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

VAMP2 can be phosphorylated by activated PKCf in vitro and the pres-ence of ProF increases the PKCf-dependent phosphorylation of VAMP2 in vitro.. B Coimmunoprecipitation assay of overexp

protein kinase Cf

Thorsten Fritzius, Alexander D Frey*, Marc Schweneker, Daniel Mayerand Karin Moelling

Institute of Medical Virology, University of Zurich, Switzerland

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,

provi-ding a protein-binprovi-ding 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 1 (IGF-1) [1] Because of this stimulation-dependent bind-ing 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

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

Department of Virology, Institute for

Medical Microbiology and Hygiene,

University of Freiburg, Germany

(Received 20 December 2006, accepted 16

January 2007)

doi:10.1111/j.1742-4658.2007.05702.x

We have recently identified a protein, consisting of seven WD repeats, pre-sumably 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 2 (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 pres-ence 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

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, target-SNARE; VAMP2, vesicle-associated membrane protein 2; Vps4, vacuolar protein sorting-associating protein 4; v-SNAREs, vesicular-SNARE.

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 4 (GLUT4) [4] The Akt substrate

of 160 kDa affects the trafficking of

GLUT4-contain-ing vesicles to the plasma membrane upon Akt

phos-phorylation [5–7] Several PKCf substrates on vesicles

have been described previously The vesicle-associated

membrane protein 2 (VAMP2) may be one of them

[8] VAMP2 belongs to the vesicular soluble

N-ethyl-maleimide-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

tis-sues, such as brain, kidney, adrenal gland, liver and

pancreas [10] VAMP2 is crucial for

stimulus-depend-ent secretion in various cell-types including

insulin-stimulated 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

VAMP2-containing vesicles with the plasma membrane is

medi-ated 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

over-expressing PKCf, which correlated with increased

GLUT4 translocation and glucose uptake [8]

Previous analyses have shown that ProF binds

spe-cifically 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

con-centration-dependent manner Furthermore, we show

that VAMP2 is directly phosphorylated by activated

PKCf in vitro and that ProF leads to increased

phos-phorylation 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-propel-ler-type structure, and a FYVE domain (Fig 1A),

des-ignated 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

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 vesicu-lar membranes via its C-terminal transmembrane domain Its central SNARE domain of 60 amino acids allows the interaction of VAMP2 with its cog-nate 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 two-hybrid screen, we first analyzed the interaction of VAMP2 with ProF by coimmunoprecipitation of over-expressed 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 copre-cipitating proteins As can be seen, Myc-tagged ProF indeed coprecipitated with Flag-tagged VAMP2 (Fig 1B, left, lane 3) and Flag-tagged VAMP2 copre-cipitated 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 FYVE-domain containing protein that is also localized to intracellular vesicles [18,19], or with the vacuolar pro-tein sorting-associating propro-tein 4, Vps4 (Fig 1B, left, lane 7), a protein involved in intracellular vesicle for-mation 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 specif-icity of the interaction of ProF with VAMP2

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

lack-ing the FYVE domain and containlack-ing only blades 1–3

(ProF 1–3) or blades 4–7 (ProF 4–7) of the

seven-bladed b-propeller (Fig 1A) Interaction of trun-cated 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

Fig 1 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 Immunopre-cipitation was performed with an antibody against the Flag-tag (left) or the Myc-tag (right) and immunoprecipitates were analyzed by immu-noblotting with antibodies against Flag, Myc, HA and GFP epitopes Direct lysates (DL) are shown as expression control (bottom) (C) Myc-ProF wild-type (wt), Myc-ProF lacking the FYVE domain (Myc-Myc-ProFDFYVE), Myc-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 immu-noblot show the interactions.

COS-7 cells and tested their interaction by

coimmuno-precipitation assays As can be seen, all ProF mutants

interacted equally well with VAMP2 (Fig 1C) In

sum-mary, 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

analy-sis showing their subcellular distribution For that,

Flag-tagged VAMP2 and Myc-tagged ProF were

coex-pressed in COS-7 cells and analyzed by confocal

micro-scopy As can be seen, a partial colocalization of

VAMP2 (green signal) and ProF (red signal) on vesicu-lar structures was detectable (Fig 2A) Colocalization

of the two proteins is indicated by the orange color, detectable in the merged picture, showing the super-position 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 confo-cal microscopy Flag-tagged VAMP2, hemagglutinin (HA)-tagged PKCf and Myc-tagged ProF were transi-ently 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 2 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

colocali-zation as visualized in yellow on the merged

picture (right) (B) COS-7 cells were

transi-ently transfected with HA-PKCf (green),

Flag-VAMP2 (red), and Myc-ProF (blue) and

analyzed by confocal microscopy using

anti-bodies against HA, Flag, and Myc epitopes.

The lower part shows detail A partial

colo-calization on cytoplasmic punctuate

struc-tures (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

spe-cific structures, while HA-PKCf (green) and

is distributed throughout the cell (D)

Confo-cal 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

immunofluores-cence analysis of 3T3-L1 pre-adipocytes,

cells were serum-starved for 2 h prior to

staining for endogenous PKCf (green),

endogenous VAMP2 (red), and stably

expressed Myc-tagged ProF (magenta) A

partial colocalization on cytoplasmic

punctu-ate structures (white) is observed (merged).

Lower part shows detail.

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

vesi-cles, 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

vesi-cles, 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

local-ization of the kinase on punctuate structures We

fur-ther substantiated these data by confocal microscopy

studies with 3T3-L1 pre-adipocyte cells, stably

expres-sing Myc-tagged ProF As can be seen, Myc-ProF,

endogenous VAMP2, and endogenous PKCf

colocal-ized on perinuclear vesicular structures (Fig 2E)

These results are in agreement with the previous

find-ings 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

Flag-VAMP2 and Flag-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

immunoprecip-itated Myc-ProF and showed coprecipitation of

Flag-VAMP2 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

immunoprecipitation of HA-PKCf As can be seen, the coimmunoprecipitation of Myc-ProF and Flag-VAMP2 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 Myc-ProF (Fig 3B, lane 4) Additionally, reciprocal immu-noprecipitation 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 there-after eluted with a PKCf-peptide and Myc-ProF (Fig 3C, lane 3) or Flag-VAMP2 was immunoprecipi-tated (Fig 3C, lane 4) A strong coimmunoprecipita-tion of HA-PKCf and a weak coimmunoprecipitacoimmunoprecipita-tion

of Flag-VAMP2 in the case of Myc-ProF (Fig 3C, lane 3) can be demonstrated by western blotting Fur-thermore, we found coimmunoprecipitation of HA-PKCf 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, Flag-VAMP2 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 HA-PKCf 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 7 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 (3.5-fold) 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 adap-tor protein-like fashion It increases the binding of PKCf to VAMP2 under optimized conditions, which

is a characteristic of adaptor proteins

Fig 3 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 Inter-action 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 immunoprecipita-tion with an anti-Myc IgG (lane 2) The complex was eluted by addiimmunoprecipita-tion of an excess of a competing Myc-peptide followed by immunoprecip-itation using an antibody directed against PKCf (lane 3) or the Flag-epitope (lane 4) Immunoprecipimmunoprecip-itations 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 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 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, 1 lg, and 4 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 1 Values represent mean± SD of three separate experiments (*P < 0.05, **P < 0.01).

Endogenous VAMP2 interacts with ProF and PKCf

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 endo-genous 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, coimmunoprecipi-tation 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 per-formed 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 competi-tion during western blotting (Fig 4B, lane 3) and immunoprepitation performed with an irrelevant anti-Myc 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 endo-genous 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 4 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

(anti-Myc, lane 1), and anti-VAMP2 IgG (lane 2 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.

Fig 5 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- 32 P-ATP in the presence of 3 lg of recombinant act-ive Akt (lane 2), 100 ng recombinant actact-ive PKCf (lanes 3 and 5) and 200 ng recombinant actact-ive PKCf (lanes 4 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 1 PKCf activity was verified by addition of the PKCf sub-strate 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 l M ) was added in lanes 5–7 as indicated VAMP2 phos-phorylation and PKCf autophosphos-phorylation were analyzed by PhosphoImager Immunoprecipitation and immunoblot were performed as indi-cated 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 1 (C) COS-7 cells, transiently overexpressing HA-PKCf, were left either unstimulated or stimulated for 10 min with 100 ngÆmL)1EGF-1, 100 n M insulin or 100 ngÆmL)1IGF-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- 32 P-ATP in the presence of GST-VAMP2 VAMP2 phosphorylation and PKCf autophosphorylation were analyzed by Phos-phoImager (top) Immunoprecipitation and immunoblot were performed as indicated A quantification of VAMP2 phosphorylation in the pres-ence 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 1 Results were obtained by densitometric scanning of PhosphoImager signal bands from three inde-pendent experiments (**P < 0.01) PKCf activity was verified by addition of MBP to immunoprecipitated PKCf (right).

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

sub-strate of activated PKCf To investigate whether

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 posi-tion 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 phosphoryla-tion The four remaining serine residues were mutated together to alanine [VAMP2 mt(1–4); Fig 1A] Three

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

GST-VAMP2 and the mutant GST-GST-VAMP2 mt(1–4) were

subjected to in vitro kinase assay using recombinant

active PKCf, expressed in bacteria, and were

subse-quently analyzed for the presence of 32

P-phosphoryla-tion (Fig 5A) We found that wild-type GST-VAMP2

was specifically phosphorylated by PKCf in a

concen-tration-dependent manner (Fig 5A, lanes 3 and 4), but

not by Akt (lane 2), while phosphorylation of

GST-VAMP2 mt(1–4) was strongly decreased (70%) when

compared with Flag-VAMP2 wild-type (lanes 6 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

PKCf-dependent VAMP2 phosphorylation by means of a

PCKf inhibitory peptide For this, we transiently

over-expressed 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

pres-ence (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

PhosphoI-mager (Molecular Dynamics, Sunnyvale, CA, USA)

As can be seen, addition of recombinant active PKCf

led to a concentration-dependent substrate

phosphory-lation of the immunoprecipitated Flag-VAMP2

Fur-thermore, 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

activa-ted 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 ngÆmL)1epidermal growth

factor (EGF)-1, 100 nm insulin, or 100 ngÆmL)1IGF-1

as indicated in order to activate PKCf Ten minutes

later, cells were lysed and HA-PKCf was

immunopre-cipitated 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

signals using PhosphoImager As can be seen, hormo-nal stimulation of the cells by epidermal growth fac-tor 1 (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 phos-phorylation 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, VAMP2 wild-type (wt) and Flag-VAMP2 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 preci-pitates were subjected to an in vitro kinase assay using recombinant active PKCf and subsequently analyzed for the presence of32P-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 1 and 2) A strongly decreased in vitro 32 P-phosphoryla-tion (90%) was found when Flag-VAMP2 mt(1–4) was used as substrate (Fig 6, lane 3), proving the spe-cificity 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 coimmu-noprecipitation of overexpressed and endogenous pro-teins 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 inter-action of v-SNARE and t-SNARE proteins, which allow docking of vesicles to their target membranes SNARE complex formation is thought to bring the

opposing membranes close enough for fusion [25].

These SNARE-dependent fusion events include a

num-ber 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

mem-brane [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

pro-teins to the formation of this complex cannot be

exclu-ded at the moment Furthermore, because ProF is able

to form oligomers [1], it is possible that one ProF

mole-cule is not simultaneously interacting with VAMP2 and

PKCf, but instead that different ProFs may

individu-ally bind to VAMP2 and PKCf Further studies using

mutants of ProF will investigate this question

Finally, we hypothesized that ProF may be import-ant 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 trans-location to the plasma membrane and increased glu-cose 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 phosphory-lation 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 pro-teins 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

Fig 6 Phosphorylation of VAMP2 by PKCf is increased in vitro by ProF Flag-VAMP2 wt and the VAMP2 mutant mt(1–4) were over-expressed either with or without Myc-ProF in COS-7 cells Flag–VAMP2 and Flag–VAMP2–Myc-ProF complexes were obtained by immuno-precipitation with an antibody against the Flag epitope Immunoimmuno-precipitations were phosphorylated by addition of 200 ng active recombinant PKCf and c- 32 P-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- medi-ated 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 sub-strate phosphorylation in the absence of ProF ¼ 1 Values represent mean± SD of three separate experiments (*P < 0.05, **P < 0.01) PKCf activity was verified by addition of MBP (bottom, right).