Optimization of the cellular import of functionally active SH2-domain-interacting phosphopeptides

Article abstract of DOI:10.1007/s00018-006-6346-6

Phosphopeptides interacting with src homology 2 (SH2) domains can activate essential signaling enzymes in vitro. When delivered to cells, they may disrupt protein-protein interactions, thereby influencing intracellular signaling. We showed earlier that phosphopeptides corresponding to the inhibitory motif of Fcγ receptor IIb and a motif of the Grb2-associated binder 1 adaptor protein activate SH2-containing tyrosine phosphatase 2 in vitro. To study the ex vivo effects of these peptides, we have now compared different methods for peptide delivery: (i) permeabilization of the target cells and (ii) the use of cell-permeable vectors, which are potentially able to transport biologically active compounds into B cells. We found octanoyl-Arg₈ to be an optimal carrier for the delivery of phosphopeptides to the cells. With this strategy, the function of cell-permeable SHP-2-binding phosphopeptides was analyzed. These peptides modulated the protein phosphorylation in B cells in a dose- and time-dependent manner. Birkhaeuser Verlag, 2006.

Full text of DOI:10.1007/s00018-006-6346-6

Cell. Mol. Life Sci. 63 (2006) 2682–2693
1420-682X/06/222682-12
DOI 10.1007/s00018-006-6346-6
© Birkhäuser Verlag, Basel, 2006
Cellular and Molecular Life Sciences
Research Article
Optimization of the cellular import of functionally active
SH2-domain-interacting phosphopeptides
Á. Kertésza, G. Váradib, G. K. Tóthb, R. Fajka-Bojac, É. Monostoric and G. Sármaya, *
a Department of Immunology, Eötvös Loránd University, Pázmány Péter s. 1c, 1117 Budapest (Hungary),
Fax: +36 1 3812176, e-mail: sarmayg@elte.hu
b Department of Medical Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged (Hungary)
c Lymphocyte Signal Transduction Laboratory, Institute of Genetics, Biological Research Center of the Hungarian
Academy of Sciences, Temesvári krt. 62. 6726 Szeged (Hungary)
Received 27 July 2006; received after revision 4 September 2006; accepted 18 September 2006
Online First 31 October 2006
Abstract. Phosphopeptides interacting with src homol- ery: (i) permeabilization of the target cells and (ii) the use
ogy 2 (SH2) domains can activate essential signaling en- of cell-permeable vectors, which are potentially able to
zymes in vitro. When delivered to cells, they may disrupt transport biologically active compounds into B cells. We
protein-protein interactions, thereby influencing intracel- found octanoyl-Arg8 to be an optimal carrier for the deliv-
lular signaling. We showed earlier that phosphopeptides ery of phosphopeptides to the cells. With this strategy, the
corresponding to the inhibitory motif of Fcγ receptor IIb function of cell-permeable SHP-2-binding phosphopep-
and a motif of the Grb2-associated binder 1 adaptor pro- tides was analyzed. These peptides modulated the protein
tein activate SH2-containing tyrosine phosphatase 2 in phosphorylation in B cells in a dose- and time-dependent
vitro. To study the ex vivo effects of these peptides, we manner.
have now compared different methods for peptide deliv-
Keywords. Cell-permeable peptide, phosphopeptide, SH2 domain, signaling, tyrosine phosphorylation.
Phosphopeptides (PPs) representing functional sequences vation [3]. A PP corresponding to the biphosphorylated
of intracellular signaling molecules offer excellent tools immunoreceptor tyrosine-based activation motif (ITAM)
to study specific signal transduction events. PPs cannot was found to activate lyn and induce protein phosphory-
penetrate the plasma membrane barrier without a mem- lation in LPC-permeabilized B cells [4], while ζ chain
brane translocalization signal. For study of their effects ITAM peptide triggered tyrosine phosphorylation of p56
on intracellular signaling, the cells must be permeabi- lck and ZAP70 in permeabilized Jurkat T cells [5]. The
lized with membrane-disrupting, pore-forming agents, or inherent limitations of cell permeabilization by mem-
the phosphotyrosine-containing motifs must be coupled brane disruption can be overcome by employing mem-
to cell-membrane-permeable carrier peptide (CPP) ve- brane-penetrating carrier peptides to deliver biologically
hicles, which then transport the peptides into the cells active compounds such as PPs into the cell. In the past
[1, 2]. The lysophosphatidylcholine (LPC)-mediated per- few years, several CPPs have been reported [1, 6–10].
meabilization of lymphocytes was previously optimized The first molecules of this kind that were described were
for study of the effects of PPs on tyrosine kinase acti- various sequences of viral proteins responsible for virus
internalization. Arg-rich peptides, such as the basic pep-
* Corresponding author.
tide derived from the human immunodeficiency virus 1

Cell. Mol. Life Sci. Vol. 63, 2006
Research Article
2683
(HIV1)Tat protein [11, 12] and oligo-arginines comprised Materials and methods
of six to eight Arg units have been reported to penetrate
the plasma membrane, thereby delivering bioactive mol- Antibodies and reagents. Monoclonal human IgM µ-
ecules into the cells [1, 7, 13]. Ex vivo characterization of chain-specific antibody (BU1) was a kind gift from Prof.
the known CPPs is not complete. The aims of this study R.Jefferis(UniversityofBirmingham,Birmingham,UK).
were to compare detergent and CPP- mediated PP deliv- Anti-SHP-2 monoclonal antibodies were from Santa Cruz
ery methods and to design, characterize and optimize the Biotechnology (Santa Cruz, Calif.). PI3-K p85 specific
procedure of PP delivery in order to analyze the effects of antibody and anti-phosphotyrosine 4G10 were from UBI
these PPs on B cell signal transduction events.
(Lake Placid, N.Y.) and anti-phosphoErk1/2 was from
The PP cargos to be transported into B cells were de- Cell Signaling Technologies (Beverly, Mass.). Enhanced
signed on the basis of the phosphorylated motifs of two chemiluminescence (ECL) reagent was purchased from
important B cell regulatory molecules: (i) The phos- Perbio UK Science (Tattenhal, UK) and other reagents
phorylated form of immunoreceptor tyrosine-based in- were from Sigma.
hibitory motif (ITIM) of Fcγ RIIb, a negative regulator Dichloromethane (DCM), dimethylformamide (DMF),
of B cells, and (ii) a C-terminal phosphorylated motif diisopropylethylamine (DIEA) methanol (MeOH), tri-
of Grb2-associated binder 1 (Gab1) adaptor/scaffold- fluoroacetic acid (TFA) and HPLC-grade acetonitrile
ing protein. FcγꢀRIIb becomes tyrosine-phosphorylated (MeCN) were purchased from Aldrich (Steinheim, Ger-
upon its co-clustering with the B cell receptor complex many) and were used without further purification. Fluka
(BCR). Subsequently, Fcγ RIIb recruits phosphatases (Buchs, Switzerland) supplied puriss grade dicyclohex-
carrying src homology (SH2) domains, such as protein ylcarbodiimide (DCC). Protected amino acid derivatives
tyrosine phosphatase (SHP-2) and phosphatidylinosi- were from Orpegen (Heidelberg, Germany). 1-Hydroxy-
tol 5-phosphatase (SHIP) to the vicinity of the signal- benzotriazole (HOBt), p-cresol and dimethyl sulfide were
ing complex, in this way terminating cell activation purchased fromAldrich. Bodipy-FL was supplied by Mo-
[14–19]. SHP-2 has a dual role in lymphocyte signal- lecular Probes (Carlsbad, Calif.). The different amidites
ing: recruited by phosphorylated ITIM (pITIM), it fa- were prepared in our own laboratories. Hydrogen fluoride
cilitates the down-regulation of signals emanating from (HF) was obtained from Gerling-Holz (Hamburg, Ger-
activatory receptors and, additionally, SHP-2 may play a many).
positive role by activating extracellular-signal-regulated
kinases (Erk1/2) via the interaction with the Gab1 adap- Peptide synthesis. The sequences of the chosen CPPs
tor/scaffolding protein [20]. Gab1 becomes phosphory- were synthesized by a solid-phase technique utilizing
lated upon ligation of antigen, cytokine and growth hor- tBoc chemistry [34]. The side-chain-protecting groups
mone receptors, and the C-terminal phosphorylated mo- were:Arg(Tos), Thr(Bzl), Ser(Bzl), Cys(Meb), Tyr(2BrZ)
tifs of Gab1 then bind to and activate SHP-2, which in and Lys(2ClZ). The peptide chains were elongated on an
turn may dephosphorylate additional phosphotyrosine MBHA resin (0.48 mmol/g) and the syntheses were car-
residues, leading to the release of the ras negative regu- ried out in an ABI 433A automatic machine. Couplings
latory molecule, rasGAP, resulting in the full activation were performed with DCC and HOBt. Amino acid incor-
of Erk1/2 [21–23].
poration was monitored with the ninhydrin test [35]. The
Tyrosine phosphorylation is involved in the modulation completed peptide resins were treated with liquid HF/di-
of the activities of target enzymes, and is also respon- methyl sulfide/p-cresol/p-thiocresol (86/6/4/2, vol/vol),
sible for the generation of signaling networks [24–26]. at 0 °C, for 1 h. HF was removed and the resultant free
Impaired signaling may lead to the development of peptides were solubilized in 10% aqueous acetic acid,
various tumors or autoimmune diseases [27–30]. Since filtered and lyophilized. The crude peptides were puri-
Gab1/SHP-2 interaction is essential for cell growth and fied by reverse-phase HPLC (Shimadzu, Kyoto, Japan)
transformation, and SHP-2 regulates signals leading using a Phenomenex (Torrance, Calif.) C-18 10 µ column
to proliferation and cell survival, Gab1/SHP-2 interac- (16ꢀ×ꢀ250 mm). The solvent system used was: 0.1% TFA
tion is an attractive target for therapy of malignant cell in water, 0.1% TFA, 80% MeCN in water, gradient: 0%-
growth. Blockade of the interaction between a function- 0% B for 15 min, and then 0–15% in 60 min, at a flow
ally relevant tyrosine-phosphorylated motif and the SH2 rate of 3.5 ml/min, with detection at 220 nm. The appro-
domain by cell-permeable PP or phosphopeptido-mi- priate fractions were pooled and lyophilized. The purified
metics may disrupt signaling and inhibit cell activation peptides were characterized by mass spectrometry with a
[31–33]. In this paper we report that SHP-2-binding, Finnigan TSQ 7000 tandem quadrupole mass spectrom-
cell-membrane-permeable PPs modulate intracellular eter equipped with an electrospray ion source.
protein phosphorylation in B cells in a dose- and time-
dependent manner.
Synthesis of fluorescent peptides. 2.4 µmol of the ap-
propriate peptide was dissolved in phosphate buffer (pH

2684
Á. Kertész et al.
Phosphopeptide delivery to B cells
Table 1. Calculated and found molecular masses of penetrating
peptides, their Bodipy-FL-conjugated forms, their conjugates with
PPs; and their HPLC retention time.
6.5, 100 mmol, 6 mol guanidine hydrochloride) together
with 1 mg (2.4 µmol) Bodipy-FL (B 10250). After stir-
ring of the solution for 1–2 h, the resulting construct was
purified by HPLC.
Code
Calc M
Found M
HPLC Rt
R8C
1368.68
1607.09
1495.09
1909.31
2284.76
2698.98
2943.48
3357.70
3047.83
3462.05
3063.83
3478.05
3289.95
5335.36
1369.1
1607.5
1495.2
1909.2
2283.6
2698.4
2942.4
3357.6
3047.6
3463.4
3063.0
3477.0
3290.2
5333.7
10.84 mina
7.90 minb
9.77 minc
8.08 mind
13.17 mine
9.423 minf
7.37 ming
12.16 minf
13.70 minh
8.567 mini
8.834 minh
8.281 minj
11.07 mink
8.85 minl
PP synthesis. Peptide synthesis was carried out on Rink
amide resin using the standard Fmoc protocol (DCC/
HOBt coupling, ABI 433 synthetizer). The side protec-
tion used wasAsp(OBut), Lys(Boc), Glu(OBut), Thr(But),
Ser(But) and His(Trt). The hydroxy groups of the serine
or tyrosine, which were the subject of phosphorylation,
were unprotected. Phosphitylation was carried out in
THF at room temperature for 30 min with 1H-tetrazole
as activator and O-tert-butyl-O-β-cyanoethyl-N,N-diiso-
propyl phosphoramidite in the case of serine and di-tert-
butyl-N,N-diisopropyl phosphoramidite in the case of
tyrosine [36]. The subsequent oxidation was performed
with 15% tert-butyl hydroperoxide in THF. Simultane-
ous cleavage of the β-cyanoethyl and Fmoc groups was
performed with 20% DBU in DCM [37]. After the DBU
cleavage, the peptide chain elongation was completed
P-R8C
O-R8C
Bodipy-O-R8C
C-TP10
Bodipy-C-TP10
C-transportan
Bodipy-C-transportan
C-MPG-α
Bodipy-C-MPG-α
C-MPG-α-ox
Bodipy-C-MPG-α-ox
O-R8C-GDLD(Pe)
O-R8C-RKHP(Pe)
and the PP was detached from the resin with TFA/water C stands for Cys, a maleinimidocaproyl linker.
(95/5) solution.
HPLC conditions for the analytical control (column dimension
4ꢀ×ꢀ250 mm, flow rate 1.2 ml/min): a gradient 5% → 35% B in
20 min, b gradient 45% ꢀ→ 60% B in 15 min, c gradient 20% → 60%
B in 20 min, d gradient 32% → 47% B in 15 min, e gradient
Conjugation of the PPs to CPPs. 8 µmol of the ma-
leinimidocaproyl derivative of the appropriate PP was
dissolved in phosphate buffer (pH 6.5, 100 mmol, g gradient 50% → 65% B in 15 min, gradient 50% → 100% B in
6 mol guanidine hydrochloride) together with 11.96 mg
(8 µmol) of O-R8C, where the C-terminal cysteine amide
moiety binds to the double bond of the PP via Michael
40% → 80% B in 20 min, f gradient 53% → 73% B in 20 min,
h
25 min, i gradient 71% → 86% B in 15 min, j gradient 65% → 80%
B in 15 min, k gradient 25% → 40% B in 15 min, l gradient
25% → 45% B in 20 min.
addition. After stirring of the solution for 3–4 h, the re-
sulting construct was purified by HPLC. Details of the 2 mM EGTA] and permeabilized with L-α-lysophospha-
MS and HPLC analysis of the synthesized cell-permeable tidyl choline at 50 µg/ml for 1 min on ice as previously
peptides are given in Table 1.
described [3]. Permeability was assessed using trypan
blue exclusion in each experiment.
Cell culturing, stimulation of intact cells. BL41 Burkitt Stimulation of permeable cells was initiated by adding
lymphoma cells were cultured in RPMI 1640 medium peptides and/or anti-IgM for 3 min at 37 °C at the con-
supplemented with 10% fetal calf serum, 105 U/l penicil- centrations indicated for each experiment. The reaction
lin and 50 µg/l streptomycin at 37 °C in a 5% CO2-con- was stopped with ice-cold 2ꢀ×ꢀlysis buffer.
taining atmosphere. The cells were washed twice in se-
rum-free RPMI 1640 and incubated at 37 °C for 30 min. Preparation of detergent cell extracts, SDS-PAGE and
5 ×ꢀ107 cells/sample were stimulated with 2 µg/ml human Western blot. 2 ×ꢀ106 cells/sample were solubilized in
µ-chain-specific monoclonal antibodies (BU1). After a 2- 0.1 ml lysis buffer containing 50 mM HEPES (pH 7.4),
min stimulation at 37 °C, the cells were centrifuged for 1% Triton X-100, 100 mM NaF, 10 mM EDTA, 2 mM
20 s and immediately frozen in liquid nitrogen. BL41 sodium orthovanadate, 10 mM sodium pyrophosphate,
cells were pretreated with the cell-membrane-permeable 10% glycerol, 10 µg/ml aprotinin, 5µg/ml leupeptin,
PPs for various periods of time and then stimulated with 0.2 mM phenylmethylsulfonyl fluoride and 10 mM β-
anti-µ.
glycerophosphate. Samples were incubated on ice for
45 min and then centrifuged at 15,000ꢀg for 20 min, and
Cell permeabilization and stimulation of permeabi- the postnuclear supernatants were used for further ex-
lizedcells.Cellswereharvestedbycentrifugation, washed aminations. Cell extracts were diluted in 40 µl reducing
in RPMI 1640 without supplements and incubated in SDS/PAGE sample buffer and boiled for 5 min at 95 °C.
RPMI for 5 min at 37 °C. Cell pellets were resuspended The samples were separated on 8.5% or 10% SDS/PAGE
in ice-cold permeabilization buffer [40 mM HEPES pH gels, and the proteins were then transferred to nitrocel-
7.4, 10 mM MnCl2, 10 mM Mg(OAc)2, 296 µM CaCl2, lulose membranes (BioRad), and consecutively probed

Cell. Mol. Life Sci. Vol. 63, 2006
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2685
Table 2. Sequences of PPs used in LPC-permeabilized cells.
with various antibodies. Immunoblots were developed
using HRPO-conjugated second antibodies, followed by
ECL detection. Some membranes were stripped accord-
ing to the manufacturer’s instruction and reprobed with a
different antibody.
Code
PPs used in LPC-permeabilized cells
AENTITY(p)SLLMHP
pITIM
pITIM
TEIINPNY(p)MGVG(AG)15 AENTITY(p)SLLMHP
chimera
EL-PN
GD-LD
ELDENY(p)VPMNPN
GDKQVEY(p)LDLDLD
Confocal microscopy. BL41 cells were incubated with
10 µM labeled CPP conjugates at 4 or 37 °C for 20 min.
Cellular uptake was followed by confocal laser scanning
microscopy, using the 488-nm line of an argon ion laser
(Olympus FluoView 500).The lipophilic fluorescent probe
1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine ence intracellular protein tyrosine phosphorylation, B
perchlorate (DiI-C18) was used to stain the cell membrane, cells were permeabilized with LPC and then treated with
while acidic lysosomes were detected with 100 nM Lyso- the PPs either in the presence or the absence of an anti-
Tracker Red probe (Molecular Probes). The latter probes IgM stimulus. Anti-IgM triggered tyrosine phosphoryla-
were excited by the 543-nm He-Ne laser line. The Pearson tion in both LPC-permeabilized and non-permeabilized
coefficient was calculated using Image J software and Im- (control) (Fig. 1a) cells. Unphosphorylated ITIM, either
age Correlator Plus plug in.
alone or in combination with anti-IgM, had no effect on
protein phosphorylation (Fig. 1a). In contrast, its phos-
Flow cytometry. Cells were harvested and washed in PBS phorylated counterpart, pITIM itself, induced tyrosine
containing 1% FCS and 0.5 mg/ml NaN3, and incubated phosphorylation of proteins at 30, 50 and 66–70 kDa;
for 5 to 60 min at 4 °C or at 37 °C (5ꢀ×ꢀ105 cells/sample) this also occurred when pITIM was used in combina-
with different amounts of Bodipy-FL-conjugated cell- tion with anti-IgM (Fig. 1a). SHP-2 is activated by the
permeable carrier or PP constructs. Then, after the cells simultaneous engagement of its two SH2 domains by a
had been washed twice in the same buffer, the samples bisphosphorylated activation motif [2, 38]. Accordingly,
were analyzed on a FACSCalibur cytofluorimeter (Bec- we also tested the effects of a chimeric bisphosphorylated
ton Dickinson, Franklin Lakes, N.J.) using CellQuest peptide consisting of pITIM and a phosphotyrosine-con-
software. In some experiments, the cells were also stained taining peptide of SHIP combined with a flexible linker
with the DNA-intercalating dye propidium iodide to de- [TEIINPNY(p)MGVG(AG)15 AENTITY(p)SLLMHP,
tect non-viable cells.
chimera] [39]. This chimeric peptide induced the tyro-
sine phosphorylation of proteins at 30, 50 and 66–70 kDa
Phosphatase activity assay. Serial dilutions of BL41 cell in a dose-dependent manner (Fig. 1b) and enhanced anti-
lysate samples (equivalent to 105 cells) and of recombi- IgM-triggered Tyr phosphorylation. The Gab1 PPs, GD-
nant SHP-2 protein were prepared in 96-well plates and LD and another Gab1 motif [ELDENY(p)VPMNPN,
preincubated with 0.5 µM biotinylated PPs/well at 37 °C EL-PN] reacting with the SH2 domain of the regulatory
for 1 h in a phosphatase buffer containing 20 mM Hepes subunit of PI3-K, did not induce detectable tyrosine phos-
(pH 7.0), 5% glycerol, 0.05% Triton X-100, 2.5 mM phorylation in LPC-permeabilized B cells (Fig. 1c). Our
MgCl2, aprotinin (10 µg/ml) and leupeptin (10 µg/ml). earlier peptide pull-down experiments followed by West-
The samples were harvested and transferred onto an ern blot revealed that the major molecule interacting both
ELISA plate precoated with avidin, and were further with pITIM, the chimera and GD-LD PPs was SHP-2 [15,
incubated at 4 °C overnight. After several rinsings, the 39] indicating that these PPs may regulate the activity of
plates were developed with phosphotyrosine-specific an- SHP-2 phosphatase ex vivo.
tibodies followed by peroxidase-conjugated anti-mouse
IgG and orthophenylene diamine as substrate. Optical Selection of an optimal cell-penetrating peptide for use
densities in the wells were measured at 450 nm.
as a vector for signaling peptides. To transport the PPs
into the cells under more physiological condition, i.e. by
cell-penetrating peptides, we first compared the effects
of a series of CPPs. Several membrane-non-permeable
peptides coupled covalently to CPP exhibited evidence of
Results
The effects of pITIM and Gab1 peptides on pro- internalization and biological activity in cell cultures [1].
tein tyrosine phosphorylation in LPC-permeabi- However, the various CPPs have not yet been compared
lized B cells. To study whether PPs (see list of PPs in a homogenous system on the same target cell type. On
in Table 2) corresponding to the pITIM of FcγRIIb the basis of known sequences, basic octa-arginin (R8) and
[AENTITY(p)SLLMHP] or to the SHP-2-binding mo- its modified versions, palmitoyl-(P-R8) and octanoyl-oc-
tif of Gab1 [GDKQVEY(p)LDLDLD] (GD-LD) influ- taarginine (O-R8), transportan, and a shorter version of

2686
Á. Kertész et al.
Phosphopeptide delivery to B cells
Figure 1. PP-induced protein tyrosine phosphorylation in LPC-permeabilized BL41 cells. (a) The permeabilized cells were treated with
250 µM ITIM, or pITIM peptides, alone, or before anti-IgM stimuli. (b) 50, 100, 250ꢀand 500 µM pITIM chimera peptide composed of
the SHIP phosphorylated motif and pITIM was added to the cells; last lane, 250 µM SHIP-pITIM chimera was added before anti-IgM.
(c) 250 µM GD-LD or EL-PN PP was added with or without anti-IgM.
transportan (TP10), MPG and an oxidized form of MPG interval 0.4–0.5% for nearly all CPPs, indicating a partial
(MPGox), were synthesized and tested for cell penetra- co-localization; the only exception was MPGox, where it
tion in the BL41 Burkitt lymphoma cell line (Table 3).
was 0.06%.
The CPPs were labeled with Bodipy FL N-(2-amino- CPPs were further analyzed by flow cytometry. The time
ethyl)maleinimide and their cellular uptake was followed dependence of CPP uptake is depicted in Figure 3. All
by confocal microscopy. The lipophilic fluorescent probe, CPPs translocated into the cells within the first 5 min of
DiI-C18 was used to stain the cell membrane, while acidic incubation, and in most cases the penetration continued
lysosomes were labeled with a LysoTracker Red probe. for up to 60 min. In the transportan- and TP10-treated
All CPPs except MPGox penetrated the cell membrane samples, a cell group with a lower and a higher positiv-
and were detected in the cytoplasm as discrete granules ity was observed. The transport of CPPs was temperature
after a 20-min incubation at 37 °C. MPGox displayed a independent, since all the CPPs penetrated equally well at
very low uptake, and was not detectable with confocal 4 and 37 °C (data not shown).
microscopy under these conditions (Fig. 2). The correla- Bodipy FL-conjugated CPPs (FL1) and propidium iodide
tion between the localization of the peptide-containing staining (FL3) were applied simultaneously to detect dead
vesicles and acidic lysosomes was calculated using the cells among the peptide-positive population in order to
Pearson coefficients. These values appeared to lie in the determine the cytotoxicity of CPPs. All the CPPs except
Table 3. Sequences of cell-membrane-penetrating peptides.
Code
Cell-permeable peptides
R8
RRRRRRRR-C(BODIPY) (-NH2)
P-R8
palmitolyl -RRRRRRRR-C (-NH2)
O-R8
octanoyl -RRRRRRRR-C (BODIPY) (-NH2)
MPG
C(BODIPY)-GALFLAFLAAALSLMGLWSQPKKKRKV (-NH2)
C(BODIPY)-GALFLAFLAAALSLM(O)GLWSQPKKKRKV (-NH2)
C(BODIPY)- GWTLNSAGYLLGKINLKALAALAKKIL (-NH2)
C(BODIPY)-AGYLLKINLKALAALAKKIL (-NH2)
octanoyl-RRRRRRRR-C(BODIPY) -GDKQVEY(p)LDLDLD(NH2)
octanoyl-RRRRRRRR-C- RKKRISALPGPANPTNPDEADKVG- AENTITY(p)SLLMHP
MPGox
Transportan
TP10
O-R8-GDLD(Pe)
O-R8-RKHP(Pe)

Cell. Mol. Life Sci. Vol. 63, 2006
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2687
Figure 2. Confocal laser scanning microscopic analysis of the penetration of Bodipy Fl-labeled carrier peptide to BL41 cells. The cell
membrane was stained with DiI-C18, and the acidic lysosomes were detected with LysoTracker Red. The cells were treated with the pep-
tides at 37 °C for 20 min.
Figure 3. Time dependence of CPP uptake by BL41 cells at 37 °C. The cells were treated with the CPPs for 5, 20 and 60 min, washed and
measured.

2688
Á. Kertész et al.
Phosphopeptide delivery to B cells
Figure 4. Uptake and cytotoxicity of cell-permeable peptides. BL41 cells were treated with Bodipy-FL-conjugated CPPs at 37 °C for
20 min, and then stained with propidium iodide. The numbers in the lower-right quadrants show the percentages of Bodipy-FL-positive
cells.
MPGox stained the cells to an extent of over 90% (Fig. 4).
MPG, TP10 and transportan were partially toxic to the
B cells, since 42, 74 and 75% of the cells, respectively,
died upon peptide treatment for 20 min at 37 °C. Most
importantly, R8- and OR8-treated samples fully retained
their viability (Fig. 4).
To test the effects of CPPs on cell viability, BL41 cells
were further analyzed with respect to the concentration
(10 and 50 µM CPPs) and time (1, 6 and 24 h) dependen-
cies. As shown in Figure 5, all CPPs were toxic at 50 µM
during co-culture with the cells for 24 h. R8 and O-R8
had no significant effect on the cell viability at 10 µM,
even after treatment for 24 h. In contrast, MPG, MPGox
and TP10 were significantly toxic in as short a treatment
Figure 5. Cell viability in the presence of CPPs. BL41 cells were
treated with various CPPs for 1, 6 and 24 h, and then stained with
propidium iodide. The proportion of dead cells was calculated after
period as 6 h, and 80% of BL41 cells died after incuba-
tion for 24 h with P-R8 and transportan (Fig. 5). In view
of the efficiency of the transport and the toxic effect of
the CPPs on the cells, we selected O-R8 as a carrier for
further experiments.
flow cytometric analysis.
Delivery of PPs into BL41 cells by O-R8 carrier. To test
whether the carrier CPPs are indeed capable of delivering
PPs into cells, O-R8 was conjugated to the Gab1 PP, GD-
LD, and the uptake of this construct by BL41 cells was
then tested by means of confocal microscopy and flow
cytometry. The intracellular localization of the construct
was the same as shown for the carrier CPP (Fig. 2): the
PP penetrated the plasma membrane and was detected in
the cytoplasm as discrete vesicles, exhibiting a partial co-
localization (Pearson coefficient: 0.45) with acidic lyso-
somes (Fig. 6). The time and temperature dependence of
peptide delivery were tested by flow cytometry. The PP
conjugate penetrated dose-dependently into the cells and
was detected inside the cells after a 5-min incubation at
either 4 or 37 °C (Fig. 7). Interestingly, the O-R8-GD-
LD conjugate demonstrated maximal engulfment as early
as 5 min after addition, in contrast with the CPP carrier
without cargo, when this was increased up to 60 min. The
Figure 6. Confocal laser scanning microscopic analysis of the de-
livery of Bodipy-Fl-labeled GD-LD PP into BL41 cells by O-R8
CPP. The cells were treated with the conjugate at 37 °C for 20 min.
The cell membrane was stained with DiI-C18; acidic lysosomes
were detected by LysoTracker Red.

Cell. Mol. Life Sci. Vol. 63, 2006
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2689
Figure 7. Time, temperature and dose dependencies of PP deliv-
ery into BL41 cells. The cells were treated for 5 or 20 min with
10 µM cell-permeable PP at 4 or 37 °C (upper panels). The cells
were incubated in the presence of increasing doses of cell-perme-
able PP for 5 or 20 min (lower panels). The samples were analyzed
by cytofluorometry.
temperature independence of peptide penetration indi-
cated that the cell-permeable PP was not translocated to
the B cells by endocytosis.
Effects of O-R8-GD-LD and O-R8-pITIM on intra-
cellular signaling. To test whether the SHP-2-binding
cell-permeable PP decoy molecules influence the activity
of SHP-2 ex vivo, BL41 cells were pretreated with O-R8-
conjugated PPs for different intervals of time, the cells
were then stimulated with anti-IgM, or left untreated,
and the protein tyrosine phosphorylation was analyzed in
the cell extracts by Western blot. A longer PP was used
here than for LPC-permeabilized cells, containing pITIM
(bold letters) completed with an N-flanking terminal se-
quence) [O-R8-RKKRISALPGPANPTNPDEADKVG-
AENTITY(p)SLLMHP, RK-HP], in order to mimic
better the intracellular sequence of FcγRIIB1. Figure 8a
shows that RK-HP alone induced the tyrosine phosphory-
lation of several proteins in a dose- and time-dependent
manner. RK-HP also enhanced the anti-IgM induced
Figure 8. The effects of cell-permeable PPs on protein phosphory-
lation in BL41 cells. The cells were treated with O-R8-pITIM (a)
or O-R8-GD-LD (b) at 37 °C for the indicated time period, and then
anti-IgM was added for 2 min. The cells were spun down, instantly
frozen in liquid nitrogen, then lysed and separated by SDS-PAGE.
The samples were blotted into nitrocellulose membrane and ana-
lyzed by Western blot using phosphotyrosine-specific antibodies.
Dephosphorylation of PPs by BL41 cell lysate samples and by
recombinant SHP-2 protein (c). The biotinylated PPs were treated
with various dilutions of cell lysate samples or recombinant SHP-
2 protein for 1 h at 37 °C, then the samples were transferred onto
a streptavidin-coated plate. After an overnight incubation at 4 °C,
the remaining phosphotyrosine contents of the bound peptides were
detected by ELISA using phosphotyrosine-specific antibodies.
tyrosine phosphorylation of proteins at 40–43, 50 and treatment of the cells with O-R8-GD-LD for the same
55 kDa. The enhancement was detected only at 20 min, time resulted in a considerably elevated level of tyrosine
and faded after incubation with the peptide for 60 min.
phosphorylation of several proteins in anti-IgM stimu-
The cell-permeable Gab1 PP OR8-GD-LD alone induced lated samples (Fig. 8b). This effect was seen much ear-
the tyrosine phosphorylation of proteins at about 55, 68 lier, after treatment for 2 min, at a higher dose (10 µM)
and 98 kDa after incubation for 20 and 60 min, while pre- of O-R8-GD-LD, and faded rapidly with time.

2690
Á. Kertész et al.
Phosphopeptide delivery to B cells
To check the possible fate of PPs within the cell, phospha- We earlier reported that the GD-LD motif of Gab1 and a
tase activity assays were performed. PPs AE-HP, RK-HP chimeric bisPP consisting of the pITIM of FcγRIIb and
and GD-LD were efficiently dephosphorylated in vitro, a tyrosine-phosphorylated motif of SHIP bind to SHP-2
although to different extents, by both cell lysates and re- SH2 domains with a Kd = 1.5 nM and Kd = 9 nM, respec-
combinant SHP-2, indicating that these PPs can indeed tively, and activate the phosphatase in vitro [39]. Thus,
be dephosphorylated by intracellular phosphatases, and our aim was to test whether these PPs have an ex vivo
among these, by SHP-2 (Fig. 8c).
effect on BCR-mediated signaling when delivered into
These data confirm that the O-R8 cell-permeable peptide BL41 cells. We first compared the effects of pITIM, the
is capable of delivering PPs into B cells and the CPP-PP chimera and Gab1 GD-LD and EL-PN peptides on the
conjugate can regulate intracellular signaling events.
protein tyrosine phosphorylation signals in LPC- permea-
bilized B cells. pITIM and the biphosphorylated chimera
peptide induced the tyrosine phosphorylation of several
proteins and enhanced the BCR-stimulated phosphoryla-
tion signals, while GD-LD and EL-PN PPs had no de-
Discussion
The interactions between SH2 domains and tyrosine- tectable effect. The two peptides containing FcγRIIb
phosphorylated proteins are essential in signal transduc- sequences probably bind to SHP-2 and compete with
tion. Phosphotyrosine-containing motifs bind specifi- the natural, intracellular phosphatase substrates, hence
cally to certain SH2 domains; they consequently generate allowing phosphorylation. As expected, EL-PN, which
signaling complexes and may regulate the activities of does not bind to SHP-2, does not influence tyrosine phos-
kinases and phosphatases [24, 25, 27, 28]. A number of phorylation events. Surprisingly, the Gab1 PP, GD-LD,
attempts have been focused on interrupting the signaling does not modulate intracellular tyrosine phosphorylation,
cascade at the level of protein-protein interactions, such although it has previously been shown to bind to and ac-
as those involving SH2 domains. Numerous synthetic tivate SHP-2 [39]. A possible explanation is that a 3-min
PPs corresponding to the SH2-domain-binding sites are treatment period was too short for the peptide to exert
able to block the interactions of phosphorylated proteins its effect; alternatively, some other signaling molecule is
with SH2-containing protein targets specifically in vitro required for the regulatory function of GD-LD, which is
[31, 40–45]. However, PPs are unable to penetrate the lost on permeabilization of the cells.
cell membrane as a consequence of the negative charge To turn to a more physiological PP delivery, our goal has
of the phosphate groups at physiological pH [46]. This been to find an optimal cell-permeable vector, which can
problem can be overcome by permeabilization of the cell translocate PPs into intact cells without causing a toxic
membrane or by applying membrane-permeable carrier effect. Cell-membrane permeable arginine- and lysine-
peptides as delivery vectors. The specificity of the phos- rich peptides, such as the transactivation (Tat) domain
photyrosine motifs for certain SH2 domains suggests from HIV1 and the homeodomain of the Antennapedia
that tyrosine-phosphorylated cell-permeable peptides transcription factor from Drosophila (Penetratin) have
can serve as models for the design of peptide-mimetic, been found to penetrate cell membranes directly [1, 2,
small-molecule inhibitors, targeting protein interactions 13]. Membrane-translocating sequences often have nu-
of SH2 domains [47].
merous positive charges. The minimal peptide of HIV1
SHP-2 tyrosine phosphatase is essential in signaling Tat protein contains eight positive charges: six Arg and
downstream of growth factor, cytokine and antigen two Lys residues [2, 6]. Oligoarginines composed of
receptors [20, 21]. It exerts both positive and negative more than six Arg residues have been found to penetrate
regulatory effects on various signaling pathways oper- cell membranes efficiently; furthermore, stearylation of
ating in immune cells. The interactions of SHP-2 with oligoarginine increased the transfection efficiency 100-
Gab1/Gab2 adaptor/scaffolding molecules are indis- fold in COS-7 cells [56].
pensable for the full activation of Erk1/2 upon cytokine Another group of cell-permeable peptides do not contain
and antigen receptor signaling [48], but SHP-2 may also arginine. The best characterized representative of this
mediate the signal transduction of inhibitory receptors group is transportan. This is a 27-amino-acid chimeric
[49]. pITIMs of inhibitory receptors such as Fcγ RIIb peptide consisting of a combination of an N-terminal
[15, 18], programmed death 1 [50], the T cell inhibi- fragment of a neuropeptide, galanin, and the membrane-
tory receptor, CTLA-4 [51], killer inhibitory receptors interacting peptide of the wasp venom, mastoparan [57].
on natural killer cells [52, 53] and the platelet endothe- MPG, a recently developed peptide-based gene delivery
lial cell adhesion molecule-1 (CD31) [54, 55] all recruit system, derived from the fusion peptide domain of the
SHP-2, beside other SH2-domain-containing phospha- HIV1 gp41 protein and the nuclear localization sequence
tases such as SHP-1 and SHIP. Various data suggest that of the SV40 large T antigen, has been reported to form
SHP-2 is required for the functioning of most inhibitory non-covalent complexes with nucleic acids and deliver
receptors.
them into a large panel of cell lines [11, 58].

Cell. Mol. Life Sci. Vol. 63, 2006
Research Article
2691
These previous studies led us to compare Arg-containing in the cytoplasm of BL41 cells, we suggest that the non-
and non-containing peptide delivery vectors in B cells; toxic carriers R8 and O-R8 are internalized by inverted
additionally, we synthesized and tested new CPPs, such micelle formation. The toxic effect of the other CPPs (P-
as palmitoylated and octanoylated octa-arginine, and an R8, MPG and transportan) might be explained by their
oxidized form of MPG. The latter was a side-product of capacity to form pores through the cell membrane, which
the synthesis of MPG sequences, and displayed surprising BL41 cells are not able to repair.
chromatographic behaviors, suggesting a drastic three-di- To check that the CPP (O-R8), which is optimal for BL41
mensional structure change after the modification of the cells, can deliver biologically active PPs into the cells,
methionine residue.
O-R8 was coupled to the SH2-binding PPs, an extended
The B cells were highly permeable for most of these vec- ITIM of FcγRIIb and the Gab1 motif. These PP conju-
tors. Octa-arginine and octanoyl-octa-arginine penetrated gates are internalized in a similar way as the carrier and
all BL41 cells and also primary B cells (data not shown), are located in the cytoplasm after just 5 min. The trans-
and the cell viability was over 80% after incubation for location is dose dependent, temperature independent and,
24 h. R8 was also tested after conjugation to a longer fatty interestingly, the internalization of O-R8-GD-LD does
acid chain (palmitolyl-R8), but this construct was surpris- not increase further after a longer incubation time, as
ingly highly toxic for BL41 cells. MPG and transportan is the case for the O-R8 carrier. This suggests that the
(likewise penetrating all cells) drastically decreased the translocation of O-R8-GDLD is rapid and irreversible,
cell viability in a time- and concentration-dependent and may be mediated by a slightly different mechanism
manner. MPGox did not penetrate the cells, probably due compared with the lone vector.
to the drastic conformational change indicated by various One of the major points of this paper involves determina-
physicochemical methods [our unpublished data]. Trans- tion of the effects of signaling PPs, once they have been
portan exerted the highest toxic effect: 80% of BL41 cells delivered into the cells. The tyrosine phosphorylation
were dead after incubation with this peptide for 1 h. TP10 events were therefore studied using CPP-PP conjugates.
containing only the minimum sequence of transportan, In activated lymphocytes, SHP-2 is recruited to the phos-
which was reported to be still active in membrane trans- phorylated scaffolding/adaptor molecule Gab1 and/or
location [59], was less toxic than transportan. Since the Gab2; consequently, their two N-terminal phosphory-
CPPs used here in BL41 cells were previously applied lated motifs activate the phosphatase [20, 38]. We have
as efficient vectors in different cell types and showed no previously reported that the most efficient activator of
toxic effect, these data together suggested that the optimal SHP-2 is the PP GD-LD of Gab1. Upon co-clustering of
targeting vector had to be chosen circumspectly for vari- BCR and FcγRIIb, the ITIM of the latter becomes phos-
ous types of cells.
phorylated and recruits SHP-2 and SHIP. SHIP is also a
CPPs can translocate into cells by different mechanisms target of tyrosine kinases and may co-operate with pITIM
[7, 60, 61]. Penetratin and R8 peptides have been shown in activating SHP-2 [15, 17, 30, 39]. When delivered to
to have distinct internalization mechanisms in HeLa BL41 cells by O-R8, both the GD-LD and the pITIM-
cells. R8 is taken up by macropinocytosis, while pen- containing peptides modulate the phosphorylation of cer-
etratin translocation is less sensitive to the inhibitors of tain intracellular proteins. These data indicate that these
macropinocytosis and cytochalasin B, an inhibitor of ac- decoy peptides may interact with SHP-2 and activate the
tin rearrangement [60]. Other investigators have shown phosphatase, which in turn may dephosphorylate, for ex-
that Arg-rich peptides can be internalized even at 4 °C, ample, inhibitory tyrosyl residues on src kinases or on its
suggesting that the transportation cannot be explained by negative regulators, such as PAG/CPB [21], resulting in
typical endocytosis [13]. An alternative mechanism for kinase activation and the phosphorylation of target pro-
peptide internalization is that the CPPs, containing many teins. In the presence of anti-IgM stimuli, GD-LD has a
positive charges, may interact with the negatively charged biphasic effect. During the early activation signal, the PP
heads of membrane phospholipids, which may induce a may compete with the natural SHP-2 substrates, while
transient formation of inverted micelles that would engulf later it may operate in the manner described above and
the CPP and its cargo. The micelles may reopen inside induce tyrosine kinase activation. This effect may be lost
the cells, releasing the peptides into the cytoplasm [9, 10, due to the inactivation of the PP by SHP-2.
61]. Some micelles may also fuse with the lysosomes. In conclusion, we have shown that CPPs effectively medi-
The third model is based on the interaction of hydropho- ate the delivery of functional signaling peptides into liv-
bic residues of CPPs with membrane lipids, forming a ing cells. Finding the optimal CPP vector for different
channel through oligomerization. This would allow the types of cells is essential for delivery of the peptide or
efficient passage of hydrophilic molecules, but pore for- secondary modified peptide cargo into the cells. Hence,
mation would also be a threat for cell survival [9]. As all we have shown that PPs that interact with SHP-2 are de-
the CPPs that we have used are internalized at 4 °C as livered into B cells by O-R8, and such conjugates can
efficiently as at 37 °C and are detected as distinct vesicles modulate the events leading to signal transduction. Fur-

2692
Á. Kertész et al.
Phosphopeptide delivery to B cells
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Acknowledgements. The authors are grateful for support from the
National Research and Development Fund (NKFP-1A/040/2004);
Economic Competitiveness Operative Program, GVOP 3.1.1.2004-
05-0183/3.0; OMFB-00541/2004 and Asboth Oszkar grant XTTP-
SRT1 from the National Office for Research and Technology
and the Hungarian National Science and Research Development
Foundation (OTKA) T048848 and K60760. We thank Dr. H. Gu
(Boston, Ma.) for the generous gift of the expression vector pGEX-
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