Supplemental Data
Results
The h-prune-nm23 interaction has been conserved throughout evolution.
In silico searches of genome databases, and the translation of several ESTs in the X.
tropicalis and D. rerio EST databases that match sequences of the h-prune protein,
revealed that the aspartate residues that correspond to the “signature” of the four domains
characteristic of the DHH phosphoesterases superfamily are conserved (Fig. 1S-E), thus
suggesting that the core structure is present in other species. Due to the high percentage
of identity between nm23-H1 and nm23-H2 and their orthologs found in different species
(80% to 92%), we wanted to determine if the h-prune-nm23 complex has been retained
throughout evolution. We used transient transfections of COS-7A cells for the
overexpression of wild-type h-prune (human) and nm23 isolated from D. rerio (nm23Z1; AAF20910) and X. laevis (nm23-X1; X97902.1) (Ouatas et al., 1997) by means of
cDNA/protein expression, with subsequent co-immunoprecipitation. The nm23 proteins
were thus cloned from extracts of unfertilized eggs of X. laevis and from embryo extracts
of D. rerio 48 h after fertilization (Fig. 1S-A, B). Within the same strategy, we also
isolated the nm23 protein homologs from C. elegans and D. melanogaster. The results
obtained indicated that h-prune binds nm23-X2 and nm23-Z1; conversely, h-prune does
not bind the nm23 proteins obtained from the C. elegans and D. melanogaster cDNAs
(Fig. 1S-A-D). Of note, these last two proteins both lack the serine at position 122, as
shown by multiple alignment using ClustalW software (Fig. 1B). We additionally show
1
that the h-prune-nm23 interaction was impaired with nm23-H1-S125A (Fig. 2S-A, B),
nm23-H1-S120G (Reymond et al. 1999) and nm-23H1-S122P (Supplemental data, Fig.
2S-C, D), thus indicating that the region between N115 and E127 is important for the hprune-nm23 interaction.
These results were produced both by transient transfection of cDNA in pCDNA
vectors in COS-7A cells and by co-immunoprecipitation, thus confirming that the most
conserved and externally exposed nm23-H1 region shows impaired the binding with hprune once mutated and is a potential site for phosphorylation by CKI, as reported in the
Results section.
Influence of phosphorylation by CKI on nm23 NDPK activity
As it has been reported that CKII can phosphorylate nm23-H1 and nm23-H2 and
negatively affect their NDPK activities (Biondi et al., 1996), we asked if the
phosphorylation of these residues by CKI is also able to affect their NDPK activities.
Figure 3S shows that in contrast with what was seen by Biondi et al. (1996), the
phosphorylation of nm23-H1 and nm23-H2 by CKI, with both of these proteins
expressed in E. coli, increased their NDPK activities by about 20% in each case. This
thus suggests that the residues phosphorylated by CKI are different from those modified
by CKII, due to these different effects on NDPK activity. We also tested this with the
mutants (S120, S122, S125), where there was no influence on their NDPK activities,
confirming also that the S120 residue should be important only for the downstream
phosphorylation of S122 and S125. Of note, the NDPK activity could be excluded as the
determinant for the interaction of nm23 with h-prune because the S120 and S122 mutants
2
are known to show impaired interactions with h-prune (Reymond et al., 1999) (and
hereby presented as supplemental data), while retaining their NDPK activities (Kim et al.,
2003), although the latter showned a mild alteration to its kinetics (Schaertl et al., 1999)
IC261 and the competitive permeable peptide alter cellular localization of h-prune
in breast cancer cells.
To determine whether IC261 and the competitive permeable peptide have any effects on
the morphology or cellular subcompartment localization of nm 23 and h-prune in breast
cancer
cells
through
their
ability
to
inhibit
cell
motility,
we
performed
immunofluorescence analyses of the MDA-MB-435-c100 wild-type cells and the clones
overexpressing h-prune, the MDA-MB-h-prune #4 (D'Angelo & Zollo, 2004). The results
shown in Figure 4S show that treatment with both IC261 (Fig. 4S-A) and the competitive
peptide (Fig. 4S-B), the localization of h-prune is changed to a subcellular compartment
just below the plasma membrane, where it co-localized with F-actin (Fig. 4S). The nm23
staining using the unpurified K73 antibody was seen as diffused nuclear and cytoplasmic
in the MDA-MB-h-prune #4 IC261-treated cells, as was seen for the untreated cells. The
non-complete co-localization with h-prune in the untreated cells appears to be due to
recognition by the antibody of both nm23 isoforms (nm23-H1 and nm23-H2), as it is
known that nm23-H2 localizes preferentially to the nucleus (Kraeft et al., 1996). Thus
nm23-H1 and h-prune co-localize in untreated cells, and this co-localization appears to be
partially impaired in the presence of IC261. Therefore, we believe that the lowered PDE
activity of h-prune in cells treated with IC261 is supported by a further role for h-prune in
this alternative cellular compartment, where it co-localizes with F-actin. This is not
3
surprising considering the recent findings of h-prune co-localization with GSK3- in
focal adhesions in colon cancer cells (Kobayashi et al., 2006) and the recent identification
of another h-prune protein partner, gelsolin, an ATP-dependent actin polymerization and
severing protein (Garzia et al., 2006).
Material and Methods
Cell culture, transfection and immunoprecipitation
COS-7A cells and MDA-435 c100 and the h-prune MDA-435 stable clone #3 and #4
cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine
serum, at 37 °C in 5% CO2; the stable clones were supplemented with 2 g/ml
puromycin,. Except where specifically indicated, the standard treatment with IC261 was
at a final concentration of 50 M for 8 h; all of the “untreated” controls were treated with
the DMSO solvent alone. CKI-7 (Sigma-Aldrich) and D4476 (Calbiochem) were used at
50 and 400 M for 8 h, unless otherwise indicated. Compound C (Calbiochem) and
KT5720 (Sigma-Aldrich) were used at 20 M and 10 M, respectively, and DMAT (a
kind gift from L.A. Pinna) was used at 10 M. Treatment with the competitive or
scrambled peptides were performed with 1 mM of each for 12 h. Preparative
immunoprecipitations for the mass spectrometry analysis were performed using COS-7A
cells (2 x 107) transfected with 20 g plasmid DNA using the Polyfect system
(Invitrogen); for standard co-immunoprecipitation experiments, 3 x 106 cells were used
for each construct. The cells were harvested 48 h after transfection and the total cell
lysates were obtained with the standard protocol of lysing the cells in 20 mM Tris-HCl, 2
4
mM MgAc, 0.3 mM CaCl2, 1 mM DTT, 2 g/ml pepstatin, 2 g/ml aprotinin, 2 g/ml
leupeptin, 1 mM NaF, 1 mM NaVO3. The immunoprecipitation was performed with antiHA, anti-FLAG monoclonal antibodies (Sigma and Roche, respectively), or a polyclonal
A59 anti-h-prune antibody, in quantities of 1 g per 400 g of total cell extract. The
preparative
immunoprecipitates
were
separated
by
standard
SDS-PAGE
on
polyacrilamide gels and then stained with Coomassie brilliant blue. The gel slice
containing the immunoprecipitated nm23 protein was excised for the MALDI-TOF
analysis. The analytic co-immunoprecipitation experiments were then analyzed by
standard Western blotting.
In situ protein digestion and MALDI analysis
Trypsin, dithiothreitol and iodoacetamide were purchased from Sigma. Trifluoroacetic
acid (TFA) (HPLC grade) was from Carlo Erba. All other reagents and solvents were of
the highest purities available and were obtained from Baker.
The MALDI-TOF mass spectrometry was performed on the Coomassie-bluestained proteins after their excision from the SDS gels, which were washed twice in
milliQ-grade water. The excised spots were washed first with acetonitrile (ACN) and
then with 0.1 M ammonium bicarbonate (AMBIC). This solution was then removed and
this washing was repeated twice. The protein samples were reduced by incubation in 10
mM dithiothreitol for 45 min at 56 °C. The reducing buffer was removed by
ACN/AMBIC washing, as above. The free cysteines were alkylated by an incubation in
55 mM iodoacetamide for 30 min at room temperature in the dark. The gel particles were
then washed with AMBIC and ACN.
5
The enzymatic digestion was carried out with trypsin (12.5 ng/µl) in 10 mM
ammonium bicarbonate, pH 8.0. The gel pieces were incubated at 4 °C for 4 h. The
trypsin solution was then removed, and a new aliquot of the buffer solution was added;
the samples were then incubated for 18 h at 37 °C. A minimum reaction volume, enough
for the complete rehydration of the gel, was used. The peptides were then extracted by
washing the gel particles in 10 mM AMBIC and 0.1% TFA in 50% ACN at room
temperature.
The MALDI-TOF mass spectra were recorded using an Applied Biosystems
Voyager DE-PRO instrument and a brand new Voyager MALDI TOF/TOF mass
spectrometer. A mixture of the peptide solution and alfa-cyano-hydroxycinnamic acid (10
mg/ml in ACN/ 0.1% TFA in water; 2:1, v/v) was applied to the metallic sample plate
and dried at room temperature. Mass calibration was performed using external standards.
The raw data were analysed using the computer software provided by the manufacturer,
and reported as monoisotopic masses.
In vitro phosphorylation, interaction assay and phospho-nm23 antibody generation
Recombinant purified nm23 and its mutants (100 ng of each) were incubated for 1 h at 30
°C in a standard reaction buffer for CKI. In vitro kinase assays for the measurement of
phosphate incorporation with the CKI isoforms were performed using recombinant
nm23-H1 (expressed in E. coli) as substrate (250 pmol) and CKI isoforms (100 ng) in the
buffers suggested by the manufacturer (Invitrogen), supplemented with 0.1 mM ATP plus
[32P]-ATP to a final specific activity of 500 nCi/nmol. Reactions were carried out for 10
min at 30 °C and stopped by adding 5x SDS-PAGE loading buffer; the samples where
6
then denatured at 95 °C and separated by SDS-PAGE. The gels were stained with
Coomassie blue and the band corresponding to nm23-H1 was excised and the
incorporated radioactivity quantified by liquid scintillation counting. The reaction
conditions to obtain less than 10% substrate phosphorylation were empirically
determined and used to evaluate the kinetic parameters of the phosphorylation. Nm23-H1
concentrations ranging from 20 nM to 40 M were tested, and the reciprocal values
plotted against the observed reciprocal values of the velocities, to extrapolate the Vmax
and Km values. The phosphorylated nm23 proteins were incubated with h-prune in RIPA
buffer supplemented with 5% BSA. The h-prune protein was then immunoprecipitated
with the A59 polyclonal antibody (Apotech-Alexis.com) that was raised against the motif
III region peptide of h-prune. The binding of the different nm23 mutants to h-prune was
revealed by successive Western blotting on immunoprecipitates, performed with an anti
6xHis antibody (Qiagen) that revealed both the h-prune and the nm23 proteins. The K73
polyclonal antibody was obtained using the phosphopeptide spanning from N115 to E127
(phosphorylated in position S122) as the immunogen, and the antiserum was used both
after an IgG purification on a protein A column, which resulted in a non-selective
antibody, and after a further affinity purification on the phosphopeptide used as the
immunogen (NIIHGSDSVKSAE). This second procedure was performed by crosslinking 1 mg of the desalted phosphopeptide dissolved in DMSO with Affi-gel 25 resin
(Biorad), according to the manufacturer protocol. The coupled resin was then used to
affinity purify the IgG-purified K73 antiserum by its direct application to the Affigellinked peptide column, until the antibody was adsorbed by the column. The column was
washed with (per ml of column): 10 ml 100 mM Tris-HCl, pH 8.0; 10 ml 500 mM NaCl,
7
10 mM Tris-HCl, pH 8.0; 10 ml 10 mM Tris-HCl, pH 8.0. The elution was performed
with 0.1 M glycine, pH 3.0. In the experiments to evaluate the inhibition of
phosphorylation by the use of competitive peptides, these were added in the indicated
concentrations together with 200 ng recombinant nm23-H1, the reaction was performed
with 200 M ATP and [32P]-ATP to a final specific activity of 500 mCi/mmol.
In vitro cell motility assays
The stable MDA-MB-435-c100 clones overexpressing h-prune were obtained as
previously described (D'Angelo et al., 2004). The control MDA-MB-435-c100 breast
cancer cell line was used in “cell motility” assays, as previously described (Hartsough &
Mulder, 1997). Cell motility was determined using transwell technology, as described in
(D'Angelo et al., 2004). IC261 treatment was at 50 M for 8 h (unless otherwise
specified), where the highest reduction in phosphorylation of nm23 without adverse signs
for the cells was seen. The specific or scrambled peptides were used at 1 mM for 12 h.
Data are the means of five independent experiments, each carried out in duplicate. The
statistical analyses were performed using unpaired t-test analysis, available at
http://www.graphpad.com/quickcalcs/index.cfm. The values were considered statistically
significant when they showed P ≤ 0.05.
Immunofluorescence analysis
Translocation based protein interaction assay.
Observation of cells, quantitation, image analysis and presentation were performed
according to (Knauer & Stauber, 2005). The criteria for efficient in vivo protein
8
interactions using the translocation based protein interaction assay were that in >80% of
200 BFP- and GFP-positive cells, BFP and GFP co-localized to the nucleolus.
The MDA-MB-h-prune clone# 4 cell line was allowed to attach to permanox
slides (Lab-Tek II Chamber Slide, Nalge Nunc International). Twenty-four hours after
plating, the cells were treated with either IC261 (50 M,
mM, 12 h). The cells were then washed twice with ice-cold phosphate-buffered saline
(PBS) and treated for immunofluorescence analyses following standard techniques. The
fixed cells (4% paraformaldehyde in PBS) were incubated for 3 h at room temperature
with the primary antibody diluted in 10% porcine or goat serum in PBS. The slides were
washed three times in PBS, and incubated in 10% porcine or goat serum in PBS
containing the directly conjugated secondary antibodies (1/100). The secondary
antibodies were FITC-conjugated pig anti-mouse IgG and TRITC-conjugated goat antirabbit IgG; rhodaminated phalloidin (Molecular Probes) was included for 30 min in this
last step for F-actin staining. After labeling, the slides were washed three times in PBS
and mounted in 10% glycerol. For immunostaining, the antibodies were: monoclonal
anti-FLAG M2 antibody (Sigma) (1:100), and rabbit polyclonal anti-nm23-H1/H2
antibody (K73, Apotech-Alexis.com) produced against the synthetic peptide described
above, without the peptide affinity purification (1:100).
9
PDE activity assay versus cAMP as substrate.
PDE activity was measured with a scintillation proximity assay (Amersham-Pharmacia
Biotech). The samples included 20 g MDA-h-prune #4 total cell extract (with cell
extract from MDA c100 used as control). This was incubated at 30 °C in 100 l assay
buffer (50 mM Tris-HCl, pH 7.4, 8.3 mM MgCl2, 1.7 mM EGTA) containing the desired
concentrations of cAMP as substrate (3:1 ratio unlabeled to [3-labeled). All of the
reactions, including the buffer-only blanks, were conducted in triplicate and allowed to
proceed for an incubation time that gave <25% substrate turnover (determined
empirically). The reactions were terminated by the addition of l Yttrium silicate SPA
beads (Amersham). The enzyme activities were calculated for the amount of radiolabeled
product detected according to the manufacturer protocol. The treatment with IC261 was
the same as for the motility assays described above.
MALDI-TOF mass spectrometry
MALDI-TOF mass spectra were recorded using a Voyager DE-PRO mass spectrometer
(Applied Biosystem, Framingham, USA) operating in reflector mode. A mixture of the
peptide solution and alpha-cyano-hydroxycinnamic acid (10 mg/mL in acetonitrile: 10
mM citric acid in water, 2:1, v/v) was applied to the metallic sample plate and dried at
room temperature. Mass calibration was performed using a mixture of peptides from
Applied Biosystem, containing des-Arg1-bradykinin, angiotensin I, Glu1-fibrinopeptide
B, ACTH (1-17), ACTH (18-39) and insulin (bovine) as external standards. Raw data
10
were analysed using the Data Explorer software provided by Applied Biosystems, and
they are reported as monoisotopic masses.
nanoLC Mass Spectrometry
The peptide mixture was then directly analysed by MALDI MS. A microtip-format
procedure for micropurification of phosphorylated peptides was used prior to the mass
spectrometric analysis, to selectively enrich the phosphopeptides from the protein digest
mixtures. The peptide cleavage products were then loaded onto the Fe3+ immobilized
metal affinity chromatography column under acidic conditions (pH 2.5-3.5), with
unbound non-phosphopeptides removed from the column with an acidic wash solution,
and phosphopeptides eluted under alkaline conditions (approximately pH 10). Finally, the
phosphorylation sites were determined by tandem mass spectrometry analysis and
database searching. The eluted peptide mixture then underwent LCMS analysis using a
4000Q-Trap coupled to a 1100 nano-HPLC system. We performed the MRM experiments
using the first quadrupole in resolving mode (RF and DC voltages applied) so that a
specific m/z was allowed to pass. These ions were accelerated into the collision cell (a
quadrupole used in RF mode only) where they collided with the gas molecules and
fragmented (CID). The third quadrupole was also operated in resolving mode, so that it
passed only one of the fragment ions from the target compound. Therefore, it was
possible to probe for several predicted phosphopeptides from a known protein sequence.
A computer program was developed to simplify the process of developing an MRM
method based on the primary amino-acid sequences. The program performs a theoretical
enzymatic digest of the protein, selects peptides containing a specified type of
11
phosphorylation (S/T or Y), and calculates the Q1 and Q3 m/z values for various charge
states and fragment ions. This program was used to create a method with 45 transitions,
corresponding to the potential phosphopeptides from nm23-H1.
Prior to mass spectrometry analysis, the peptide mixtures were purified using
ZipTip MC pipettes (Millipore), following the recommended purification procedure.
Briefly, the ZipTip MC pipettes were activated by aspirating and then dispensing 200
mM FeCl3 in 10 mM HCl. The pipettes were then equilibrated by aspirating and
dispensing 30% acetonitrile, 5% acetic acid in water, ten times. The peptide mixtures
under acidic conditions (pH 2.5–3.5) were loaded onto the pipettes by multiple aspirating
and dispensing operations. The pipette were then washed using 30% acetonitrile, 0.1%
acetic acid in water, and the peptides were eluted using 20 l 2% NH3 in water.
Mixtures of the eluted peptide solutions were analysed by LCMS using a 4000QTrap (Applied Biosystems) coupled to a 1100 nano-HPLC system (Agilent
Technologies). The mixtures were loaded onto an Agilent reverse-phase pre-column
cartridge (Zorbax 300 SB-C18, 5x0.3 mm, 5 m) at 10 l/min (solvent A: 0.1% formic
acid; loading time, 7 min). The peptides were separated on an Agilent reverse-phase
column (Zorbax 300 SB-C18, 150 mm X 75m, 3.5 m) at a flow rate of 0.3 l/min with
a 0% to 65% linear gradient in 60 min (solvent A: 0.1% formic acid, 2% acetonitrile in
Milli-Q water; solvent B: 0.1% formic acid, 2% Milli-Q water in acetonitrile). A microionspray source was used at 2.5 kV with liquid coupling, with a declustering potential of
20 V, using an uncoated silica tip from NewObjectives (O.D. 150 µm, I.D. 20 µm, T.D.
10 µm). For selective detection of phosphopeptides, the 4000 QTRAP was operated in
12
MRM mode. MRM transitions (50 ms dwell time) were used to trigger dependent linear
ion trap scans: enhanced resolution and enhanced product ion (EPI) scans. The total cycle
time for this method was 3-5 s. This data-dependent method is referred to as targeted
MRM-IDA, for multiple reaction-monitoring information-dependent acquisition.
Software
The MRM transitions for nm23-H1 were calculated from the list of MRM transitions of
potential phosphopeptides that was generated either by manual calculation or by a
software script developed by Applied Biosystems. In general, the transitions were
included for all tryptic peptides (maximum one missed cleavage) containing Ser, Thr or
Tyr residues with either one or two modifications, and for doubly and triply charged
species for the Q1 mass range 400-1600 m/z. The number of MRMs is dependent on
various factors, including protein size, number of peptides following digestion, and the
number of potential phosphorylation sites. It is, however, important to optimize the cycle
time to around or below 5 s. Such a cycle time ensures that if the peak width is around
0.5 min, it is highly probable that a peptide is scanned for and analyzed at least twice as it
is eluted, and that one of these analyses will occur at, or close to, the apex of its elution
profile. The software requires the amino-acid sequence of the protein of interest, a starter
method containing the LC conditions, and an empty MRM-IDA experiment. The
software performs an in-silico digestion of the protein and creates a set of peptides, each
of which contain at least one possible site of modification. For each peptide, an MRM
transition for the calculated m/z of the precursor ion and an appropriate fragment ion is
generated. The new method, specific for the protein of interest, is saved and submitted as
13
a batch for data acquisition. The Agilent Nanoflow LC system and 4000 QTRAP were
both controlled using Analyst 1.4.1. The combined information from each MRM–IDA
experiment was used to perform Mascot searches against the SwissProt database.
Supplemental Figure legends
Figure 1S. Similarities and protein-protein interactions of the nm23 and h-prune
proteins through evolution.
A. Protein-protein interactions revealed by transient transfection in COS-7A cells and coimmunoprecipitation. The full-length h-prune protein and nm23 protein isoforms were
cloned into the pcDNA-derived vectors pReyzo and HA-pcDNA, respectively. Coimmunoprecipitation with the nm23-X2 cDNA/protein from X. laevis. B. As for (A), as
co-immunoprecipitation with the nm23-Z1 cDNA/protein from D. rerio (zebra fish). C.
As for (A), as co-immunoprecipitation with the nm23 cDNA/protein from C. elegans. D.
As for (A), as co-immunoprecipitation with the nm23-awd cDNA/protein from D.
melanogaster. E. ClustalW multiple protein sequence alignment between the h-prune
motifs of the DHH family proteins. The protein sequences from X. tropicalis and in D.
rerio were obtained by translation from EST sequences found in genome databases for X.
tropicalis and D. rerio, respectively (X97902.1, ENSDART09127). Grey shading
indicates the identical residues that are conserved across these species.
Figure 2S. Protein-protein interactions between h-prune, nm23-H1 mutants and
CKI / in COS-7A cells.
14
Protein-protein interactions revealed by transient transfection in COS-7A cells and coimmunoprecipitation using protein extracts of the full-length h-prune protein (h-pruneFlag) (A-D) and human CKIand CKI cDNA/protein (E, F), with the nm23-H1 mutated
isoforms HA-nm23-H1-S125A (A, B) and HA-nm23-H1-S122P (C, D), and with HAnm23-H1 (E, F). A. Co-immunoprecipitation of h-prune with the artificially mutated
nm23-H1-S125A cDNA/protein. B. Co-immunoprecipitation of h-prune with an anti-Flag
epitope antibody. C. Co-immunoprecipitation of h-prune with the artificially mutated
nm23-H1-S122P cDNA/protein. D. Co-immunoprecipitation of h-prunewith an anti-Flag
epitope antibody. E, F. Co-immunoprecipitation of nm23-H1 with the human CKI and
CKI proteins.
Figure 3S. Effect of phosporylation on nm23 NDPK activity.
NDPK activity assay of non-phosphorylated and phosphorylated forms of nm23-H1 and
nm23-H2 and their mutants on S120, S122 and S125, as indicated.
Figure 4S. Immunofluorescence analysis showing cellular localization of endogenous
h-prune and nm23-H1/H2.
A. Immunofluorescence analysis of h-prune and nm23 in the MDA-MB-435-h-prune #4
clone cells in the absence and presence of 50 M IC261. B. Immunofluorescence analysis
of h-prune and F-actin filaments (rhodaminated phalloidin) in MDA-MB 435-h-prune #4
clone cells treated with 50 M IC261 and the specific competitive cell-permeable
peptide.
15