m4:01634 revised gem gtpase and tau: morphological changes

M4:01634 Revised

Gem GTPase and tau: morphological changes induced by Gem GTPase in CHO cells

are antagonized by tau

Fumitaka Oyama1,3, Svetlana Kotliarova3, Akihiro Harada2,4, Mamoru Ito5, Haruko

Miyazaki3, Yoshito Ueyama5,6, Nobutaka Hirokawa2, Nobuyuki Nukina3, and Yasuo

Ihara1*

1Department of Neuropathology and 2Department of Cell Biology and Anatomy,

Faculty of Medicine, University of Tokyo, Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo,

Japan; 3Laboratory for Structural Neuropathology, Brain Science Institute, RIKEN,

Wako-shi, Saitama, Japan; 4Laboratory of Cellular and Molecular Morphology,

Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-

machi, Maebashi, Gunma, Japan; 5Central Institute for Experimental Animals,

Kawasaki, Kanagawa, Japan; 6Department of Pathology, School of Medicine, Tokai

University, Isehara, Kanagawa, Japan

Running Title: Gem GTPase and tau

Correspondence to Yasuo Ihara, M.D.

Department of Neuropathology, Faculty of Medicine, University of Tokyo, 7-3-1

Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

TEL: +81-3-5841-3541; FAX: +81-3-5800-6852; E-mail: [email protected]

A series of observations have indicated that tau, one of the major microtubule-

associated proteins, is involved in neuronal cell morphogenesis and axonal maintenance.

Tau is also the major component of paired helical filaments found in the brain affected

1

JBC Papers in Press. Published on April 15, 2004 as Manuscript M401634200

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by Alzheimer’s disease. To explore an as yet unidentified role of tau in vivo, ~11,000

mRNAs were profiled from tau-deficient mouse brains, and compared with those from

control brains at the same ages. The expression of Gem GTPase, a small GTP-binding

protein of the ras superfamily, was significantly increased in the brain of tau-deficient

mice at eight weeks of age. Because Gem GTPase is a negative regulator of the Rho-

Rho kinase pathway for cytoskeletal organization, this protein was transiently

overexpressed in CHO cells that do not express tau. Overexpression of Gem GTPase

induced a marked elongation of CHO cells, and simultaneous expression of tau

eliminated this effect, although tau did not bind directly to Gem GTPase. This anti-

elongation activity of tau was attributed to its microtubule (MT)-binding domain, and

homologous domains of MAP2 and MAP4 exhibited similar antagonistic activities.

Taken together, the present results indicate that the level of Gem GTPase and its cell

elongation activity are modulated by tau, and suggest that tau may be involved in a Gem

GTPase-mediated signal transduction pathway.

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Tau, a microtubule-associated protein (MAP) that is predominantly expressed in

neurons, promotes tubulin assembly into microtubules (MTs) and stabilizes them in

vitro, and presumably also in vivo. It is mainly localized in axons and forms short

cross-bridges between individual MTs (1-4), and has been thought to play an important

role in maintaining neuronal morphology (5-7). However, the central nervous system of

tau-deficient mice produced by gene targeting appeared to be normal, although the

number of MTs was significantly reduced in some small caliber axons (8). In addition,

according to one report (9), axonal elongation of the neurons cultured from tau-

deficient brains is not affected.

The hyperphosphorylated form of tau is the major constituent of paired helical

filaments (PHFs), unusual fibrils that accumulate in subsets of neurons in brains

affected by Alzheimer’s disease (AD) (10). PHF-like fibrils are also one of the striking

characteristics of frontotemporal dementia and parkinsonism linked to chromosome 17

(FTDP-17), which is caused by a number of missense and intronic mutations in the tau

gene (11-13). Because abundance of the unusual fibrils well correlates with the extent

of neuronal loss, these findings suggest that tau is involved in the cascade to neuronal

death in AD and FTDP-17.

Gem GTPase is a small GTP-binding protein of the ras superfamily, the major

function of which has remained to be determined (14,15). Overexpression of mouse

Gem GTPase induced invasive pseudohyphal growth in Saccharomyces cerevisiae (16).

Two functions of Gem GTPase have recently been reported: the inhibition of voltage-

gated calcium-channel activity and a negative regulation of the Rho-Rho kinase

pathway for cytoskeletal organization. Gem GTPase regulates the expression of α-

subunit of voltage-gated calcium channels at the plasma membrane by binding to ß-

subunit (17). Gem GTPase also regulates the cytoskeletal organization mediated by Rho

kinase (ROK) ß. Overexpression of Gem GTPase inhibits ROK-mediated stress fiber

formation, focal adhesion, and neurite retraction (18). In addition to ROKß, Gem

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GTPase has been shown to bind to KIF-9, a kinesin-like protein (19), and to Gmip, a

RhoGAP-containing protein (20).

To clarify an as yet unidentified role(s) of tau, gene expression was compared

between the brains of wild-type (tau+/+) and tau-deficient (tau-/-) mice, using a high-

density oligonucleotide array. We found that the expression of Gem GTPase is

significantly increased in the brains of eight-week-old tau-deficient mice. Here, we

report that the overexpression of Gem GTPase induces elongation of CHO cells, and

that tau antagonizes this effect, the activity of which is attributable to the MT-binding

domain.

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EXPERIMENTAL PROCEDURES

Animals—Male tau-deficient mice (8) that were backcrossed to C57Bl/6J mice for

seven generations and wild-type (wt) control mice were killed at one week, eight

weeks, and ten months of age. Tau-deficient mice grew normally, but displayed some

muscle weakness at ~10 weeks of age (21).

RNA extraction and array hybridization—RNA was extracted from the cerebrum

using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s

recommended protocol. RNA from the cerebral cortices of five tau-deficient mice or

five age-matched wt mice was combined into one sample (~250 µg of total RNA) for

each time point. Poly (A)+RNA was isolated from the RNA samples using an Oligotex

mRNA isolation kits (Takara Bio, Kyoto, Japan). Biotinylated cRNAs were prepared

according to the Affymetrix protocol. Mu11K oligonucleotide arrays (Affymetrix, Santa

Clara, CA) were prehybridized, hybridized, washed, and stained as recommended by the

manufacturer, using a GeneChip Fluidics Station 400 (Affymetrix).

Criteria for selection of abnormally expressed genes—Analysis of the array data

was performed essentially as described previously (22). The present study was designed

to minimize potential experimental confounds as follows. First, a pooled sample was

subjected to the GeneChip analysis, and subsequently the result was confirmed by

examining individual variability. Second, each sample was measured in duplicate to

minimize false positives caused by chip hybridization variability. Four comparisons of

control vs. tau-deficient mice were performed using two hybridization replicates for

each of the pooled samples for wt and tau-deficient groups (22). Finally, some of the

findings were confirmed by quantitative RT-PCR. Using the Affymetrix algorithm (23)

and multiple analysis comparison software for assessing gene expression differences,

mRNAs that were increased or decreased in tau-deficient mice relative to wt mice in all

four comparisons (see ref 22; high stringency analysis) were identified. Default setting

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in the GeneChip3.1 software was used to assign a particular mRNA to be increased,

decreased or unchanged.

Verification of altered expression by reverse transcriptase-coupled PCR (RT-

PCR)—Total RNA was extracted from the brains of eight-week-old wt and tau-deficient mice

using the TRIzol reagent. Contaminating genomic DNA was removed with RQ1

RNase-Free DNase I (Promega, Madison, WI). Quantitative RT-PCR was carried out

to confirm the altered gene expression, as described previously (24), except for the use

of [α-32P] dCTP as tracer. Primers were designed to yield ~350-500 bp cDNA

fragments. The nucleotide sequences of the primers are given in Table S1.

Complementary DNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was

simultaneously amplified as a control. Each PCR cycle consisted of 1 min at 94 ÚC, 1

min at 55 ÚC, and 1 min at 72 ÚC. PCR amplification was performed for 22~24 cycles

for the mRNAs suggested as candidates by GeneChip analysis, and 17 cycles for

GAPDH mRNA. These ranges of cycling provided the exponential amplification

required for quantification of each mRNA. After amplification, the products were

separated on a polyacrylamide gel (6%). The gel was then dried and exposed to an

imaging plate, which was subjected to laser image analysis (Fujix BAS 2000; Fuji Film,

Tokyo, Japan). All values obtained were normalized against the levels of GAPDH

mRNA.

Mammalian expression vectors—Total mouse brain RNA was reverse-transcribed

using random hexamers as primers, as described elsewhere (24). Gem GTPase cDNA

was amplified by PCR from cDNAs thus prepared, using oligonucleotide primers

anchored with EcoRI (5’end) and SalI (3’end) restriction sites. The forward primer was

5’-CCCCAGGAATTCCCACTCTGAATAATGTCACCATGCG-3’, which is in frame

with the carboxyl terminal region of GFP, and the reverse primer was 5’-

GCTCGAGTCGACCTAGAGCACAGACAGGTCATGGC. PCR was carried out for

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35 cycles with each consisting of 1 min at 94 ÚC, 1 min at 55 ÚC, and 4 min at 72ÚC.

The PCR products were then digested with EcoRI and SalI, and subcloned into the

expression vector pEGFP-C3 (BD Biosciences, Palo Alto, CA). The entire nucleotide

sequence of Gem GTPase cDNAs was determined by the dideoxynucleotide termination

method.

A tau-red fluorescent protein (RFP) expression construct was generated as follows.

The full-length wt and mutant human tau cDNAs (four-repeat tau cDNAs) lacking

exons 2 and 3 (0N4R), incorporated into the EcoRI and BglII sites in pCXN2, were

prepared as described previously (25). The tau-encoding sequence without stop codon

was amplified by PCR using the forward primer 5’-

GCTTCCATGGCTCTGAATAATGTCACCATG-3’ and the reverse primer 5’-

TTTTGTCGACTAGAGTACAGAGAGGTCA-3’. The PCR product was then digested

with EcoRI and XhoI, and subcloned into the EcoRI and XhoI sites of pDsRed-N1 (BD

Biosciences). Likewise, cDNAs encoding the amino-terminal half (NTH; residues 2-

220 of the 441-residue isoform; GenBank accession number NP_005901) and the

carboxyl-terminal half (CTH; residues 251-419) of tau, the carboxyl-terminal

fragment of MAP2 (CTF; residues 1668-1805 of the 1827-residiue MAP2; GenBank

accession number NP_002365) and the CTF of MAP4 (residues 1001-1136 of the

1152-residue MAP4; GenBank accession number AAA59553) were also prepared by

PCR. These cDNAs were cloned into pDsRed1-N1 in frame with the DsRed1-coding

sequence.

Transient transfection and fluorescence microscopy—CHO cells were maintained at

37 °C in 5% CO2 in Ham’s F-12 medium (Invitrogen) supplemented with 10% fetal

bovine serum. The cells were co-transfected with cDNA encoding EGFP-C3 alone or

EGFP-C3 fused to Gem GTPase, and cDNA encoding Ds-Red-N1 alone or Ds-Red-

N1 fused to tau, CTF of MAP2, or CTF of MAP4. Cells (250 µl) and each plasmid

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DNA (1 µg) encoding GFP-Gem GTPase and tau-RFP were combined with

Lipofectamine (Invitrogen), essentially according to the manufacturer’s instructions. For

standard fluorescence microscopy, cells on coverslips were fixed for 15 min with 4%

paraformaldehyde in phosphate-buffered saline (PBS) and mounted onto a slide.

Labeled cells were examined under a Zeiss Axioskop microscope (Carl Zeiss, Tokyo,

Japan).

Confocal immunofluorescence microscopy—The cells on chamber slides (Nalge

Nunc International, Nperville, IL) were fixed with cold methanol or 4%

paraformaldehyde, followed by permeabilization with 0.1% Triton X-100 in PBS. The

fixed cells were probed with rat anti-tubulin monoclonal antibody (MAB1864, 1 µg/ml,

Chemicon international, Temecula, CA), followed by incubation with goat anti-rat IgG

(H+L) conjugated with Alexa Fluor 647 (1:1000; Molecular Probes, Eugene, OR), as

described previously (25). Specimens were observed under a confocal microscope (TCS

SP2; Leica Microsystems, Wetzlar, Germany).

Brain protein preparation—Control mouse brain at age of 8 weeks was

homogenized in 10 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing

150 mM NaCl and protease inhibitors (Complete EDTA-free, Roche Diagnostics,

Mannheim, Germany) using a Teflon/glass homogenizer. The homogenate was

centrifuged at 105,000 x g for 15 min at 4 ÚC, and the supernatant was saved for

biochemical analyses. For preparing heat-stable fraction, the supernatant was heated to

100 ÚC for 10 min and cleared by centrifugation at 105,000 x g for 15 min. Protein

concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA).

Interaction of recombinant Gem GTPase and tauSoluble and heat-stable fractions

prepared from mouse brain homogenates were incubated at 4 ÚC for 1 h with

glutathione-Sepharose beads that were prebound to 50 µg glutathione-S-transferase

(GST) or GST-Gem GTPase as described previously (18). Whether tau binds to GST or

GST-Gem GTPase on glutathione-Sepharose was examined by western blotting using

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tau 1 (Chemicon international).

Results

Gem GTPase is upregulated in the brains of eight-week-old tau-deficient

mice—To identify transcripts with altered expression, the levels of ~11,000 mRNAs in the

brains of tau-deficient mice were compared with those from the brain of control mice of

the same ages. To minimize potential confounding factors, pooled RNA samples from

control and tau-deficient mice were subjected to analysis of expression differences on

Affymetrix GeneChip, as described in Experimental Procedures. This initial microarray

screening suggested that the levels of 74 mRNAs including tau are significantly altered

in the brains of eight-week-old tau-deficient mice (Supplemental Table S2).

Quantitative RT-PCR using five wt and five tau-deficient mice indicated that, of these

transcripts, the levels of 13 mRNAs encoding transcription factors, cytokines, a ras-

related GTP-binding protein, protease inhibitors and a membrane protein were indeed

increased (Supplemental Table S3).

Of those genes with upregulated expression, we selected Gem GTPase for further

analysis, because it was significantly and consistently increased in tau-deficient mice.

The levels of Gem GTPase mRNA were most elevated at eight weeks of age in the

brains of tau-deficient mice (P < 0.001, Student’s t-test; Fig. 1), and did not differ

significantly from those of control mice at one week or eight months of age (Fig. 1).

Overexpression of Gem GTPase induces long cellular processes from CHO

cells—Although the major function of Gem GTPase has not yet been determined, this protein

was recently shown to be a negative regulator of the Rho-Rho kinase pathway for

cytoskeletal organization (18). To gain more insight into the function of Gem GTPase,

we examined the effects of its overexpression on the morphology of CHO cells. The

cells were transfected with GFP-tagged Gem GTPase and/or RFP-tagged tau, and

processed for fluorescence microscopy as described in Experimental Procedures.

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First, we examined the intracellular localization of exogenous Gem GTPase and tau

in CHO cells. When cells were fixed with paraformaldehyde, the fluorescence of GFP

or RFP alone was distributed uniformly in the cytoplasm with occasional aggregation,

suggesting that neither GFP nor RFP is bound to cytoskeletal components, such as MTs

(Fig. 2A). In contrast, tau-RFP mainly colocalized with MTs (Fig. 2B and Fig. 1SA).

To confirm that tau binds to MTs, the cells were fixed with cold methanol to wash away

cytosolic tau-RFP. This fixation protocol showed that most tau-RFP colocalized with

MTs (Fig. 1SB), which suggested that the tau in the cytoplasm represents “overflowing”

tau. In many cells transfected with tau-RFP cDNA, MT bundles were observed (Fig. 2B

and Fig. 1S), but no other distinct morphological features were noted compared with

non-transfectant cells (Fig. 2A and B). Thus, overexpression of GFP, RFP, and

especially tau-RFP, induces no morphological changes in CHO cells.

In contrast, CHO cells overexpressing GFP-Gem GTPase and RFP displayed a

remarkable alteration in their morphology. Each cell became highly elongated and often

had one very long process (Fig. 2C). Because this effect was not observed in the GFP-

or RFP-expressing control cells (Fig. 2A), we concluded that Gem GTPase induced this

morphological change in CHO cells.

Surprisingly, the cell elongation induced by Gem GTPase was completely

suppressed by co-transfection with tau-RFP cDNA (Fig. 2D). To localize the active

region of tau that antagonizes the effect induced by Gem GTPase, either the amino-

terminal half (NTH) or the carboxyl-terminal half (CTH) of tau that is fused to RFP

was co-expressed with Gem GTPase. As shown in Fig. 2E and F, whereas the NTH of

tau did not suppress the Gem-GTPase-induced cell elongation, the CTH of tau

suppressed it completely. Thus, this suggests that the antagonistic effect is attributable

to the MT-binding domain of tau.

One hundred CHO cells expressing both GFP and RFP were chosen in an unselected

way and subjected to the quantification. The ratio of long axis (X)/ short axis (Y) of the

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cell was taken as an index for cell elongation; higher values indicate higher degrees of

cell elongation. As shown in Fig. 3, the mean X values for Gem GTPase-

overexpressing cells were remarkably high, compared with those for the control cells

expressing only GFP or RFP (P < 0.01, Student’s t-test). This effect was completely

abolished by the co-expression of tau (P < 0.01).

A number of isoforms of tau are transcribed from a single gene by alternative

splicing. Each isoform is generated by the following combinations: no insert, inclusion

of exon 2-encoded insert or exons 2- and 3-encoded insert in the amino-terminal half

(referred to as S, M, and L, respectively), and exclusion/inclusion of exon 10-encoded

second repeat in the carboxyl-terminal half [referred to as three-repeat (3R) and four-

repeat (4R) tau, respectively]. This rather complicated splicing is developmentally

regulated, and the species S3R that is expressed exclusively in the fetal brain is rapidly

replaced by 4R isoforms after birth in rodent (26-28). We examined whether the effects

of the Gem GTPase are isoform-dependent. The L and M4R isoforms showed similar

inhibitory activities against the effect induced by Gem GTPase (data not shown). The

three-repeat tau also inhibited Gem-GTPase-induced cell elongation (data not shown).

These results are consistent with the assumption that the MT-binding domain of tau has

the activity that antagonizes Gem-GTPase-induced cell elongation.

Antagonizing activity is attributable to the MT-binding domain of MAPs—The

MT-binding region of tau is composed of highly conserved 18-residue-long binding

elements, each having a distinctive Pro-Gly-Gly-Gly motif (29-31). The MT-binding

motifs are also found in the CTFs of MAP2 and MAP4 (Fig. 4A and B). We examined

whether these two homologous MT-binding regions suppress Gem-GTPase-induced

cell elongation. When GFP-Gem GTPase and MAP2 CTF-RFP were co-expressed,

the elongated cell morphology was no longer observed (Fig. 4C and D). The MT-

binding domain of MAP4 had also a similar inhibitory effect (Fig. 4E). Whereas the C-

terminal extensions of tau and MAP2 are highly homologous, that of MAP4 beyond the

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MT-binding domain is not homologous with the former (Fig. 4B). These results

indicate that the MT-binding domains but not the C-terminal extensions have these

antagonistic activities, and suggest that those MAPs as well as tau may regulate the

function of Gem GTPase in vivo.

To clarify whether the inhibitory effect of tau on cellular elongation is inherent to

CHO cells, other cell lines were examined. HeLa, Neuro2a, or PC12 cells were

transfected with either a GFP-Gem GTPase- or a GFP- (control) expressing construct,

and the cell shape was evaluated 24 h later. Overexpression of Gem GTPase in HeLa

cells caused a morphological change similar to that observed in CHO cells, but no such

phenotype was observed in Neuro2a or PC12 cells (data not shown). We do not yet

know whether this negative effect is due to the presence of low levels of tau in these cell

lines.

Tau does not directly bind to Gem GTPase—Finally, we asked whether there is a

direct molecular interaction between Gem GTPase and tau. We sought to detect a

potential interaction using recombinant glutathione S-transferase (GST)-Gem GTPase

and tau in mouse brain extracts. Further, we used the tau in heat-treated mouse brain

extract, because heat-treated tau must be free of tau-interacting proteins. In either case,

we were unable to find any noticeable interaction between Gem GTPase and tau (Fig.

5A and B). In addition, the intracellular distribution of GFP-Gem GTPase and tau-RFP

in transfected CHO cells was carefully assessed using a confocal microscope. When

fixed with paraformaldehyde, the GFP-Gem GTPase was distributed diffusely in the

cytoplasm, consistent with a previous report (18). In contrast, tau-RFP almost

completely colocalized with MTs (Fig. 5C). Similar results were also obtained in the

methanol-fixed cells (Fig. 5D). All of the above results strongly suggest that there is no

direct molecular interaction between Gem GTPase and tau.

Discussion

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DNA microarray technology allows global analysis of neural responses to tau

deficiency in vivo, and offers comprehensive analysis of the altered or compensatory

gene expression involved in tau deficiency. This screening suggests that the expression

of Gem GTPase, a member of the ras superfamily, is significantly increased in the

brains of eight-week-old tau-deficient mice, which was confirmed by quantitative RT-

PCR.

Because the function of Gem GTPase has not yet been determined, we looked for a

possible cell-biological relationship between Gem GTPase and tau. We first found that

Gem GTPase induces profound changes in the cell morphology in CHO cells. A similar

cellular effect of Gem GTPase was reported in other cells, including human umbilical

vein endothelial cells (HUVEC) and HEK-293 cells (19). In addition, Ges, another

Rad/Gem/Kir (RGK) family member and a human ortholog of mouse Rem1, was

reported to induce endothelial cell sprouting (32). Tau is indeed able to antagonize the

effects induced by Gem GTPase, and this activity is localized to the MT-binding

domain of tau (Fig. 2E and F). The motifs of the domain are composed of highly

conserved 18-amino acid repeat identified in tau (29-31), which are also found in

MAP2 and MAP4 (Fig. 4A and B). Either of these MT-binding regions similarly

antagonized the Gem GTPase–induced effect (Fig. 4). These results indicate that

cellular elongation, a Gem GTPase-induced phenotype, can be modulated by MT-

binding domains of tau, MAP2, and MAP4.

A number of exonic and intronic mutations in the tau gene have been identified in

patients affected by frontotemporal dementia and parkinsonism linked to chromosome

17 (FTDP-17). This indicates that tau is involved in neuronal death in subsets of

neurons in this disease, and presumably this is the case with Alzheimer’s disease.

Because the mutant forms of tau associated with FTDP-17 destabilize MTs to a certain

extent in CHO cells (25), the effects of several mutations on the cell elongation induced

by Gem GTPase were evaluated in CHO cells. Co-expression of GFP-Gem GTPase

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and a tau variant, G272V, P301L, V337M, or R406W completely suppressed the

morphological changes (data not shown). Thus, at least these four mutant tau molecules

have similar degrees of the antagonistic activity.

A syndrome known as hereditary dysphasic disinhibition dementia 2 (HDDD2) is

similar in neuropathology to a certain form of FTDP-17 and maps to 17q21-22, but no

mutation in the tau gene has thus far been identified in the members of this family (33).

Recent studies revealed almost complete loss of tau in several HDDD2 brains, despite

apparently normal levels of tau mRNA (34). These results may suggest that HDDD2 is

a distinct tauopathy, possibly caused by a profound reduction in the protein levels of

tau. If so, it would be of particular interest to examine whether Gem GTPase is

upregulated in HDDD2 brains.

The cytoskeletal changes that alter cellular morphology and motility depend upon a

complicated interplay of the molecules that regulate actin, myosin, and other

cytoskeletal components. The Rho family of GTP-binding proteins is an important

upstream mediator for the cytoskeletal organization. Gem GTPase is a negative

regulator on the Rho-Rho kinase pathway, which is involved in stress fiber formation

and neurite retraction (18). Gem GTPase interacts with the Rho pathway through its

association with the Rho effecter, ROKß. Therefore, Gem GTPase may affect cell

morphology by antagonizing the actions of ROKß. Recently, it was shown that

phosphorylation of serines 261 and 289, located in the carboxyl terminal extension of

Gem GTPase, is required for Gem GTPase-mediated cytoskeletal reorganization (35).

Thus, the phosphorylation of serines 261 and 289, which are outside the GTP-binding

region of Gem GTPase, regulates its inhibition of ROKß and associated changes in the

cytoskeleton. The present results provide evidence that one function of Gem GTPase

related to the cell morphology is modulated by the MT-binding region of tau. This

raises the possibility that tau may exist upstream in the Gem GTPase-mediated signal

transduction pathway. Further studies should provide a better understanding of the

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molecular basis of the tau-modulated, Gem GTPase-mediated signal transduction

pathway.

Acknowledgments: We thank Dr. T. Yamazaki for valuable suggestions on fluorescence

microscopy, and Dr. M. Morishima-Kawashima for critical reading of this manuscript.

We also thank Mr. K. Fukumoto for the help for GeneChip analysis and Dr. Y.V.

Kotliarov for providing us a multiple analysis comparison software.

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34. Zhukareva, V., Vogelsberg-Ragaglia, V., Van Deerlin, V. M., Bruce, J., Shuck, T., Grossman, M., Clark, C. M., Arnold, S. E., Masliah, E., Galasko, D., Trojanowski, J. Q., and Lee, V. M. (2001) Ann Neurol 49, 165-175

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Footnotes

* This work was supported by Grants-in-Aid for Scientific Research (No. 14580722)

and for Scientific Research on Priority Areas (C), Advanced Brain Science Project,

from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Abbreviations used: AD, Alzheimer’s disease; CTF, carboxyl-terminal fragment; CTH,

carboxyl-terminal half; FTDP-17, frontotemporal dementia and parkinsonism linked to

chromosome 17; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green

fluorescent protein; MAPs, microtubule-associated proteins; MT, microtubule; GST,

glutathione S-transferase; NTF, amino-terminal fragment; NTH, amino-terminal half;

PHFs, paired helical filaments; RFP, red fluorescent protein; ROK, Rho kinase; RT-

PCR, reverse transcriptase-coupled PCR; wt, wild-type

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Figure legends

Fig. 1. Confirmation by RT-PCR of the upregulation of Gem GTPase mRNA in the

tau-deficient mouse brain. Quantitative RT-PCR was performed using individual wild-

type (Wt) and tau-deficient mice (KO) at one week, eight weeks and eight months of

age. Gem GTPase mRNA levels are normalized against those of GAPDH. Values are

given as mean ± SD (n = 5). The levels of Gem GTPase mRNA were significantly

elevated at eight weeks of age in the brains of tau-deficient mice (P < 0.001, Student’s

t-test), but did not differ significantly from those of control mice at one week or eight

months of age.

Fig. 2. Effect of overexpressed Gem GTPase and tau on the morphology of CHO cells.

CHO cells on glass coverslips were co-transfected with cDNAs encoding GFP alone or

GFP fused to Gem GTPase, and RFP alone or RFP fused to tau, and further cultured for

24 h. The cells were then fixed, and the GFP (left) and RFP (right) fluorescence was

observed under a fluorescence microscope. The transfected cDNAs are as follows: A,

GFP and RFP cDNAs; B, GFP and tau-RFP cDNAs; C, GFP-Gem GTPase and RFP

cDNAs; D, GFP-Gem GTPase and tau-RFP cDNAs; E, GFP-Gem GTPase and the

amino-terminal half of tau (tau NTH)-RFP cDNAs; F, GFP-Gem GTPase and the

carboxyl-terminal half of tau (tau CTH)-RFP cDNAs. Bar, 50 µm.

Fig. 3. Quantification of the cellular elongation induced by Gem GTPase. One hundred

transfected cells were unselectedly chosen from each specimen, and the length of the

long axes (X in µm), and short axes (Y in µm), and their ratio (X/Y) are given as means

± SD. The X values for Gem-GTPase-overexpressing cells were markedly shifted to

the right (data not shown), compared with those of control cells expressing only GFP or

RFP. Co-expression of tau or its CTH abolished the effect.

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Fig. 4. The MT-binding domains of MAP2 and MAP4 suppress Gem-GTPase-induced

cell elongation. Sequence similarities between CTH of tau (169 residues) and CTF of

MAP2 (138 residues) or CTF of MAP4 (136 residues) are shown according to NCBI 2

blast algorithm. A, CTF of MAP2 is highly homologous to CTH of tau [Identities =

90/136 (66%), Positives = 110/136 (80%)]. It has been known that, like tau, MAP2

contains either three or four tandem repeats in the MT-binding domain (36). Each

repeat of MAP2 consists of highly conserved 18 residues. Only the CTF from three-

repeat MAP2 is shown here. B, The CTF of MAP4 has also MT-binding motifs

[Identities = 61/120 (50%), Positives = 78/120 (64%), Gaps = 31/120 (25%)]. The 18-

residue-long MT-binding motifs of tau are indicated by thick lines. C, transfection of

GFP-Gem GTPase and RFP cDNAs; D, GFP-Gem GTPase and MAP2 CTF-RFP

cDNAs; and E, GFP-Gem GTPase and MAP4 CTF-RFP cDNAs. The expression of

CTFs of MAP2 and MAP4 abolished the cell elongation (D, E). Bar, 50 µm.

Fig. 5. Lack of direct interaction of Gem GTPase and tau. Mouse brain extract (A) or

heat-treated mouse brain extract (B) was incubated with glutathione-Sepharose beads

alone (lanes 1 and 4), recombinant GST (lanes 2 and 5)- or GST-Gem GTPase (lanes 3

and 6)-bound glutathione-Sepharose beads. The bound and unbound fractions were

subjected to western blot analysis with anti-tau antibody (tau 1). The tau-reactive band

was undetectable in the bound fractions (A, B). CHO cells were co-transfected with

GFP-Gem GTPase and tau-RFP, and fixed with paraformaldehyde (C) or methanol

(D), followed by immunostaining with tubulin. Specimens were observed under a

confocal microscope. Tau apparently colocalized with MTs in the cells. Bar, 50 µm.

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0

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Fig. 1. Oyama et al.

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50 mm

A. GFP / RFP B. GFP / tau-RFP

C. GFP-Gem / RFP D. GFP-Gem / tau-RFP

E. GFP-Gem / tau NTH-RFP F. GFP-Gem / tau CTH-RFP

GFP │ FP

GFP-Gem │ FP

GFP-Gem tau NTH-RFP

GFP

GFP-Gem

GFP-Gem

tau-│ FP

tau-│ FP

tau CTH-RFP



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-RFP

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-Gem

/ tau

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GFP

/RFP

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/RFP

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-Gem

/ tau

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-Gem

/ tau

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-RFP

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/RFP

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-Gem

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C. GFP-Gem / RFP

D. GFP-Gem / MAP2 CTF-RFP

E. GFP-Gem / MAP4 CTF-RFP

50 mm

GFP-Gem RFP

GFP-Gem

GFP-Gem

MAP2 CTF-RFP

MAP4 CTF-RFP


A. Tau-CTH vs. MAP2-CTF

tau-CTH :DLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCDL NV+SK GS DNIK+ P GG VQIV K +DLS VTSKC

MAP2-CTF:DLKNVKSKIGSTDNIKYQPKGGQVQIVTKKIDLSHVTSKC

tau-CTH :GSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHGSL NI H+PGGG+V+++S KLDFK++ Q+K+GSLDN H

MAP2-CTF:GSLKNIGHRPGGGRVKIESVKLDFKEKAQAKVGSLDNAHH

tau-CTH :VPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDVPGGGN KI++ KL FRE+AKA+ DHGAEI+ +SP S

MAP2-CTF:VPGGGNVKIDSQKLNFREHAKARVDHGAEIITQSPGRSSV

tau-CTH :TSPRHLSNVSSTGSIDMSPR LSNVSS+GSI++

MAP2-CTF:ASPRRLSNVSSSGSINL

B. Tau-CTH vs. MAP4-CTF

tau-CTH :LKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGLKNV+SK+GSTEN+KHQPGGG+VQI NKK+D+S V SKCG

MAP4-CTF:LKNVRSKVGSTENIKHQPGGGRVQIQNKKVDISKVSSKCG

tau-CTH :SKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPSK NIK HKP

MAP4-CTF:SKANIK-------------------------------HKP

tau-CTH :GGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIEGGG V+++S+KL+FK++ Q+K+GSLDN+ H+P GG K E

MAP4-CTF:GGGDVKIESQKLNFKEKAQAKVGSLDNVGHLPAGGAVKTE

MT-binding motif 2

MT-binding motif 3 MT-binding motif 4

MT-binding motif 1

MT-binding motif 2 MT-binding motif 3

MT-binding motif 4


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moc

kG

ST GST

-Gem

moc

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ST GST

-Gem

A. Brain extract

1 2 3 4 5 6

BoundUnbound

10075

5037

kDa

moc

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STG

ST-G

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GST

-Gem

B. Brain heat-stable fraction

1 2 3 4 5 6

BoundUnbound

10075

5037

GFP-Gem tau-RFP

tubulin merge

50 mm

C. Paraformaldehyde fixation D. Methanol fixationGFP-Gem tau-RFP

tubulin merge

50 mm


Glutathione-Sepharose Glutathione-Sepharose


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Supplemental Table S1. The nucleotide sequences of the primers for quantitative RT-PCRmRNA name Forward primer Reverse primerFosB protein (fosB) tcaaccagcacaaccaccagtggacctgtg gtctaaagagtttacagagcaagaagggagc-fos(p55) and p15 E reading frames acttgaagatgagaagtctgcgttgcagac cacagggccagcagcgtgggtgagctcagg prostaglandin synthase tgagtcattcaccagacagattgctggccg tcagtagacttttacagctcagttgaacgcsuppressor of cytokine signalling-3 (SOCS-3) aagcgagtaccagctggtggtgaacgccgt gctccttaaagtggagcatcatactgatccmacrophage inflammatory protein (MIP) atgaaggtctccaccactgcccttgctgtt gtctacctagaatagctgtcaccaaacagtinterferon beta (type 1) coding for the c-terminal part gtcactgtttgtggacggatcaggtcctcg gctgctgtctgatagttctttatttcaccamacrophage inflammatory protein (MIP) ccatgaagctctgcgtgtct catcagggctgaggaggcctPTX3 cttcccaatgcgttcgaagaagatttttgg aacacttaagaaacatactgggctcctccgC10-like chemokine agatgaagccttttcatactgccctctcct gtcattgaggtcagcttcagaccttccaggplatelet-derived growth factor-inducible protein (JE) cagctctctcttcctccaccaccatgcagg ggcatcacagtccgagtcacactagttcacctla-2-alpha atggtttctatctgtgaacagaagctgcag ttaatcttacatgaattcaatgttacactatranscription factor LRG-21 tggccgctctctggaccctggccgccccga cctctgcttagctctgcaatgttccttctttumor-associated membrane protein (TMP) ctctactcagaagtcaccaaaaagccaaga acacagtgactcctgcttccctcccaggttMCP-1 related chemokine cctctagctttcatttcgaa tcagcctagacatgaaggttMyD118, a myeloid differentiation primary response gene cagcgtggtcttgtgcctcc tctcagcgttcctctagagaplasminogen activator inhibitor (PAI-1) gaggccagcacccaccagcgcctcttccac tcaaggctccatcacttgccccatgaagagserum amyloid A (SAA) agaagctggtcaagggtctagagacatgtg tgcatttcacaagtatttattcagcacattC/EBP delta caccctagagctgtgccacgacgaactctt gccgcgcgttaccggcagtcggcgccggtgCD14 ctgcaggcgctccgagttgt atgttccttaaacaaagaggtransglutaminase (TGase) ttcgacgtgtttgcccacatcggcaacgac cccttaggccgggccgatgataacattccggly96 gcccctggctccggaggatg cataaatacataaatattaaC3H cytochrome P450 (Cyp1b1) ccaagtggcctaacccagag ccctccggcctctcacttgcspi2 proteinase inhibitor (spi2/eb4) cagcttacaaccagagaccc gtctcatttggggttggctaadipose differentiation related protein (ADFP) gggcttatcaccaggctctcagcagggtta gggtttactgagctttgacctcagactgctbrain-derived neurotrophic factor ggttcgagaggtctgacgacgacatcactg ccactatcttccccttttaatggtcagtgtinterferon beta (type 2) coding for the C-terminal part ttgatgctgcaacacttaaa catcacaaacactttatttgGADD45 protein (gadd45) aatatgactttggaggaatt ttctccagtagcagcagctcN10 gene for a nuclear hormonal binding receptor cagggcctccgccacctcca gcagccctcacaggtgcggaGem GTPase (gem) atgacagcatggacagcgactgtgaggtct gtgcctagagcacagacaggtcatggcaggAPP-S cagaattcggacatgattca gggcttagttctgcatttgcsmall heat shock protein (HSP25) tccctccaggtgtggacccc gtagcttgcaacttttatttgut-enriched Kruppel-like factor GKLF gaggagcccaagccaaagag cgtgggatttaaaagtgcctinterleukin-11 ctccggcatgtacaatggct agtcacagtcgagtctttaagrowth factor-induced protein (zif/268) tccatttaagacagaaggac ccctttagcaaatttcaattheparin-binding EGF-like growth factor precursor gaagaagagagacccatgcc agtgggagctagccacgcccsignal transducer and activator of transcription (Stat3) atgggctataagatcatgga cacatgggggaggtagcacamyelin associated-glycoprotein (MAG) ccgaggatgatggggaatactggtgtgtgg ctcagtgacaatcccgggtagagacctctcmyelin/oligodendrocyte glycoprotein (Mog) ttctattgggtcaaccccgg ccatcactcaaaaggggtttG-alpha-12 protein taacttggaccggattggccagctgaacta gtctctcactgcagcatgatgtctttcaggGAPDH gtggaagggctcatgaccacagtccatgcc tcttactccttggaggccatgtaggccatg


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Supplemental Table S2. Altered expression of mRNAs in eight weeks old tau-deficient mice by GeneChip analysisProbe Set Fold Change Descriptionx14897_s_at 23.9 Mus musculus FosB protein (fosB) gene, complete cdsv00727_s_at 16.0 Provirus of a replication defective murine sarcoma virus with c-fos(p55) and p15 E reading framesm64291_s_at 11.3 Mus musculus prostaglandin synthase mRNA, complete cds.U88328_at 10.0 Mus musculus suppressor of cytokine signalling-3 (SOCS-3) mRNAm58691-2_s_at 8.5 Mouse TIS11 primary response gene, complete cdsx12531_s_at 8.2 Mouse mRNA for macrophage inflammatory protein (MIP).v00755_s_at 7.4 Messenger RNA fragment for mouse interferon beta (type 1) coding for the c-terminal part.X83601_s_at 7.0 M.musculus PTX3 mRNAu19482_s_at 6.7 Mus musculus C10-like chemokine mRNAw82380_s_at 6.7 Mus musculus cDNA clone 404017 5, mRNA sequencem19681_s_at 6.2 Mouse platelet-derived growth factor-inducible protein (JE) geneX15591_s_at 6.1 Mouse ctla-2-alpha mRNA, homolog. to cysteine protease proregionD67076_s_at 5.9 Mouse mRNA for secretory protein containing thrombospondin motifsm35590_s_at 5.9 Mouse macrophage inflammatory protein 1-beta (MIP-1) mRNAu19118_s_at 5.9 Mus musculus transcription factor LRG-21 mRNAMsa.570.0_g_at 5.5 Mouse connexin (Cx26) geneu25633_s_at 5.4 Mus musculus tumor-associated membrane protein (TMP) mRNAu50712_s_at 5.1 Mus musculus MCP-1 related chemokine mRNAMsa.34555.0_s_at 5.1 Homologous to sp P06685: SODIUM/POTASSIUM-TRANSPORTING ATPASE ALPHA-1 CHAIN M33960_s_at 4.7 Mouse plasminogen activator inhibitor (PAI-1) mRNA, complete cdsX54149_s_at 4.7 Mouse mRNA for MyD118, a myeloid differentiation primary response genex03479_s_at 4.0 Mouse mRNA fragment for serum amyloid A (SAA) 3 proteinX61800_s_at 3.9 M.musculus mRNA for C/EBP deltaC77421_rc_f_at 3.8 Mus musculus cDNA clone J0030G04 3 similar to Mouse B10.VL30LTR geneM55154_s_at 3.6 Mouse transglutaminase (TGase) mRNAx13333_s_at 3.6 Mouse CD14 mRNA for myelid cell-specific leucine-rich glycoprotein.X15592_s_at 3.6 Mouse ctla-2-alpha mRNA, homolog. to cysteine protease proregionMsa.2173.0_at 3.5 M.musculus mRNA for inhibin beta-A subunitaa111276_s_at 3.4 Mus musculus cDNA clone 557308 5 similar to ALPHA-2-MACROGLOBULIN PRECURSOR (HUMAN)X67644_s_at 3.3 M.musculus gly96 mRNAAA289952_at 3.2 Mus musculus cDNA clone 752147 5, mRNA sequence.u03283_s_at 3.2 Mus musculus C3H cytochrome P450 (Cyp1b1) mRNAMsa.31964.0_f_at 3.2 Homologous to sp P05197: ELONGATION FACTOR 2 (EF-2)Msa.7498.0_s_at 2.9 Homologous to sp P24522: GROWTH ARREST AND DNA-DAMAGE-INDUCIBLE PROTEIN GADD45 MIP1-B_at 2.7 #N/AMsa.3437.0_f_at 2.7 Homologous to sp P42930: HEAT SHOCK 27 KD PROTEIN (HSP 27).x51834_f_at 2.7 Murine gene for osteopontinM64086_s_at 2.6 Mouse spi2 proteinase inhibitor (spi2/eb4) mRNAMsa.1376.0_f_at 2.6 Mouse mRNA for early T-lymphocyte activation 1 protein (ETa-1)M93275_s_at 2.5 Mouse adipose differentiation related protein (ADFP) mRNAl28177_s_at 2.4 Mus musculus GADD45 protein (gadd45) genev00756_f_at 2.4 Messenger RNA fragment for mouse interferon beta (type 2) coding for the C-terminal partX55573_s_at 2.4 M.musculus brain-derived neurotrophic factor mRNAx16995_s_at 2.3 Mouse N10 gene for a nuclear hormonal binding receptoraa407278_rc_s_at 2.2 EST02275 Mouse 7.5 dpc embryo ectoplacental cone cDNA library u10551_s_at 2.2 Mus musculus Gem GTPase (gem) mRNAMIP1-B_at 2.2 #N/AMsa.10643.0_s_at 2.2 Homologous to sp P12023: Alzheimer's disease amyloid A4 protein homolog precursorMsa.5082.0_s_at 2.2 Homologous to sp P41220: G0/G1 SWITCH REGULATORY PROTEIN 8.l07577_s_at 2.1 Mus musculus small heat shock protein (HSP25) gene,.aa120653_s_at 2.0 Mus musculus cDNA clone 574724 5 similar to gb:D21261 SM22-ALPHA HOMOLOG (HUMAN)U03421_s_at 2.0 Mus musculus interleukin-11 mRNAu20344_s_at 2.0 Mus musculus gut-enriched Kruppel-like factor GKLF mRNAm22326-2_s_at 1.9 Mouse growth factor-induced protein (zif/268) mRNAx62600_s_at 1.9 Mouse alpha-1-acid glycoprotein (AGP/EBP) mRNAaa184871_s_at 1.8 mu51f10.r1 Soares mouse lymph node NbMLN Mus musculus cDNA clone 642955 5c78546_rc_s_at 1.8 similar to moesin homolog [mice, teratocarcinoma F9 cells, mRNA, mRNA sequence.aa426892_s_at 1.7 Mouse tissue plasminogen activator mRNA, complete cds (MOUSE)l07264_s_at 1.7 Mus musculus (clone lambda mouse 1) heparin-binding EGF-like growth factor precursor mRNAMsa.31166.0_s_at 1.7 Homologous to sp P10644: cAMP-dependent protein kinase type-1 alpha regulatory chainu06922_s_at 1.6 Mus musculus signal transducer and activator of transcription (Stat3) mRNAl21027_s_at -1.6 Mouse A10 mRNAw83858_g_at -1.6 mf26a04.r1 Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA clone 406158 5Msa.24554.0_s_at -1.7 Homologous to sp P28656: BRAIN PROTEIN DN38 Msa.2867.0_s_at -1.9 M.musculus mRNA for astrocytic phosphoprotein, PEA-15m31811_s_at -2.0 Mouse myelin associated-glycoprotein (MAG) mRNAu64572_s_at -2.1 Mus musculus myelin/oligodendrocyte glycoprotein (Mog) mRNAET63463_s_at -2.1 2-HYDROXYACYLSPHINGOSINE 1-BETA-GALACTOSYLTRANSFERASE PRECURSOR aa013647_s_at -2.2 Mus musculus cDNA clone 442176 5, mRNA sequence.Msa.10603.0_at -2.3 Homologous to sp P32076: neuron specific calcium-binding protein hippocalcin (P23K)Msa.22035.0_s_at -2.3 Homologous to sp P48547: POTASSIUM CHANNEL PROTEIN KV3.1/KV4 (NGK2).M63659_s_at -3.0 Mouse G-alpha-12 protein mRNAm18776_s_at -6.8 Mus musculus Him OF1 SPF microtubule-associated protein Tau isoform 23 mRNA


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Supplemental Table S3. Altered expression of 13 mRNAs confirmed by quantitative RT-PCRAccession No mRNA Fold change Functionv00727 c-fos 2.6 cellular oncogene; regulation of transcriptionv00756 IFN beta tpe 2 1.9 interferon-related developmental regulator u10551 Gem GTPase 1.9 GTP binding protein (gene overexpressed in skeletal muscle)m22326 Zif/268 1.7 growth factor-inducible protein; transcription factoru20344 GKLF 1.7 negative regulator of cell growth; regulation of transcriptionX54149 MyD118 1.6 a novel myeloid differentiation primary response geneM64086 spi2 1.6 serine proteinase inhibitor; serpin; serum protein l28177 GADD45 1.5 activation of MAPKK; protein biosynthesis; cell differentiationX15592 ctla-2 1.4 cysteine protease; proteasex13333 CD14 1.4 CD14 antigen; inflammatory response; immune responseu25633 TMP 1.3 tumor-associated gene; a membrane glycoproteinu06922 Stat3 1.3 signal transducers and activators of transcriptionv00755 IFN beta tpe 1 1.2 interferon-related developmental regulator


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50 mm

GFP tau-RFP

tubulin merge

A. Paraformaldehyde fixation

50 mm

GFP tau-RFP

tubulin merge

B. Methanol fixation

Supplemental Fig. 1S. CHO cells were co-transfected with GFP and RFP-taggedtau and fixed with paraformaldehyde (A) or methanol (B) and immunostained withanti-tubulin antibody. Samples were observed using a confocal microscope. Tau is apparently colocalized with MTs in the cells.


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Yoshito Ueyama, Nobutaka Hirokawa, Nobuyuki Nukina and Yasuo IharaFumitaka Oyama, Svetlana Kotliarova, Akihiro Harada, Mamoru Ito, Haruko Miyazaki,

are antagonized by tauGem GTPase and tau: morphological changes induced by Gem GTPase in CHO cells

published online April 15, 2004J. Biol. Chem.

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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M401634200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/04/15/jbc.M401634200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2004/04/15/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/04/15/jbc.M401634200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2004/05/05/M401634200.DC1

http://www.jbc.org/

m4:01634 revised gem gtpase and tau: morphological changes

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