94 Kelly Tilleman1 Ilse Stevens1 Kurt Spittaels2 Chris Van den Haute2 Stefan Clerens3 Gert Van den Bergh3 Hugo Geerts4 Fred Van Leuven2 Frans Vandesande3 Luc Moens1 1
Department of Biochemistry, University of Antwerp, Antwerp, Belgium 2 Experimental Genetics Group, Center for Human Genetics, K.U.Leuven, Leuven, Belgium 3 Laboratory of Neuroendocrinology and Immunological Biotechnology, K.U.Leuven, Leuven, Belgium 4 Janssen Research Foundation Beerse, Belgium
Proteomics 2002, 2, 94–104
Differential expression of brain proteins in glycogen synthase kinase-3â transgenic mice: A proteomics point of view One of the landmarks of Alzheimer’s disease are neurofibrillary tangles (NFT) in the brain. NFT mainly consist of a hyperphosphorylated form of the protein tau, which is responsible for stabilisation of the neuronal cytoskeleton by microtubule binding and is unable to function properly in its hyperphosphorylated form. Glycogen synthase kinase-3b (GSK3b) is able to phosphorylate tau in a cellular context which could play a role in the formation of these NFT. In order to learn more about the effect of GSK-3b in the brain, two-dimensional electrophoresis patterns of cerebrum extracts of GSK3b[S9A] transgenic mice and wild type mice were compared quantitatively. Fiftyone spots were identified as being different in integrated intensity by at least a factor 1.5. The spots were subsequently identified by mass spectrometry. Identification of several proteins linked to signal transduction pathways in which GSK3b plays a role, indicates that our population of identified proteins includes some down stream proteins of GSK3b. This study may contribute to filling the gaps between GSK3b, its substrates and finally the phosphorylation of tau. Keywords: Glycogen synthase kinase / Alzheimer’s disease / Phosphorylation
1 Introduction Glycogen synthase kinase-3b (GSK3b) was originally isolated as a serine/threonine protein kinase phosphorylating glycogen synthase, thus inhibiting the glycogen synthesis [1]. GSK3b is expressed in many tissues, but is most abundant in the brain. Its expression is widespread in the brain, especially in gray matter of the adult brain. This suggests that in the adult brain, GSK3b might be involved in metabolic pathways and/or signal transduction cascades present in many neurons [2]. Actually, GSK3b is entangled in the regulation of several physiological processes by phosphorylating many substrates including cyclic AMP responsive element binding protein (CREB), c-Myc, c-Jun and b-catenin [1, 3, 4]. It also acts on neuronal cell adhesion molecules, neurofilament and synapsin I [1, 5, 6]. Furthermore, this kinase is capable of phosphorylating enzymes like ATP-citrate lyase and ATP-dependent kinase [7]. GSK3b also phosphorylates adenomatous polyposis coli (APC) tumor suppressor protein [8] and thereby enhances the binding of APC to b-catenin. Phosphorylation of pyruvate dehydrogenase Correspondence: Luc Moens, PhD., Department of Biochemistry, Universiteitsplein 1, B-2610 Antwerp, Belgium E-mail:
[email protected] Fax: +32-3-820-22-48 Abbreviation: GSK-3â, glycogen synthase kinase-3b
ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002
PRO 0119
by GSK3b inactivates the conversion of pyruvate into acetyl-CoA. This causes a dysfunction of the mitochondria, contributing to neuronal death through failure of energy metabolism and leading to reduced levels of acetylcholine due to a reduced level of acetyl-CoA [7]. Recent studies report a requirement for GSK3b as an activator for the transcription factor NF-kB and cell survival [9]. GSK3b may also be implicated in Alzheimer’s disease (AD). It was described as an in vitro and in situ kinase for protein tau [5, 10, 11]. This microtubule-associated protein binds to microtubules and is responsible for stabilizing the cytoskeleton of neurons. Experimental phosphorylation of protein tau prevents its binding to the microtubules and destabilizes the cytoskeleton of transfected cells [12, 13]. Since the phosphorylation of protein tau by GSK3b is enhanced by other kinases, as well as by heparin and tubulin [14], it is likely that GSK3b is a prime candidate for a physiological tau kinase in neurons, which has recently been demonstrated in the brain of GSK3b transgenic mice [15]. Hyperphosphorylated tau is one of the main constituents of neurofibrillary tangles (NFT) found in AD [16, 17]. Many in vitro and in situ studies have been undertaken to understand and reveal the mechanism that leads to these intracellular deposits. The activated form of GSK3b has been found to be colocalized with tangle-bearing neurons in AD patients [18]. Much still remains unknown however, especially what the in vivo relationship between GSK3b and tau, tau phosphorylation and NFT formation is. 1615-9853/02/0101–94 $17.50+.50/0
Proteomics 2002, 2, 94–104
Proteomics study of GSK-3b transgenic mouse brain proteins
This paper describes a study on transgenic mice overexpressing a constitutively active form of GSK3b (GSK3b[S9A]) in neurons of the brain. We used a proteomics approach based on 2-DE and mass spectrometry, in order to gain more in vivo information on the function of this enzyme by identifying proteins whose level of expression is affected.
2 Materials and methods 2.1 Chemical and biological materials Chemicals were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich (Steinheim, Germany). DTT was obtained from ICN Biomedicals (Aurora, Ohio, USA). Organic solvents including HPLC grade ACN were supplied by BDH (Poole, Dorset, England). Acrylamido buffer solutions pK 3.6, 6.2, 7.0, 8.5 and 9.3 were obtained from Fluka (Buchs, Switzerland). Long nanoelectrospray capillaries (cat. no. ES381) were purchased from Protana (Odensk, Denmark). Mice transgenic for GSK3b were constructed and characterized by Spittaels et al. (submitted). A mutated, constitutively active GSK3b, i.e. GSK3b[S9A], was exclusively expressed in neurons of the central nervous system. The enzymatic activity showed approximately a two-fold increase relative to nontransgenic mice. The onset of GSK3b[S9A] expression starts after day P7. Female and male mice subjected to proteomic analysis were 70 and 82 days old, respectively.
2.2 2-DE 2.2.1 Sample preparation Mice brain tissue was rapidly dissected and immediately stored at –807C after freezing in liquid nitrogen. The proteins were isolated from one hemisphere of the cerebrum according to Bauw and Van Montagu [19]. The amount of protein was determined with the bicinchoninic acid (BCA) protein assay kit of Pierce (Rockford, USA). The amount of protein required for 2-DE (500 mg) was precipitated with nine volumes of cold acetone for minimum of 4 h at –207C. After centrifugation at 4000 g, the protein pellet was air dried.
2.2.2 2-DE 2-DE was performed according to Görg et al. [20] with minor adjustments. Briefly, 500 mg of the total mouse brain extract was dissolved in 400 mL lysis solution containing 7 M urea, 2 M thiourea, 2% CHAPS, 100 mM DTT
95
and 2% Pharmalyte 3–10 [21]. The sample was immediately incorporated in a home made linear IPG strip pH 3–10, which was rehydrated for at least 6 h [22]. After rehydration, the strips were focussed on a Multiphor II (Amersham Pharmacia Biotech, Uppsala, Sweden) at 150 V for 30 min, 300 V for 1 h, 1500 V for 1 h and finally at 3500 V for 10 h 54 min so as to obtain approximately 40000 Vh. After IEF, the strips were equilibrated by gently shaking for 2615 min in a solution containing Tris-HCl buffer (50 mM, pH 6.8), 6 M urea, 30% w/v glycerol, 2% w/v SDS. DTT (2% w/v) was added to the first step, and 2.5% w/v iodoacetamide to the second step. After equilibration, the IPG strips were placed on top of a SDS-polyacrylamide gel (12.7% T; 1.6%C) and run in sets of six in the vertical Protean II xi Multi-Cell (Bio-Rad, Hercules, CA, USA) at 40 mA/gel at 107C. The gels were Coomassie stained according to Bauw and Van Montagu [19]. During the second dimension, molecular weight standards (broad range, Bio-Rad) were run alongside the proteins. Their molecular weight values were logarithmically interpolated to the protein spots with the use of the BioImage 2-D analyzer software V 6.1 (Genomic Solutions, Ann Harbor, Michigan, USA). The theoretical pIs of some identified proteins were linearly interpolated to determine the exact nature of the pH gradient of the IPG strips. The stained gels were digitized (Molecular Dynamics Personal Densitometer) and analyzed with the BioImage 2-D analyzer software V 6.1 (Genomic Solutions). A digitized master image of both the GSK3b[S9A] transgenic and the wild type group was prepared for use in our statistical analysis. These master images were composed by matching three gels of wild type male or female mice with each other and afterwards matching the masters of the two different sexes to a final master for the wild type mice. The same was done for the GSK3b[S9A] transgenic mice. In summary, six gels were used for the master image of the wild type mice and six gels were used for the master image of the GSK3b[S9A] transgenic mice. After preparing the master images, all gels of each group were normalized to the same total integrated intensity of all spots. The final master gels are in fact composite images of a set of 2-D gels taken into consideration the variation in position (x, y) and intensity (i) of each spot on each gel. The integrated intensities of all spots appearing on the wild type and GSK3b[S9A] master images were compared. Only the spots of which the integrated intensity differed by a factor of 1.5 minimum and occuring on all gels were further analyzed statistically. For this analysis, the F-test was used to determine if two data sets were significantly different. If the variances were significantly
96
K. Tilleman et al.
different, we used the Welch modified t-test to test the significance of the means, otherwise we used the classical t-test. All the statistical calculations were done in Excel (Microsoft Office 97). Means were concluded as significantly different if pvalue # a. Three significance levels were taken into account: a = 0.1, a = 0.05, a = 0.01.
2.3 N-terminal amino acid sequencing Following 2-DE, the mouse brain proteins were electroblotted onto the PVDF membrane in 10 mM CAPS, pH 11 at 50 V for 3 h and stained with CBB R-250 (0.1% CBB R-250, 40% methanol and 1% acetic acid) for 1 min and destained in 50% methanol. The membranes were stored in distilled water at 47C or air dried. Sequencing of protein spots was performed on an ABI 471-B Sequencer (Applied Biosystems, Foster City, CA, USA) operated in the pulsed liquid mode as recommended by the manufacturer.
2.4 Mass spectrometry 2.4.1 Sample preparation Three to six spots, depending on the intensity of the spot, were pooled, cut into smaller pieces and digested according to Jensen et al. [23]. Prior to MS analysis, the tryptic peptides were dissolved in 40 mL 1% formic acid, desalted and concentrated by ZipTip C18 (Millipore, Bedford, MA, USA). After subsequent washing steps of the ZipTip column matrix with 100% ACN, 50% ACN/0.1% TFA and 0.1% TFA, the sample was loaded onto the ZipTip, equilibrated with 0.1% TFA and washed with 0.1% formic acid. The sample was eluted in 4 mL 60% ACN/ 1% TFA, of which 1–2 mL was loaded in the nanoelectrospray capillary for MS analysis.
2.4.2 Electrospray ionisation tandem mass spectrometry ESI-TOF MS and ESI-TOF MS/MS were performed on a Q-TOF quadrupole TOF mass spectrometer (Micromass, Manchester, UK) fitted with a Z spray dual orthogonal sampling API source (Micromass). ESI was obtained in continuous flow mode using a nanoelectrospray capillary from Protana. The capillary voltage was set to 800 V and the cone voltage to 35 V. For the acquisition of MS data, the quadrupole operates in a wide bandpass mode. The radio frequency (RF) mode was set to scan over a m/z range of 300 to 1900. During MS/MS measurements, the quadrupole is used to select the parent ions operating in RF/direct current mode. Collision energy was manually optimized for each peptide to obtain a complete fragmentation spectrum of the peptide.
Proteomics 2002, 2, 94–104
2.4.3 Protein identification Raw MS/MS data was submitted to the MASCOT (www.matrixscience.com) search engine which was set to query the OWL database. The following search parameters were used: monoisotopic molecular masses, enzyme specificity (trypsin), all species allowed, one missed cleavage site. Peptide mass and MS/MS mass tolerance was set to 0.4 Da. If the results were nonsignificant, the MS/MS spectra were processed manually using the MassLynx PepSeq software (Micromass) and submitted to the BLITZ search engine (www.ebi.ac.uk/ bic_sw) which was set to query the SWALL nonredundant protein sequence database using default parameters.
3 Results 3.1 Quantitative variation in brain proteins of GSK3â[S9A] transgenic mice The digitized master gel is composed of a total of six gels; three gels originating from female mice and three from male mice studied at an age between 70 and 90 d. The master gel of the wild type and GSK3b[S9A] transgenic mice, revealed 718 and 623 spots, respectively. After statistical analysis of the integrated intensity of the corresponding spots, 51 were found to be different by a factor 1.5. Only spots that occurred on all six 2-D gels were analysed. Since we used the master gels derived from three males and three females in our analysis, the results are not sex specific. The intensity of 34 spots was significantly increased in the GSK3b[S9A] transgenic mice. Taking into account the three different significance levels, we found 2, 6 and 26 spots up-regulated with a = 0.1, 0.05 and 0.01, respectively. In addition, the intensity of 17 spots was decreased in the GSK3b[S9A] transgenic mice; 3 spots with a = 0.1, 6 spots with a = 0.05 and 8 spots were down-regulated with a significance level of 0.01 (Fig. 1a–c).
3.2 Identification of the proteins Table 1 shows the identification of the 51 proteins by N-terminal sequencing and by mass spectrometry.
3.2.1 ESI MS/MS We were unable to identify spot 46 and spot 149 due to contamination of keratin. The intensity of these spots was very poor and even by combining several spots, the signal was still insufficient to allow identification. We were able to identify all other spots, except spot 1775. Although
Proteomics 2002, 2, 94–104
Proteomics study of GSK-3b transgenic mouse brain proteins
97
Figure 1. (A) Digitized master gel of soluble brain proteins of wild type mice. 500 mg was loaded onto the gel (IPG 3–10, 12.5% SDS-PAGE). Visualisation by CBB R-250. Region A and B of Fig 1(a) are enlarged by a factor of 2.5 and are represented in Fig. 1(B) and (C) respectively. Spots that are upregulated are numbered in black, pink and blue color, those that are down-regulated are numbered in red, green and orange corresponding to their significance levels, respectively a = 0.1, a = 0.05, a = 0.01. Coloured reprints of the figures can be obtained on request.
FASC_MOUSE
ENOA_MOUSE
Ser/thr protein phosphatase 2B catalytic a isoform T-complex protein 1, theta subunit
Dihydropyrimidinase related protein-2
Dihydropyrimidinase related protein-2 Dihydropyrimidinase related protein-2 No results Tubulin a1 chaina) Tubulin a1 chaina)
Fascin
a enolaseb)
Glutamine sythetase Mitogen-activated protein kinase 1
Isocitrate dehydrogenase
Aldose reductase
124
140
146
149 181 191
219
280
285 344
372
389
147
126
Vesicular fusion protein
cytoplasmic
mitochondrial
ALDR_MOUSE
IDHA_HUMAN
GLNA_MOUSE MK01_MOUSE
TBA1_MOUSE TBA1_MOUSE
DPY2_MOUSE
DPY2_MOUSE
DPY2_MOUSE
TCPQ_MOUSE
P2BA_MOUSE
NSF_MOUSE
P45376
P50213
P15105 P27703
P17182
Q61553
P02551 P02551
O08553
O08553
O08553
P42932
P20652
P46460
P02768
Accession SWISS-PROT
35601.05
36640.21
42145.63 41275.6
46993.19
54273.62
50135.63 50135.63
62170.5
62170.5
62170.5
59555.48
58643.79
82565.4
66472.21
38943
40562
44773 42308
47893
55557
58136 57984
70916
70916
70104
66601
67531
81926
912983 76325
6.79
5.71
6.47 6.5
6.36
6.21
66474 4.94 4.94
5.95
5.95
5.95
5.44
5.58
6.52
5.67
D
D
E
pI
Molecular mass (Da)
6.594
5.547
6.42 6.528
6.254
6.175
6.097 5.05 5.016
5.97
5.97
6.097
5.47
5.47
6.463
5.91 5.57
E
TIGGGDDSFNTFFSETGAGK AVFVDLEPTVIDEVR TIGGGDDSFNTFFSETGAGK LIGQIVSSITASLR YLAPSGPSGTLK FLVVAHDDGR YLAPSGPSGTLK FVEGLPINDFSR DATNVGDEGGFAPNILENK IGAEVYHNLK GVSKAVEHINK LLNETGDEPFQ YK LFPNADSK ELIFEETAR GQVFDVGPR IAEFAFEYAR APIQWEER TIGVSNFNPLQIER RQDLFIVSK
LSQTFPNADFAEITK LGEYGFQNAILVR VLDDGELLVQQTK LLDYVPIGPR IITEGASILR LFEVGGSPANTR NVGLDLEAEVPAVK LFVTNDAATLLR LVPGGGATELELAK (DV)DEVSSLLR IVLEDGTLHVTEGSGR QIGENLIVPGGVK SAAEVIAQAR ISVGSDADLVIWDPDSVK GSPLVVISQGK ISVGSDADLVIWDPDSVK
Peptide Sequence
K. Tilleman et al.
cytoplasmic
cytoplasmic
cytoplasmic
cytoplasmic
cytoplasmic
cytoplasmic
cytoplasmic
ALBU_MOUSE
98
extracellullar
No results Serum albuminb)
ID SWISS-PROT
46 76
Subcellular location
Proteins identified
Spot no.
Table 1. List of identified proteins that are significantly different between GSK3b[S9A] transgenic mice and wild type littermates 98 Proteomics 2002, 2, 94–104
Phosphatidyl inositol transferase a isoform Anti-oxidant protein 2
Hypoxanthine guaninephosphoribosyl transferaseb) Glutathione S-transferaseb) Complexin Nucleoside diphosphate kinase A Dual specificity mitogenactivated protein kinase kinase 1 NADH-ubiquitin dehydrogenase 24 kDa subunit Precursor NADH-ubiquitin oxidoreductase 49 kDa subunit D-3-phosphoglycerate dehydrogenase
420
530
P31938
MPK1_MOUSE
HBA_MESAU
Hemoglobin a chain
NADH-ubiquitin oxidoreductase 23 kDa subunit Synaptonal associated protein Unidentified Ser/thr protein phosphatase 1 catalytic g subunit Transcriptional associated protein purine rich Single stranded DNA binding protein alfa
1700
1731
1850
1746 1775 1799
1646
PP1G_MOUSE PUR_MOUSE
cytoplasmic nuclear
SN25_HUMAN
NUIM_BOVIN
VAB2_MOUSE NFL_MOUSE
endomembrane
Vacuolar ATP synthase subunit b, brain isoform Neurofilament triplet L proteinb)
1604
CORO_MOUSE
Coronin like protein P57 fragment
P42669
P13795 Unidentified P36873
P42028
P01945
P08551
P50517
O89053
Q61753
P17694
NUCM_BOVIN
miochondrial inner membrane, matrix side SERA_MOUSE
P19234
NUHM_RAT
mitochondrial
nuclear and cytoplasmic
O14810 P15532
GTM_MOUSE CLX1_HUMAN NDKA_MOUSE
cytoplasmic
P00493
O08709
P53810
Accession SWISS-PROT
HPRT_MOUSE
AOP2_MOUSE
PPI_MOUSE
ID SWISS-PROT
cytoplasmic
cytoplasmic
Subcellular location
1597
1594
1548
1461
1005
605 612
528
Proteins identified
Spot no.
Table 1. Continued
34883.73
36983.79
23315.08
20195.95
15248.34
61448.34
56584.9
13427
51448.55
49174.47
23902.36
43342.85
15029.98 17207.8
24439.18
24739.45
31762.2
41532
29482 32799 38412
21269
9685
77821
59393
61526
59913
45209
28340
44640
29413 18475 17357
29413
28699
34846
6.07
6.12
4.66
5.21
8.07
4.63
5.57
6.5
6.51
5.95
5.07
6.26
4.93 6.84
6.25
5.72
5.98
D
D
E
pI
Molecular mass (Da)
5.887
4.71 5.15 5.704
5.05
7.88
4.48
5.43
5.995
6.097
5.835
5.337
5.966
6.463 4.936 6.489
6.463
6.096
6.044
E
FFFDVGSNK
EIFISQPILLELEAPIK
AWGNNQDGVVASQPAR
AGTGVDNVDLEAATR QADVNLVNAK GTIQVVTQGTSLK DAGPLLISLK QTTWDSGF AVVQVFEGTSGIDAK IPQSTLSEFYPR SAYSSYSAPVSSSLSVR SFGYDPYF TYFPHFDVSHGSAQVK IGGHGAEYVAEALER LCEAVCPAQAITIEAPR
DIEEIIDELK VLFGEITR
DSDSILETLQR
LTQSNAILR EAEAQAAMEANS EGSLTRPK EISLWFQPEELVEYK TFIAIKPDGVOR IPEQILGK ISELGAGNGGVVFK
PGGLLLGDEAPNFEANTTIGR LSILYPATTGR VIGGDDLSTLTGK
VILPVSVDEYQVGQLYSVAEASK
Peptide Sequence
Proteomics 2002, 2, 94–104 Proteomics study of GSK-3b transgenic mouse brain proteins 99
Peanut-like protein
Transcriptional associated protein purine rich Single stranded DNA binding protein alfab) Creatine kinase b chaina),b)
Creatine kinase b chaina),b) Fascin
T-complex protein 1 (b subunit)
Mitochondrial matrix protein P1 precursor b) a-internexin b) Tubulin a1 chain a) Tubulin b1 chain a) Transformation sensitive protein IEF SSP 3521 Phosphoglucomutase T-complex protein 1 (e subunit)
1852
1854
1893 1912
1916
1925
Vesicular fusion protein
Succinate dehydrogenase flavoprotein subunit Precursor Neurofilament triplet L proteinb)
Heat shock protein HSP 90 b
1977
1980
2018
HS9B_MOUSE
NFL_BOVIN
DHSA_MOUSE
NSF_MOUSE
P11499
P08851
P31040
P46460
P46660
AINX_MOUSE
P38652 P80316
PGMU_RAT TCPE_MOUSE P19226
P46660 P02551 P07437 P31948
AINX_MOUSE TBA1_MOUSE TBB1_MOUSE IEFS_HUMAN
P60_MOUSE
P19226
P80314
Q04447 Q61553
Q04447
P42669
Q99719
Accession SWISS-PROT
CH60_MOUSE
TCPB_MOUSE
KCRB_MOUSE FASC_MOUSE
KCRB_MOUSE
PUR_MOUSE
PNL1_HUMAN
ID SWISS-PROT
83194.09
61448.34
72691.51
82565.4
55869.98
57925.78
21271.95 59624
55869.98 50135.63 49759 62639.26
60955.49
57447.21
42713.26 54273.62
42713.26
34883.73
42776.99
94491
77822
74257
82262
64527
64527
70759 66523
63573 58270 57648 70759
63573
55538
48932 50450
48547
42398
43121
4.97
4.63
7.06
6.52
5.16
5.35
6.32 5.72
5.16 4.94 4.75 6.4
5.91
5.97
5.4 6.21
5.4
6.07
6.21
D
D
E
pI
Molecular mass (Da)
5.05
4.5
6.63
6.36
5.31
5.31
6.41 5.7
5.36 4.99 4.95 6.41
5.36
5.992
5.312 6.36
5.39
5.81
5.887
E
AQLQDLNDR SAYSSYSAPVSSSLSVR NPDDITQEEYGEFYK IDILPNPQER
LSGTGSAGATIR IADGYEQAA LGFAGVVQEISFGTTK TVIIEQSWGSPK LVQDVANNTNEEAGDGTTTATVLAR VGEGFEETLGEAVISTK FANLNEQAAR VLDDGELLVQQTK QSIINPDWNFEK LGANSLLDLVVFGR
VLTPELYAELR GTGGVDTAAVGGVFDVSNADR LAVEALSSLDGDLSGR YWTLTATGGVQSTASTK YLAPSGPSGTLK LSSFIGAIAIGDLVK LALVTGGEIASTFDHPELVK TVIIEQSWGSPK NAGVEGSLIVEK FANLNEQAAR TIGGGDDSFNTFFSETGAGK NSSYFVEWIPNNVK LAYINPDLALEEK
ESAPFAVIGSNTVVEAK INQTVEILK FFFDVGSNK LIDDYGVEEEPAELPEGTSLTVDN KRR
Peptide Sequence
Columns labelled as D and E represent molecular masses or pI obtained from the database or experimentally measured from the 2-D gels, respectively. Identification of proteins labelled by a) are also identified by N-terminal sequencing. Proteins labelled with b) represent proteins also identified in mouse brain by Gauss et al. [25].
cytoplasmic
membrane
mitochondrial inner
cytoplasmic
mitochondrial matrix
cytoplasmic
cytoplasmic
mitochondrial matrix
cytoplasmic
cytoplasmic
cytoplasmic
nuclear
Subcellular location
K. Tilleman et al.
2003
Mitochondrial matrix protein P1 precursor a-internexin
1967
1962
1933 1935 1949
1892
Proteins identified
Spot no.
Table 1. Continued 100 Proteomics 2002, 2, 94–104
Proteomics 2002, 2, 94–104
Proteomics study of GSK-3b transgenic mouse brain proteins
sequence information of 10 peptides was generated for this protein, its identification by database searching was unsuccessful, suggesting that this protein may be a new, for now, unknown protein. In spot 530 we identified two proteins; hypoxanthine guanine phosphoribosyl transferase and glutathione S-transferase. For the latter we were unable to determine the correct isoform. We found both GTM1_MOUSE and GTM2_MOUSE. The presence of serum albumin and hemoglobin a chain probably originates from residual blood present in the brain. The protein species identified can be assigned to several categories (compared to Tsugita et al. [24]) and are summarized in Table 2. Tubulin, one of the major constituents of microtubuli, and the identified neuronal intermediate filaments (a-internexin and neurofilament) were all downregulated. Apparantly, we identified two isoforms of a-internexin. One was up-regulated, the other downregulated. In contrast, fascin and coronin, two actin binding proteins, were up-regulated. Complexin and synaptonal associated protein (SNAP-25) are crucial proteins for the regulation of neurotransmitter release and are downregulated. However, vesicular fusion protein, a cargo protein for all protein material that is transported to the Golgi apparatus was up-regulated. With regard to the chaperones, various subunits of the T-complex protein were up-regulated (+1.59), whereas the mitochondrial chaperone matrix protein P1 precursor is down-regulated (–1.54 to –2.11). Phosphoglucomutase, participating in glucose metabolism, and a-enolase, participating in glycolysis, were upregulated. Proteins responsible for the disposal of the oxygen radicals, antioxidant protein 2 (+1.58) and glutathione S-transferase (+1.89) were also up-regulated. Only two identified proteins were part of the TCA cycle; one up(succinate dehydrogenase flavoprotein, +1.98) and one down- (isocitrate dehydrogenase, –1.62) regulated. In contrast with the overall up-regulation of enzymes active in glycolysis and the glucose metabolism, we found that two identified proteins responsible for the energy transduction (vacuolar ATP synthase subunit b, creatine kinase b chain) were down-regulated in GSK3b[S9A] transgenic mice. Three subunits of the NADH-ubiquitin dehydrogenase complex, a protein responsible for electron transfer to the respiratory chain, were also down-regulated (–1.53 to –1.8). Proteins playing a role in signal transduction cascades (mitogen-activated protein kinase I, phosphatidyl inositol transferase a isoform, dual specificity mitogen-activated protein kinase kinase 1, ser/thr protein phosphatase 1
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(g subunit), ser/thr protein phosphatase 2B (a isoform), dihydropyrimidinase related protein-2; an intracellular signal transducer in development of the central nervous system) were up-regulated. Also proteins playing a role in amino acid synthesis (glutamine synthetase, D-3-phosphoglycerate dehydrogenase) were up-regulated. The expression of hypoxanthine guanine phosphoribosyl transferase, contributing to the purine salvage pathway, was found to be up-regulated (+1.89). Other identified proteins not in one of the categories mentioned above, were all up-regulated.
4 Discussion In this paper we report a proteomics study in which we compare GSK3b[S9A] transgenic mice and wild type animals. Our extraction method obtained only the soluble proteins of the mouse cerebrum. This procedure was chosen because it included a phenol extraction removing the lipids occurring in brain tissue, thus preventing horizontal streaking in the second dimension of the 2-DE. This effect is due to lipids clotting to the proteins and altering their electrophoretic behavior. The 2-D pattern of the mouse brain we analysed, is comparable to the one obtained by Gauss et al. [25], taking into account the differences in sample preparation and 2-DE. Proteins identified in our study that were also identified by Gauss et al. [25] are indicated in Table 1 with b). The integrated intensity of 51 spots differed significantly when brain homogenates of wild type and GSK3b[S9A] transgenic mice were compared. The threshold, at least 1.5 times up- or down-regulation, was chosen arbitrarily to exclude proteins that differ in integrated intensity due to small variations occurring randomly during the experimental setup. Careful considerations have to be taken into account when characterizing the proteins as up- or downregulated. If you consider that phosphorylation or any other additional post-translational modification of a protein results in a shift of the spot on a 2-D gel, it could be possible that the identified spot is a phosphorylated or modified form of the protein and it would be incorrect to assume that the unmodified form of the protein is up- or down-regulated. On the other hand, it is hardly plausible that such a shift can be visualised on a wide pH gradient (IPG 3–10). Since we analysed different spots resulting in the identification of the same protein, it is likely that we identified different isoforms of a protein. How this protein was modified was not examined. The phenotypic characterization of the GSK3b[S9A] transgenic mice was performed by Spittaels et al. [15, submitted]. Briefly, the major phenotype of GSK3b[S9A]
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Table 2. Identification of proteins that are up- or downregulated, sorted by function. A comparison between GSK3b[S9A] transgenic and wild type mice.
Table 2. Continued Spot No.
Protein identification
Spot No.
420
Phosphatidyl inositol transferase a isoform Dual specificity mitogenactivated protein kinase kinase 1 Ser/thr protein phosphatase 1 catalytic g subunit Ser/thr protein phosphatase 2B catalytic a isoform Dihydropyrimidinase related protein-2 Dihydropyrimidinase related protein-2 Dihydropyrimidinase related protein-2
Protein identification
Cytoskeleton 181 Tubulin a1 chain 191 Tubulin a1 chain 1933 Tubulin a1 chain 1935 Tubulin b1 chain 1646 Neurofilament triplet L protein 2003 Neurofilament triplet L protein 1967 a-internexin 1925 a-internexin Actin binding proteins 219 Fascin 1912 Fascin 1597 Coronin like protein P57 fragment
Protein expression up (a)
1.87 (0.05) 1.54 (0.05) 1.97 (0.01) 2.79 (0.05) 2.16 (0.01) 2.36 (0.01) 2.11 (0.1)
Chaperones 126 T-complex protein 1 (theta subunit) 1916 T-complex protein 1 (b subunit) 1962 T-complex protein 1 (e subunit) 1925 Mitochondrial matrix protein P1 precursor 1967 Mitochondrial matrix protein P1 precursor 2018 Heat shock protein HSP 90 b
1.58 (0.1) 2.16 (0.01) 1.55 (0.01)
124 140
up (a)
down (a)
1.5 (0.01) 2.1 (0.01)
1.51 (0.05) 1.51 (0.01) 1.88 (0.01) 2.55 (0.01) 2.12 (0.01)
Amino acid synthesis 285 Glutamine synthetase 1594 D-3-phosphoglycerate dehydrogenase
1.84 (0.01) 2.14 (0.01)
Purine salvage 530 Hypoxanthine guanine phosphoribosyl transferase
1.89 (0.1)
1.56 (0.01)
Redoxprotein 1461 NADH-ubiquitin dehydrogenase 24 1.8 (0.01) kDa subunit precursor 1548 NADH-ubiquitin oxidoreductase 49 1.61 (0.05) kDa subunit 1731 NADH-ubiquitin oxidoreductase 23 1.53 (0.01) kDa subunit 389 Aldose reductase 1.69 (0.05)
1.62 (0.01) 1.6 (0.01)
Detoxication 530 Glutathione S-transferase 528 Anti-oxidant protein 2
1.89 (0.1) 1.58 (0.01)
Blood related 76 Serum albumin 1700 Hemoglobin a chain
1.51 (0.05) 2.48 (0.01)
2.21 (0.01) 1.63 (0.01) 1.54 (0.05) 1.99 (0.01)
1.54 (0.01) 2.11 (0.1) 1.59 (0.05) 1.84 (0.01)
Glucose metabolism 1949 Phosphoglucomutase
1.87 (0.01)
Signal transduction 344 Mitogen-activated protein kinase 1
1799
147 1.86 (0.01) 1.89 (0.01) 2.08 (2.08)
Glycolyse 280 a enolase
TCA cycle 372 Isocitrate dehydrogenase 1980 Succinate dehydrogenase flavoprotein subunit precursor
1005
146
1.62 (0.01)
Energy metabolism 1604 Vacuolar ATP synthase subunit b. brain isoform 1892 Creatine kinase b chain 1893 Creatine kinase b chain Vesicular transport 98 Vesicular fusion protein 1977 Vesicular fusion protein 605 Complexin 1746 Synaptonal associated protein
down (a)
Protein expression
Other 612 1852 1850
1854 1.62 (0.1) 1.98 (0.01) 1949 1.5 (0.05)
Nucleoside diphosphate kinase A 2.01 (0.05) Peanut-like protein 1.78 (0.01) Transcriptional associated protein 1.64 (0.01) purine rich single stranded DNA binding protein alpha Transcriptional associated protein 1.52 (0.1) purine rich single stranded DNA binding protein alpha Transformation sensitive protein IEF1.87 (0.01) SSP 3521
The protein expression level is expressed in fold regulated, the figures represented by (a) express the significance level.
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Proteomics study of GSK-3b transgenic mouse brain proteins
transgenic mice was found to be microcephaly, although the total number of neurons in the brain remained the same, resulting in an increase in neuronal density. However, the somatodendritic compartment of the neurons in the brain of the GSK3b[S9A] transgenic mice were significantly reduced. These observations could be explained by down-regulation of the tubulin and two identified intermediate filaments; neurofilament and a-internexin. It has been reported by Marcus et al. that GSK3b inhibits the function of NeuroD and therefore could prevent neuronal differentiation at a relatively late stage in the developmental pathway [26]. Overexpression of GSK3b could lead to the down regulation of tubulin, identified in our population of proteins. Spittaels et al. also found a decrease in expression of N-tubulin (submitted). In contrast, two actin binding proteins, coronin and fascin were up-regulated. Interestingly, it appeared that both proteins are related to GSK3b through distinct signal transduction pathways. Fascin interacts with b-catenin in a noncadherin complex in vivo [27]. b-catenin plays a role in the Wnt developmental signalling pathway, where it is phosphorylated by GSK3b and directed to the ubiquitin-mediated degradation pathway. Decreased levels of b-catenin do not allow binding to transcription factors and the expression of Wnt-target genes is inhibited [28]. Recent studies report a sensitivity of a pool of mammalian coronin to PI3-kinase [29]. GSK3b is inhibited by PI3/Akt, important mediators of cell survival, upon phosphorylation [30]. The PI3/Akt pathway is involved in cell survival where Akt may function as inhibitor of death-promoting proteins [31]. This pathway also regulates cell size in Drosophila [32], Spittaels et al. discussed this pathway to interpret the obtained microcephaly in their GSK3b transgenic mice (submitted). Spittaels et al. (submitted) also reported a decrease in MAP2 and a two-fold increase in enzymatic activity of GSK3b. MAP2 (mass = 199 kDa, pI = 4.8) was not identified in this study. It is most likely that MAP2 is too large to enter or to exit the IPG strip after IEF, or the concentration of the protein is too small that it cannot be visualised by CBB. In addition, no GSK3b was found in our population of proteins. GSK3b could not be seen on our 2-D pattern because the pI of GSK3b is 8.98. Apparently our IPG 3–10 only reaches to approximately 8.15. To optimize our proteome analysis of mouse soluble brain proteins, we are currently working with IPG pH 6–12 and hope to identify more proteins to conclude our proteome study of the GSK3b transgenic mice. We are also trying to identify the phosphorylated proteins in our population by labelling brain slices with 32P and analyzing these data according to the setup described in this study.
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5 Concluding remarks In conclusion, this proteomics study identified 34 proteins that were up-regulated and 17 proteins which were downregulated in GSK3b[S9A] transgenic mice. Some of the phenotypic characterizations found in these mice could be traced to proteins found in this study. If and how GSK3b is directly responsible for the difference in protein expression observed in this study has not been determined. However, the identification of several proteins linked to signal transduction pathways where GSK3b has been mentioned to play a role, indicates that our population of identified proteins could possess some down stream proteins of GSK3b and may contribute to filling the gaps between GSK3b, its substrates and eventually the phosphorylation of tau. This work was supported by the fund for scientific research Flanders (FWO) grant 144.33.29. Stefan Clerens and Gert Van den Bergh are research assistants of the fund for scientific research Flanders (FWO). Received May 20, 2001
6 References [1] Plyte, S. E., Hughes, K., Nikolakaki, E., Pulverer, B. J., Woodgett, J. R., Biochem. Biophys. Acta 1992, 1114, 147– 162. [2] Leroy, K., Brion, J. P., J. Chem. Neuroanat. 1999, 16, 279– 293. [3] Nikolakaki, E., Coffer, P. J., Hemelsoet, R., Woodgett, J. R., Defize, L. H., Oncogene 1993, 8, 833–840. [4] de Groot, R. P., Derua, R., Goris, J., Sassone-Corsi, P., Mol. Endocrinol. 1993, 7, 1495–1550. [5] Mandelkow, E. M., Drewes, G., Biernat, J., Gustke, N., et al., FEBS lett. 1992, 314, 315–321. [6] Yang, S. D., Song, J. S., Hsieh, Y. T., Liu, H. W., Chan, W. H., J. Protein Chem. 1992, 11, 539–546. [7] Hoshi, M., Takashima, A., Noguchi, K., Murayama, M., et al., Proc. Natl. Acad. Sci. USA 1996, 93, 2719–2723. [8] Rubinfeld, B., Albert, I., Porfiri, E., Fiol, C., et al., Science 1996, 272, 1023–1026. [9] Hoeflich, K. P., Luo, J., Rubie, E. A., Tsao, M.-S., et al., Nature 2000, 406, 86–90. [10] Lovestone, S., Reynolds, C. H., Latimer, D., Davis, D. R., et al., Curr. Biol. 1994, 14, 1077–1086. [11] Hong, M., Chen, D. C., Klein, P. S., Lee, V. M., J. Biol. Chem. 1997, 272, 25326–25332. [12] Michel, G., Mercken, M., Murayama, M., Noguchi, K., et al., Biochim. Biophys. Acta 1998, 1380, 177–182. [13] Wagner, U., Utton, M., Gallo, J.-M., Miller, C. C. J., J. Cell Sci. 1996, 109, 1537–1543. [14] Moreno, F. J., Munoz-Montano, J. R., Avila, J., Mol. Cell Biochem. 1996, 165, 47–54. [15] Spittaels, K., Van Den Haute, C., Van Dorpe, J., Geerts, H., et al., J. Biol. Chem. 2000, 275, 41340–41349. [16] Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y. C., et al., J. Biol. Chem. 1986b, 261, 6084–6089.
104
K. Tilleman et al.
[17] Wood, J. G., Mirra, S. S., Pollock, N. J., Binder, L. I., Proc. Natl. Acad. Sci. USA. 1986, 83, 4040–4043. [18] Pei, J. J., Braak, E., Braak, H., Grundke-Iqbal, I., et al., J. Neuropathol. Exp. Neurol. 1999, 58, 1010–1019. [19] Bauw, G., Van Montagu, M., in: Hanson, E., Harper, G. (Eds.), Differentially Expressed Genes in Plants: A Bench Manual, Taylor & Francis, London 1997, pp.105–118. [20] Görg, A., Obermaier, C., Boguth, G., Harder, A., et al., Electrophoresis 2000, 21, 1037–1053. [21] Görg, A., Boguth, G., Obermaier, C., Posch, A., Weiss, W., Electrophoresis 1995, 9, 531–546. [22] Rabilloud, T., Valette, C., Lawrence, J. J., Electrophoresis 1994, 15, 1552–1558. [23] Jensen, O. N., Wilm, M., Shevchenko, A., Mann, M., in: Link, A. L. (Ed.), Methods in Molecular Biology, vol. 112: Proteome Analysis Protocols, Humana Press, Totowa, NJ, 1999, pp. 513–525.
Proteomics 2002, 2, 94–104 [24] Tsugita, A., Kawakami, T., Uchida, T., Sakai, T., et al., Electrophoresis 2000, 21, 1853–1871. [25] Gauss, C., Kalkum, M., Löwe, M., Lehrach, H., Klose, J., Electrophoresis 1999, 20, 575–600. [26] Marcus, E. A., Kintner, C., Harris, W., Mol. Cell. Neurosci. 1998, 12, 269–280. [27] Tao, Y. S., Edwards, R. A., Tubb, B., Wang, S., et al., J. Cell. Biol. 1996, 134, 1271–1281. [28] De Farrari, G. V., Inestrosa, N. C., Brain Res. Rev. 2000, 33, 1–12. [29] Didichenko, S. A., Segal, A. W., Thelen, M., FEBS Lett. 2000, 485, 147–152. [30] Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., Hemmings, B.A., Nature 1995, 378, 785–789. [31] Datta, S. R., Brunet, A., Greenberg, M. E., Genes Dev. 1999, 13, 2905–2927. [32] Scanga, S. E., Ruel, L., Binari, R. C., Snow, B., et al., Oncogene 2000, 19, 3971–3977.
