hyperphosphorylation of proteins associated with alzheimer's disease: the case of jnk-interacting

97 Pages·2005·1.54 MB·English

MCP Papers in Press. Published on September 29, 2005 as Manuscript M500226-MCP200
HYPERPHOSPHORYLATION OF PROTEINS ASSOCIATED WITH
ALZHEIMER’S DISEASE: THE CASE OF JNK-INTERACTING PROTEIN 1

Chiara D’Ambrosio 1*, Simona Arena 1*, Gabriella Fulcoli 1*, Meir H. Scheinfeld 2,
Dawang Zhou 2, Luciano D’Adamio 2,3 and Andrea Scaloni 1

1 Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, 80147 Naples, Italy;
2 Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;D
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3 Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, ad
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Ceinge Biotecnologie Avanzate, 80145 Naples, Italy h
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* These authors contributed equally to this work p
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Running title: Hyperphosphorylation of JNK-interacting protein 1. u
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Keywords: JNK-interacting protein 1, phosphorylation, post-translational modification, proteomics, A
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mass spectrometry, Alzheimer’s disease , 20
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Correspondence: Andrea Scaloni
Proteomics and Mass Spectrometry Laboratory
ISPAAM, National Research Council
via Argine 1085, 80147 Naples, Italy
Tel.: ++39-81-5966006; Fax: ++39-81-5965291
Email: [email protected]
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
SUMMARY
The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases are activated by
pleiotropic signals including environmental stresses, growth factors, and hormones. JNK-interacting
protein 1 (JIP1) is a scaffold protein that assembles and facilitates the activation of the mixed lineage
kinase-dependent JNK module, and also establishes a still functionally not characterized interaction with
β-Amyloid Precursor Protein (APP). Here we show that, similarly to other proteins involved in various
neurological diseases, JIP1 become hyperphosphorylated following activation of stress and MAP
kinases. By immobilized metal affinity chromatography and a combined µLC/MALDI-TOF/ESI-IT
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mass spectrometry approach, we have identified thirty-five sites of mitotic phosphorylation within JIP1, lo
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among which eight present within Ser/Thr-Pro sequence. This motif is modified by various kinases also fro
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in aggregates of the microtubule-associated protein tau, which generate typical intra-neuronal lesions ://w
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occurring in Alzheimer’s disease. Most of the post-translational modifications found were located within .m
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the JNK, MAP-KK and RAC-α Ser/Thr protein kinase binding regions; no modifications occurred in line
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protein Src homology 3 and phospho-tyrosine interaction domains, which are essential for binding to y g
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kinesin, APP and MAP-KKK. Protein phosphorylation is known to affect stability and protein-protein n
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interactions. Thus, the findings that JIP1 is extensively phosphorylated after activation of stress and ril 2, 2
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MAP kinases indicate that these signalling pathways might modulate JIP1 signalling by regulating its
stability and association with some, but not all, interacting proteins.
ABBREVIATIONS
Aβ, amyloid β peptides; AD, Alzheimer’s disease; APP, β-Amyloid Precursor Protein; Cdks; cyclin-
dependent kinases; DHB, 2,5-dihydroxy-benzoic acid; ESI, electrospray; IMAC, immobilized metal
affinity chromatography; IT, ion trap; JNK, c-Jun N-terminal kinase; JIP1, JNK-interacting protein 1;
K, kinase; LC, liquid chromatography; MALDI, matrix assisted laser desorption ionization; MAP,
mitogen activated protein; MS, mass spectrometry; MS-MS, tandem mass spectrometry; NFT,
neurofibrillary tangles; PVDF, polyvinylidene fluoride; Q, quadrupole; TOF, time of flight.
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INTRODUCTION
Alzheimer’s disease (AD), the most common senile dementia, is characterized by amyloid plaques,
vascular amyloid, neurofibrillary tangles (NFT) and progressive neurodegeneration. Amyloid plaques
are mainly composed of Aβ peptides (Aβ40 and Aβ42), which are derived from processing of the
ubiquitous type I transmembrane β-Amyloid Precursor Protein (APP) by various secretases (1-4). A
pathogenic role for APP processing in AD has been ascertained by the finding that mutations in APP
(5) and presenilins (6-9), key components of the γ-secretase, cause AD autosomal dominant familial
forms. In AD, APP is first cleaved at plasma membrane by β-secretase (10) releasing the
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APPβ ectodomain extracellularly or into intracellular compartments, while its C-terminal fragment of loa
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99 amino acids (C99) remains membrane bound. Then, C99 is cleaved by γ-secretase, generating m
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amyloidogenic Aβ peptides and an intracellular product named APP Intracellular Domain (AID) (11- ://w
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13). In non amyloidogenic proteolytic pathway, APP is first processed by α-secretase in the Aβ p
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sequence leading to the production of APPα and a membrane bound C-terminal fragment of 83 amino rg
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acids (C83). Then, C83 is cleaved by γ-secretase into P3 and AID fragments. While Aβ is implicated in ue
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the pathogenesis of AD, AID mediates most of the APP signalling functions. In fact, AID fragment is p
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able to activate expression of various genes in combination with its binding-partner Fe65 (12) or 01
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repress it (14-15). Indeed, it has been shown that upon over-expression of APP, Fe65 and Tip60, a
repressor complex assembled on the KAI-1 promoter is replaced by a complex containing AID, Fe65,
Tip60 (16). All these reports reinforce the role for AID in gene regulation.
Another pathological feature of AD is the presence of intra-neuronal lesions known as NFT. These are
generated by hyperphosphorylated aggregates of the microtubule-associated protein tau and coincide
with cell death in several neurodegenerative diseases known as tauopathies (17, 18). Mutations in the
tau gene have been linked to NFT formation in a number of tauopathies in which no β-amyloid
deposits develop (19, 20). However, no tau gene mutations have been found in AD, and the relationship
between tau pathology, amyloid deposition, and neuronal degeneration is still not clear (21, 22).
Rampant hyperphosphorylation of tau seems to be the key event associated to NFT formation (17, 18).
A complete characterization of tau phosphorylation sites in normal and AD brain required massive
analysis by different mass spectrometric methodologies (23-25). Around thirty modified Ser/Thr
residues were identified in tau samples from AD brain, among which a dozen are present in Ser/Thr-
Pro sites. These motifs, similar to those occurring in other disease-related proteins, are subjected to
physiological isomerization and dephosphorylation events (controlled by the peptidyl-prolyl cis/trans
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isomerase Pin1 and the phosphatase PP2A, respectively), which regulate protein conformation/function n
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and NFT formation (18, 26, 27). Growing body of evidence suggests that inappropriate activation of fro
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the cell cycle, especially mitotic events, may contribute to this abnormal hyperphosphorylation, and ttp
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thereby play an important role in the development of the disease (18, 28-33). .m
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We have previously identified JNK Interacting Protein-1 (JIP1) as an APP interacting protein (34, 35). lin
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JIP1 was initially identified as an inhibitor of JNK activation but was soon shown to be a scaffold b/
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protein, which bound various components of the JNK cascade including MKK7, MLK, DLK and other t o
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upstream kinases (36). The significance of this APP-JIP1 interaction is still unknown. It is tempting to ril 2
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speculate that JIP1-APP interaction could provide the molecular basis for activation of signalling 9
cascades involving mitogen activated- and stress kinases in the brain of AD patients, which may be
ultimately responsible to tau hyperphosphorylation and NFT formation (37-41). Preliminary site-
directed mutagenesis experiments demonstrated that JIP1 is also phosphorylated and its modification
regulates JNK module dynamics and activation (42). In an attempt to define a possible role of JIP1 in
hyperphosphorylation events present in AD cases, which are accompanied by activation of various
kinases, we have studied the phosphorylation of JIP1 in response to activation of MAP and stress
kinases. This investigation, based on the massive use of some recent MS approaches developed to
study protein phosphorylation, demonstrates that also JIP1 is hyperphosphorylated.

EXPERIMENTAL PROCEDURES
Reagents - Monoclonal anti-APP N-terminal antibody 22C11 was purchased from Chemicon and anti-
α-tubulin DM 1A was obtained from Sigma. Polyclonal anti-APP C-terminal antibody was purchased
from Zymed Laboratories. Polyclonal anti-JNK or phosphorylated JNK, anti-ERK or phosphorylated
ERK and anti-p38 or phosphorylated p38 were obtained from Cell Signaling Technology. Anti-Flag
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was from Sigma, anti-phospho-Tyr and anti-phospho-Thr antibodies were from Cell Signaling o
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Technology. Sequencing grade-proteolytic enzymes and phosphatases were purchased from Roche. de
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Ultrapure HPLC-grade solvents were from Baker. All chemical reagents were purchased from Sigma. h
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Cell Culture and transfection - HEK293 cells were maintained in Dulbecco's modified Eagle's medium lin
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(Invitrogen) supplemented with 10% fetal bovine serum (Biofluids) and penicillin/streptomycin. Cells by/
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were transfected with a construct coding for FLAG-tagged JIP1 by using FuGENE6 (Roche), as t o
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previously described (35). Cells were untreated (control) or treated with anisomycin (10 µg/ml) for 30 ril 2
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min, and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 70 mM NaCl, 1 mM Na VO , 1 mM NaF, 1% 9
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v/v Triton X-100) containing 2 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride. Lysates were boiled in SDS sampling buffer and subjected to SDS-PAGE and Western
blotting with the indicated antibodies.

Immunoprecipitation and immunoblotting analysis - Twenty-four to forty-eight hours following
transfection, cells were washed with ice-cold PBS, and lysed in lysis buffer containing a protease
inhibitor tablet (Roche). Lysis was allowed to continue for 10 min on ice and was then spun down at
full speed, at 4°C, for 10 min. Some lysate representing the total lysate was removed and boiled with
SDS loading buffer containing DTT, while the rest was immunoprecipitated for 2 h, at 25°C, with a
monoclonal antibody directed against the Flag epitope already bound to agarose beads (Sigma). The
beads were washed 5 times with lysis buffer and boiled with SDS loading buffer with DTT.
Immunoprepitates and total lysates were separated by SDS-PAGE and blotted onto nitrocellulose
(Biorad). Membranes were probed with the same antibodies as was used for immunoprecipitation and
HRP conjugated secondary antibodies were used. Proteins were detected using the Supersignal West
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Pico chemiluminescent system (Pierce). n
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In vivo orthophosphate labelling of cells - HEK293 cells were transfected with the indicated construct. w
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After 24 h, cells were washed 3 times with phosphate-free Dulbecco’s modified Eagle’s medium and po
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incubated at 37°C in the same medium, for 30 min. Cells were then incubated in phosphate-free .org
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Dulbecco’s modified Eagle’s medium containing 0.5% dialyzed fetal bovine serum and 1 mCi/ml [γ- u
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32P]ATP orthophosphate (Amersham Biosciences) for 4 h, at 37°C. Ap
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Phosphoamino acid analysis - Cellular extracts were resolved by SDS-PAGE and transferred to a
PVDF membrane; [32P]-labelled JIP1 was identified by autoradiography and excised from the
membrane. Blotted protein was subjected to vapour-phase hydrolysis with 6 N HCl, at 110°C, for 3 h
(43). Two-dimensional phosphoamino acid analysis was carried out on thin layer cellulose plates using
the Hunter thin-layer electrophoresis system (44). The pattern of phosphoamino acids was visualized
by exposing the dried cellulose plates to a model Storm-860 PhosphorImager plate (Amersham
Biosciences).
µLC-ESI-Q-MS analysis – Aliquots of protein extracts containing human JIP1 were analysed by using an
API-100 single quadrupole mass spectrometer (Perkin Elmer-SCIEX) equipped with an electrospray
source connected to a Phoenix 40 pump (ThermoFinnigan, USA). Protein extracts were separated on a
capillary Hypersil-Keystone Aquasil C4 Kappa column (100 x 0.32 mm, 5 µm) using a linear gradient
from 2% to 60% of acetonitrile in 0.1% (v/v) formic acid, over 80 min, at flow rate of 5 µl/min. Spectra
were acquired in the range 200-2000 m/z, as previously reported (45). Data were analyzed using the
BioMultiView 3.1 software provided by the manufacturer. Mass calibration was performed by means of
the multiply charged ions from a separate injection of horse heart myoglobin (molecular mass 16951.5
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Da). Masses are reported as average values. lo
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In-situ digestion with proteolytic enzymes - Samples containing 10-50 µg of total proteins were subjected ://w
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to electrophoresis under denaturing conditions on 12% polyacrylamide gels (46). Separated bands were c
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visualized by colloidal Coomassie G250 staining. JIP protein was excised from the gel, triturated, in-gel e.o
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reduced, S-alkylated and digested either with trypsin or endoprotease AspN, as previously reported (47). g
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Gel particles were extracted with 25 mM NH4HCO3/acetonitrile (1:1 v/v) by sonication and peptide n A
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mixtures were concentrated. , 2
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Phosphopeptides enrichment – To isolate phosphopeptides, whole digest aliquots solved in 5% (v/v)
acetic acid were loaded on SwellGel Ga3+-chelated discs columns (Pierce) and incubated for 5 min, at
25°C. Columns were centrifuged at 5000 rpm, for 10 s. Columns were washed with 0.1% (v/v) acetic
acid (once), 10% (v/v) acetonitrile in 0.1% (v/v) acetic acid (twice), 50% (v/v) acetonitrile in 0.1% (v/v)
acetic acid (twice), and finally with water. Phosphopeptides were initially eluted with 100 mM
NH HCO , pH 8 and finally with 50% (v/v) acetonitrile in 100 mM NH HCO , pH 8. Eluted solutions
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were mixed and analyzed for phosphopeptides as described below.
µLC analysis – Aliquots of whole JIP1 digests or phosphopeptide-enriched samples were resolved on a
Phoenix 40 LC system (ThermoFinnigan, USA). Peptides were separated on a microbore Jupiter Proteo
C12 column (150 x 0.5 mm, 4 µm) using a linear gradient from 2% to 60% of acetonitrile in 0.1% (v/v)
trifluoroacetic acid, over 80 min, at flow rate of 20 µl/min. Individual components were collected
manually and freeze-dried.

MALDI-TOF-MS analysis – Aliquots of phosphopeptide mixtures or individual fractions from µLC
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separation were loaded on the MALDI-TOF instrument target together with 2,5-dihydroxy-benzoic acid lo
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(30 mg/ml in 50% (v/v) acetonitrile/1% (v/v) orthophosphoric acid) as matrix (48), using the dried from
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droplet technique. Samples were analysed with a Voyager-DE PRO spectrometer (Applera). Spectra ://w
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were acquired in positive and negative polarity, using the instrument either in linear and reflectron mode. c
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Internal or external mass calibration was performed with peptides derived from enzyme autoproteolysis e.o
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or with molecular mass markers (Applera). Data were elaborated using the DataExplorer 5.1 software g
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(Applera). Masses are reported as monoisotopic values. n A
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µLC-ESI-IT-MS-MS analysis – Aliquots of whole JIP1 digests or phosphopeptide-enriched samples were
analysed by using a LCQ Deca Xp Plus mass spectrometer (ThermoFinnigan, USA) equipped with an
electrospray source connected to a Phoenix 40 pump (ThermoFinnigan, USA). Phosphopeptides were
separated on a capillary Hypersil-Keystone Aquasil C18 Kappa column (100 x 0.32 mm, 5 µm) using a
linear gradient from 2% to 60% of acetonitrile in 0.1% (v/v) formic acid, over 80 min, at flow rate of 5
µl/min. Spectra were acquired in the range 200-2000 m/z. Mass spectrometer was set up to automatically
acquire a full MS scan, followed by 3 MS2 spectra of the 3 most abundant ions in the MS spectrum.
Spectra were acquired using a dynamic exclusion of 3 min, with a mass width of ± 2.0 m/z. Data were
elaborated using the BioWorks 3.1 software provided by the manufacturer. Masses are reported as
monoisotopic values.

Alkaline phosphatase treatment – Phosphopeptides were dephosphorylated by mixing 3 µl of each
sample solved in 50 mM NH HCO , pH 8.5, with 2 U of shrimp alkaline phosphatase (Roche) solved
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in 2 µl of 50 mM NH HCO . Reaction mixtures were incubated for 60 min, at 37°C, followed by
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addition of 1 µl of 10% (v/v) formic acid. In the case of whole digests, peptide mixtures were desalted
on ZipTipC18 pipette tips (Millipore) before MALDI-TOF-MS analysis.
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Phosphopeptides subdigestion - Phosphopeptides solved in 5 µl of 50 mM NH HCO , pH 8, were fro
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subdigested with 30 ng of endoprotease AspN, chymotrypsin, trypsin or carboxypeptidase A. Reaction ://w
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mixtures were incubated for 360 min, at 37°C, followed by addition of 1 µl of 10% (v/v) formic acid. c
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Digests were directly analyzed by MALDI-TOF-MS. e.o
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Data analysis – Observed MALDI-TOF mass values were assigned to JIP1 phosphopeptides/peptides n A
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using the GPMAW 4.23 software (Lighthouse Data). This software generated a mass/fragment , 2
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database output, based on JIP sequence, protease selectivity, nature of the amino acid susceptible to
eventual post-translational modification and molecular mass of the modifying groups. Mass values
were matched to protein regions using a 0.02% mass tolerance value. Sequest software allowed
automated identification of JIP1 phosphopeptides/peptides using µLC-ESI-IT-MS-MS data (49, 50).
This software was run against an indexed database containing human JIP1 and proteases sequence, by
using Met oxidation and Ser/Thr phosphorylation as differential modifications. A value of 2.5 and 1.0
m/z was used as mass tolerance for parent and fragment ions, respectively. Candidates with a Sequest