influencing vesicle trafficking Another possibility would be that the phosphorylation of VAMP2 by PKCf influences its interaction with accessory proteins, which could up- or down-regulate SNARE–SNARE protein interactions Our results show that ProF stimulates phosphorylation of the SNARE protein by PKCf in vitro, possibly by recruitment of PKCf to VAMP2 This could be a possible mechanism by which PKCf influences vesicle trafficking A recent publication supports our idea of ProF as a regulator of vesicle trafficking Knockdown of the protein using small interfering (si)RNA was reported to affect vesicle cycling and to inhibit endocytosis in mammalian cells, as well as in the nematode Caenorhabditis elegans [39] As the expression profile of ProF suggested a broad tissue distribution, we hypothesize that a complex of ProF, VAMP2 and PKCf may occur in various tissues and may be involved in several secretory pathways Experimental procedures Antibodies and reagents Antibodies against Myc-epitope (A14, rabbit polyclonal and 9E10, mouse monoclonal), against HA (Y-11, rabbit polyclonal), PKCa (C-20, rabbit polyclonal), PKCd (C-17, rabbit polyclonal), PKCe (C-15, rabbit polyclonal), PKCf (C-20, rabbit and goat polyclonal), and PKCf-blocking peptide were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Insulin was from Novo Nordisk 1562 (Bagsvaerd, Denmark), the antibody against the GFPepitope (632377, rabbit polyclonal) was derived from BD Biosciences (San Jose, CA, USA), and the antibody against the GST-epitope (goat polyclonal) was from Amersham Pharmacia (Piscataway, NJ, USA) The mouse monoclonal and the rabbit polyclonal antibodies to VAMP2 were from Synaptic Systems (Goettingen, Germany) and ABR Affinity Bioreagents (Golden, CO, USA), respectively The antibody to Flag-epitope (M2, mouse monoclonal), EGF-1, and MBP were from Sigma (St Louis, MO, USA) IGF-1 was from Calbiochem Signal Transduction (La Jolla, CA, USA) A polyclonal peptide antibody directed against the 15 C-terminal amino acids of ProF was raised in rabbits and affinity purified on the peptide used for immunization [1] This peptide was also used for peptide competition in endogenous interaction analysis during immunoprecipitation or, later, during western blotting All secondary antibodies for western blotting and indirect immunofluorescence staining were from Amersham Pharmacia and Jackson Immuno Research (West Grove, PA, USA), respectively Yeast two-hybrid analysis A human B-cell-specific cDNA library was obtained from S J Elledge (Baylor College of Medicine, Houston, TX, USA) [40] The yeast two-hybrid analysis was performed essentially as described using full-length ProF as a bait [17] Recombinant DNA manipulation and plasmid constructs Serine to alanine point mutations were inserted into the coding sequence of Flag-VAMP2 using the Quick Change Mutagenesis Kit (Stratagene, La Jolla, CA, USA) Plasmid pEF-Flag-VAMP2 was used as a template The primers for mutagenesis were obtained from Microsynth (Balgach, Switzerland), mutation (Ser28Ala): forward, cca aac ctt act gct aac agg aga ctg, reverse, cag tct cct gtt agc agt aag gtt tgg; mutation (Ser61Ala): forward, gac cag aag ttg gcg gag ctg gat gac, reverse: gtc atc cag ctc cgc caa ctt ctg gtc, mutation (Ser75Ala): forward, gca ggg gcc gcc cag ttt gaa, reverse: ttc aaa ctg ggc ggc ccc tgc, mutation (Ser80Ala): forward, cag ttt gaa aca gct gca gcc aag ctc, reverse, gag ctt ggc tgc agc tgt ttc aaa ctg The inserted mutation is in bold in the forward primer Mutants containing four single and one mutant harboring all four mutations were constructed All mutations were verified by DNA sequencing N-Terminally Myc-tagged human ProF encoding constructs were described earlier [1]: Myc-ProF, MycProFDFYVE lacking the FYVE domain for phosphatidylinositol-3-phosphate binding, Myc-ProF 4–7 lacking blades 1–3 and the FYVE domain, and Myc-ProF 1–3 lacking blades 4–7 and the FYVE domain were used in this study FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS T Fritzius et al A HA-tagged PKCf construct was obtained from F J Johannes (Stuttgart, Germany) The plasmid encoding Flag-VAMP2 has previously been described [41] and was kindly provided by M Fukuda (RIKEN, Saitama, Japan) Cloning and expression of GST-VAMP2 The coding sequence of VAMP2 and VAMP2 mt(1–4) was excised from a pEF-plasmid containing Flag-tagged VAMP2 (kindly provided by M Fukuda, Saitama, Japan) using BamHI and EcoRI restriction enzyme sites and cloned into the pGEX-6p-2 vector (Amersham Biosciences) for expression of GST-tagged VAMP2 and VAMP2 mt(1–4) in transformed BL21 + (Invitrogen, Carlsbad, CA, USA) Escherichia coli cells Colonies were grown overnight in 20 mL of LB medium at 37 °C LB medium (400 mL) was added to this preculture and bacteria were grown at 28 °C until the A600 reached 0.6 Addition of 0.1 mm isopropyl b-d-thiogalactoside to the bacterial culture, which was further grown at 28 °C for another h, induced protein expression After h cells were collected by centrifugation at 6000 g and °C in a Sorvall RC-5B superspeed centrifuge using a Sorvall GSA rotor (Sorvall Instruments Inc., Newton, CT, USA) For extraction of recombinant proteins, 10 mL of sucrose, Tris-HC1, and EDTA (STE)-buffer was added to the bacterial pellets, containing 20 mm Tris ⁄ HCl, pH 7.5, 300 mm NaCl, mm EDTA, pH 8.0, lgỈmL)1 aprotinin, lgỈmL)1 leupeptin, and complete EDTA-free protease inhibitor tablets (Roche Diagnostics, Basel, Switzerland) Resuspended cells were lysed by two freeze–thaw cycles in liquid nitrogen, followed by three cycles of sonification (Branson sonifier 250) on ice The bacterial cell lysate was thereafter incubated with 100 lL dry volume of glutathione-Sepharose beads (Amersham Biosciences) for h at °C in a spinning wheel Beads were washed twice with wash buffer, containing 50 mm Tris ⁄ HCl, pH 7.5, 250 mm NaCl, lgỈmL)1 aprotinin, lgỈmL)1 leupeptin, and complete EDTA-free protease inhibitor tablets, to remove unbound material GST-tagged proteins were eluted from beads by addition of three times 200-lL fractions of elution buffer, containing 50 mm Tris ⁄ HCl, pH 7.5, 100 mm NaCl, and 20 mm reduced glutathione by vigorous shaking for 20 at 15 °C Fractions were pooled, dialyzed using Centricon centrifugal filter devices (Millipore, Bedford, MA, USA), aliquoted, and stored at )20 °C Retroviral transduction and generation of stably transduced 3T3-L1 fibroblasts Retroviruses containing the construct pRTP-Myc-ProF or the empty pRTP vector as control were produced using the BOSC-23 packaging cell-line as described [42,43] Early passage 3T3-L1 fibroblasts were incubated in virus-containing medium for 48 h The cells were used for immunofluorescence studies WD-FYVE protein interacts with VAMP2 and PKCf Immunoprecipitation For transient expression, the simian kidney-derived cell line COS-7 (ATCC CRL-1651) and the human embryonic kidney cell line 293T (ATCC CRL-11268) were transfected with expression vectors encoding the indicated proteins using Lipofectamine 2000 (Invitrogen) [1] Cells were lysed using a lysis buffer containing 100 mm NaCl, mm EDTA, 20 mm Tris ⁄ HCl, and 0.5% NP-40 (NETN) [1] including lgỈmL)1 aprotinin, lgỈmL)1 leupeptin, and complete EDTA-free protease inhibitor tablets (Roche Medicals) and were cleared by centrifugation for 10 at 16 000 g and °C in a 5415 R refrigerated benchtop centrifuge from Ependorf (Hamburg, Germany) Interactions of endogenous proteins were analyzed in mouse brain extracts Proteins were extracted in an extraction buffer containing 20 mm Tris ⁄ HCl, pH 7.5, 150 mm NaCl, mm EDTA, 0.2% Nonidet NP-40 and supplemented with protease inhibitor tablets, by incubating the lysates at °C for 30 with vigorous shaking Glycerol was added to a final concentration of 10% after clearing of lysates Protein concentrations were determined by Bradford assay in a microtiterplate reader at 595 nm using bovine serum albumin as standard Lysates were precleared for h using 30 lL Protein-ASepharose (Amersham Pharmacia Biotech) Cleared lysates were immunoprecipitated with lg of the appropriate antibody for at least h at °C and then for h in the presence of 10 lL of protein G-sepharose (Amersham Pharmacia Biotech) Immunoprecipitation of endogenous ProF and VAMP2 was conducted overnight at °C For immunoprecipitation of endogenous VAMP2 bringing down ProF, the anti-VAMP2 IgG (ABR Affinity Bioreagents) was covalently coupled to protein G-sepharose in order to reduce the signal of the IgG antibody heavy chain as described earlier [1] In brief, 10 lL of protein G-sepharose (Amersham) were incubated with lg of anti-ProF serum in 500 lL of washing-binding buffer (all buffers were from Pierce, Rockford, IL, USA) for 30 at room temperature, followed by washing twice with this buffer and incubating in 260 lL of cross-linking buffer with 850 lL of freshly added dimethylpimelidate Thereafter, protein G-sepharose was incubated for h at room temperature and washed twice with cross-linking buffer, incubated for 10 with blocking buffer at room temperature, washed twice with blocking buffer, washed three times with elution buffer, before boiling the samples for at 95 °C For competition studies, lg anti-ProF IgG was preincubated with 10 lg peptide on ice for 30 for competition in immunoblot and immunoprecipitate For studying the protein complexes, elution of overexpressed Myc-ProF and HA-PKCf was performed by vigorous shaking for h at 24 °C, followed by two rounds of shaking for 45 at °C in 150 lL of NETN buffer containing 900 lgỈlL)1 of C-Myc-peptide (Roche) or PKCf FEBS Journal 274 (2007) 1552–1566 ª 2007 The Authors Journal compilation ª 2007 FEBS 1563 WD-FYVE protein interacts with VAMP2 and PKCf T Fritzius et al (C-20) blocking peptide (Santa Cruz) After elution with C-myc, one-half of the eluate was used for immunoprecipitation against HA-PKCf using lg of anti-PKCf (C-20) IgG (Santa Cruz) The other half of the eluate was used for immunoprecipitation against Flag-VAMP2 using lg of anti-Flag (M2) IgG (Roche) After elution with PKCf blocking peptide, one half of the eluate was used for immunoprecipitation against Myc-ProF using lg of anti-Myc (9E10) IgG The other half of the eluate was used for immunoprecipitation against Flag-VAMP2 using lg of anti-Flag (M2) IgG Lysates and the resulting immunoprecipitates were resolved using commercial 10–20% SDS ⁄ PAGE (Invitrogen) The primary and secondary antibodies were used at a dilution of : 10 000 Western blotting of endogenous ProF was performed overnight at °C with a : 1000 dilution of the antibody All incubation and wash steps were performed in 1% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 Detection was performed using a chemiluminescence detection kit (Amersham Pharmacia Biotech) Confocal microscopy COS-7 cells were grown on glass cover slips and were transiently transfected with fugene-6 (Roche Medicals) according to the manufacturers’ instructions Twenty hours after transfection, cells were washed with NaCl ⁄ Pi, fixed with 3% paraformaldehyde and permeabilized in NaCl ⁄ Pi containing 0.25% Triton X-100 3T3-L1 cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in NaCl ⁄ Pi Afterwards, the cover slips were successively incubated with appropriate primary and secondary antibodies before they were mounted in Mowiol (Hoechst Pharmaceuticals, Frankfurt, Germany) The cells were examined by sequential excitation at 488 nm (fluorescein isothiocyanate), 568 nm (tetramethylrhodamine isothiocyanate) and 633 nm (cyanine-5) using a confocal microscope (SP2, Leica, Wetzlar, Germany) and a 40 · 1.25 oil objective (Leica) The images were processed by using photoshop (Adobe Systems, San Jose, CA, USA) In vitro kinase assays Flag-VAMP2 was transiently expressed in the presence or absence of Myc-ProF or HA-PKCf in COS-7 cells Cells were lysed as described above and lysates were immunoprecipitated using an anti-Flag IgG Immunocomplexes were washed in a kinase reaction buffer (20 mm Tris ⁄ HCl, pH 7.5, 20 mm MgCl2, mm dithiothreitol) For the kinase reaction, the buffer was supplemented with lm protein kinase A inhibitor, 20 lm ATP, and 10 lCi c-32P-ATP with a specific activity of 3000 mCiỈmol)1 (Amersham Pharmacia) The reaction was started with addition of the indicated amounts of recombinant active PKCf (Upstate 1564 Biotechnology, Lake Placid, NY, USA) to the samples The phosphorylation reaction was conducted at 30 °C for 30 and was stopped by boiling the samples in SDS ⁄ PAGE loading buffer For Fig 5C, the reaction was started without or with addition of 100 lm of PKC pseudosubstrate inhibitory peptide II (Calbiochem) In all cases, the activity of overexpressed or recombinant active PKCf was verified by addition of 0.5 lg of the PKC substrate myelin basic protein (Sigma) In vitro kinase experiments with GST-tagged proteins were performed by addition of lg of GST-VAMP2 and GST-VAMP2 mt(1–4) to 30 lL of the kinase reaction buffer (20 mm Tris ⁄ HCl, pH 7.5, 20 mm MgCl2, mm dithiothreitol), followed by addition of 200 ng PKCf For Fig 5A, lg of active recombinant Akt1 (Upstate), or 100 or 200 ng of PKCf were added to the reaction buffer and the kinase assay was thereafter performed as described above For the PKCf activity experiments, HA-PKCf was transiently expressed in COS-7 cells, which were left either unstimulated or stimulated for 10 with 100 ngỈmL)1 EGF-1 (Sigma), 100 nm insulin (Novo Nordisk) or 100 ngỈmL)1 IGF-1 (Calbiochem) before lysis as described above Lysates were immunoprecipitated using an anti-PKCf IgG, immunocomplexes were washed in kinase reaction buffer and subjected to in vitro kinase assay as described above Samples were resolved on 10–20% SDS ⁄ PAGE Expression of Flag-VAMP2 and Myc-ProF was verified by western blotting and phosphorylation was visualized using a Storm 840 PhosphoImager Statistical analysis Statistical analysis was performed using students t-test (*P < 0.05, **P < 0.01) Acknowledgements The authors thank Dr M Fukuda (Saitama, Japan) for kindly providing us the plasmid encoding FlagVAMP2, F J Johannes (Stuttgart, Germany) for the plasmid encoding HA-tagged PKCf, and B Schaub and J Rohrer (Zurich, Switzerland) for the plasmids encoding HA-Hrs and GFP-Vps4 The authors also thank the Electron Microscopy Center (EMZ) at the University of Zurich for providing the facilities for confocal microscopy and for expert help Thanks go to S Dettwiler and C Hagedorn for excellent technical assistance E Haas (Zurich, Switzerland) and G Burkard (Berne, Switzerland) were very 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interaction between VAMP2 and PKCf In order to test this, we expressed Flag -VAMP2 and HA-PKCf in... FEBS 1557 WD-FYVE protein interacts with VAMP2 and PKCf T Fritzius et al Endogenous VAMP2 interacts with ProF and PKCf Fig Interactions of endogenous VAMP2 with ProF and PKCf in brain (A) Immunoprecipitation... threonine kinases Akt and PKCf, and was located on internal vesicles via its FYVE domain These two kinases preferentially bound to ProF after WD-FYVE protein interacts with VAMP2 and PKCf hormonal