Valosin-containing protein (VCP) is a novel IQ motif-containing GTPase activating protein 1 (IQGAP1)-interacting protein

Accepted Manuscript Valocin-containing protein (VCP) is a novel IQ motif containing GTPase activating protein 1 (IQGAP1) interacting protein Norimichi Itoh, Taku Nagai, Takashi Watanabe, Kentaro Taki, Toshitaka Nabeshima, Kozo Kaibuchi, Kiyofumi Yamada PII:

S0006-291X(17)31949-6

DOI:

10.1016/j.bbrc.2017.09.159

Reference:

YBBRC 38604

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 22 September 2017 Accepted Date: 28 September 2017

Please cite this article as: N. Itoh, T. Nagai, T. Watanabe, K. Taki, T. Nabeshima, K. Kaibuchi, K. Yamada, Valocin-containing protein (VCP) is a novel IQ motif containing GTPase activating protein 1 (IQGAP1) interacting protein, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.09.159. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Valocin-containing protein (VCP) is a novel IQ motif containing GTPase

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activating protein 1 (IQGAP1) interacting protein.

3 Norimichi Itoh1,2, Taku Nagai1, Takashi Watanabe2,3, Kentaro Taki4, Toshitaka

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Nabeshima5, Kozo Kaibuchi2, Kiyofumi Yamada1.

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1. Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya

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University Graduate School of Medicine: 65 Tsurumai-cho, Showa, Nagoya,

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Aichi 466-8560, Japan

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2. Department of Cell Pharmacology, Nagoya University Graduate School of

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Medicine: 65 Tsurumai-cho, Showa, Nagoya, Aichi 466-8550, Japan

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3. Present address: Department of Pharmacology, University of North Caroline

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at Chapel Hill, School of Medicine: Chapel Hill, NC 27599 USA

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4. Division for Medical Research Engineering, Nagoya University Graduate

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School of Medicine: 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550,

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Japan

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5. Advanced Diagnostic System Research Laboratory Fujita Health University,

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Graduate School of Health Sciences and Aino University, 1-98 Dengakugakubo,

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Kutsukake-cho, Toyoake, Aichi 470-1192, Japan

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Address correspondence to: Kiyofumi Yamada, PhD.

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Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya

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University Graduate School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya

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466-8560, Japan

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Tel.: +81-52-744-2674, Fax: +81-52-744-2979

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E-mail: [email protected]

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Keywords: IQGAP1; VCP; proteomics

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Abstract Scaffold proteins play a pivotal role in making protein complexes, and

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organize binding partners into a functional unit to enhance specific signaling

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pathways. IQ motif-containing GTPase activating protein 1 (IQGAP1) is an

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essential protein for spine formation due to its role in scaffolding multiple signal

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complexes. However, it remains unclear how IQGAP1 interacts within the brain.

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In the present study, we screened novel IQGAP1-interacting proteins by a

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proteomic approach. As a novel IQGAP1-interacting protein, we identified

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valocin-containing protein (VCP) which is a causative gene in patients with

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inclusion body myopathy with Paget’s disease of bone and frontotemporal

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dementia (IBMPFD). The physiological interaction of IQGAP1 with VCP was

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confirmed by an immunoprecipitation assay. Both the N-terminal (N-half) and

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C-terminal (C-half) fragments of IQGAP1 interacted with the N-terminal region of

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VCP. Co-localization of IQGAP1 and VCP was observed in the growth corn,

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axonal shaft, cell body, and dendrites in cultured hippocampus neurons at 4

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days in vitro (DIV4). In cultured neurons at DIV14, IQGAP1 co-localized with

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VCP in dendrites. When HEK293T cells were co-transfected with IQGAP1 and

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VCP, an immunoprecipitation assay revealed that binding of IQGAP1 with

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disease-related mutant (R155H or A232E) VCP was markedly reduced

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compared to wild-type (WT) VCP. These results suggest that reduction of

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IQGAP1 and VCP interaction may be associated with the pathophysiology of

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IBMPFD.

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1. Introduction In response to extracellular signals, such as growth factors, cells

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propagate signal transduction systems by recruiting signal components

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spatiotemporally. To achieve this event, signal components should be precisely

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localized and form a complex with other signaling components. Scaffolding

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proteins contain several protein-interacting domains assembling signal

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transduction proteins to form a spatiotemporal complex [1-3]. This coordination

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of signal transduction systems by scaffolding proteins leads to several cellular

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events. Depending on the signal input, scaffolding proteins select different

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binding partners to activate specific signaling cascades. Therefore, scaffolding

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proteins play a pivotal role in organizing and enhancing specific signal cascades.

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IQ motif-containing GTPase activating protein 1 (IQGAP1) is a key

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molecule for a variety of cellular processes, such as directional migration in

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fibroblasts [4, 5], cell-cell adhesion in epithelial cells [6, 7], cell proliferation in

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cancer cells [8], and spine morphogenesis in neurons [9], through regulation of

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actin cytoskeleton and microtubules. In addition to the regulation of actin

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cytoskeletons [10], IQGAP1 serves as a scaffold protein that recruits several

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proteins such as extracellular signal-regulated kinase 1/2 (ERK1/2) [11],

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cytoplasmic linker protein p170 (CLIP-170) [4], and Rho family small GTPase

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Rac1 and Cdc42 [12-14].

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It has been reported that IQGAP1 knockout mice show cognitive

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dysfunction with impairment of long-term potentiation in the hippocampus [15].

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Each IQGAP domain region has a different function in the regulation of spine

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density and morphology [9]. The actin-binding CHD domain is required for spine

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head formation, while the Ras-binding GRD domain is responsible for stalk spine

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extension. The C-terminal region in IQGAP1 contains the RasGAP-C domain,

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which several proteins bind to. The role of NR region in spine formation was not

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shown. Although the role of IQGAP1 is not yet fully understood, comprehensive

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proteomic analysis has increased our understanding of the novel physiological

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and pathophysiological functions of IQGAP1 in the central nervous system.

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Here, we performed comprehensive proteomic analysis using a

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combination of affinity column chromatography and LC-MS/MS to clarify the

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regulation mechanism of IQGAP1 in the brain. We identified valocin-containing

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protein (VCP) as a novel IQGAP1-binding protein, and suggested a possible

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association of IQGAP1 with neurodegenerative disorders such as inclusion body

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myopathy with Paget’s disease of bone and frontotemporal dementia (IBMPFD)

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and Amyotrophic lateral sclerosis (ALS).

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2. Material and methods

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2.1. Reagents and chemicals The cDNA encoding mouse IQGAP1 was obtained as previously

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described [4]. Anti-myc polyclonal antibody was obtained from Cell Signaling

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Technology (Beverly, MA). Anti-GFP monoclonal antibody was from MBL

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(Nagoya, Japan). Anti-IQGAP1 polyclonal antibody was from Santa Cruz

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Biotechnology (Santa Cruz, CA). Monoclonal anti-VCP antibodies were

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purchased from Transduction Laboratories (Lexington, KY) and Abcam

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(Cambridge, UK). Other materials and chemicals were obtained from

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commercial sources.

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2.2. Plasmid construction

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Full-length and deletion mutants of VCP were amplified by RT-PCR

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from HeLa total RNA, and inserted into a pCRII-blunt TOPO vector (Invitrogen,

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Eugene, OR). After the sequence was confirmed, cDNA was subcloned into a

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pCAGGS-myc vector at the EcoRI site. Plasmids containing IQGAP1 deletion

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mutants were as previously described [4].

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To create mutant VCP, PCR was performed using a PrimeStar

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mutagenesis kit (Takara, Japan). Primers used in this experiment are as follows;

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VCP R155H-F, 5’-CTTGTCCATGGTGGGATGCGTGCTGTG-3’; VCP R155H-R,

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5’-CCCACCATGGACAAGAAAAATGTCTCC-3’;

VCP

A232E-F,

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5’-TTTAAGGAAATTGGTGTGAAGCCTCCT-3’;

VCP

A232E-R,

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5’-ACCAATTTCCTTAAAGAGGGCAGGATG-3’.

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2.3. Purification of recombinant proteins and affinity column chromatography

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The purification of glutathione S-transferase (GST)-fusion proteins from

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E.coli

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chromatography was performed as previously described [14]. Briefly, rat brain

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lysate was loaded on a column packed with GSH beads coated with GST-fusion

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proteins. After washing with buffer A (20 mM Tris-HCl (pH7.4), 1 mM EDTA, 1

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mM DTT, and 50 mM NaCl), bound proteins were eluted with 1M

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NaCl-containing buffer followed by glutathione-containing buffer. The eluate was

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subjected into SDS-PAGE and stained with SilverQuest silver staining kit

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(Thermo Fischer Scientific, San Jose, CA), according to the manufacturer’s

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instructions.

performed

as

previously

described

[4].

Affinity

column

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Interacting proteins were identified as previously described [16].

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Peptides and proteins were searched against the Swiss Prot and NCBI

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databases by Mascot (Matrix Science, Boston, MA) and X!Tandem. Search

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parameters for Mascot were as follows: Variable modifications-Carbamidomethyl

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and Oxidation; Mass values: Monoisotopic; Protein Mass: Unrestricted; Peptide

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Mass Tolerance: ±10 ppm; Fragment Mass Tolerance: ±0.8 Da; Max Missed

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Cleavages: 1; Instrument type: ESI-TRAP. Scaffold (Proteome Software,

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Portland, OR) was used for validation. Candidate proteins contain at least two

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identified peptides with greater than 95% probability.

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2.4. Cell culture and transfection

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HeLa cells and HEK293 cells were cultured in DMEM (Sigma-Aldrich,

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St. Louis, MO) containing 10% fetal bovine serum in an atmosphere of 5% CO2.

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Cells were transfected with Lipofectamine LTX (Invitrogen), according to the

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manufacturer’s instructions. Preparation of primary cultured hippocampal

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neurons was performed as previously described [17].

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4 2.5. Immunoprecipitation assay

Immunoprecipitation assay was performed as previously described [16].

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The bond proteins were subjected to western blot analysis with the indicated

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antibodies.

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2.6. Immunofluorescence

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Cells were grown on coverslips coated with PDL. Cells were fixed with

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4% formaldehyde, and permeabilized with 0.2% TritonX-100 containing PBS.

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Cells were immunostained with the indicated antibodies (IQGAP1 1:100, VCP

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1:200). Cells were observed with a laser scan microscope LSM-700 (Carl Zewitz,

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Jena, Germany).

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2.7. Statistics

Results were reported as the means and the standard error (SE).

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Comparison of the difference between the control group and other groups for

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more than three groups was assessed by one-way analysis of variance

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(ANOVA) followed by Bonfferoni post-hoc tests. A P value of less than 0.05 was

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considered to be significant.

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Results

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3.1. Identification of VCP as a novel IQGAP1 interacting protein. IQGAP1 has several characteristic domains within its molecules

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(Figure 1A), and serves as a scaffold protein to regulate signal transduction. The

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IQ-repeat-containing N-terminal region (NR) has been reported to interact with

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several interacting partners [18]. We speculated that the interacting proteins in

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this region may play an important role in the regulation of neural functions. To

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understand the role of IQGAP1, we explored novel IQGAP1 interacting proteins

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using a proteomic approach. Glutathione beads coated with GST or

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GST-IQGAP1-NR were incubated with brain lysate extracted from rats on

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postnatal days 2-5. The bond proteins were eluted with 1M NaCl-containing

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buffer, and eluate was subjected to SDS-PAGE (Figure 1B). Several proteins

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specifically eluted from the GST-IQGAP1 affinity column were observed.

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LC-MS/MS analysis revealed that IQGAP1 binds to several proteins in the NR

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region (Supplementary Table 1). Among them, Transitional Endoplasmic

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Reticulum ATPase (TERA), also named as VCP, was identified with high score

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and much more coverage than others. As VCP is involved in diverse cellular

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processes, we focused on the interaction between IQGAP1 and VCP. To confirm

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this result, a portion of the eluate was subjected to immunoblotting using an

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anti-VCP antibody. As a result, the immunoreactivity of VCP was observed only

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in the sample from GST-IQGAP1 (Figure 1C).

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3.2 Interaction of IQGAP1 with VCP To further confirm the results obtained by proteomic analysis, the

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interaction between IQGAP1 and VCP was examined by an immunoprecipitation

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assay using HeLa cell lysate. VCP was co-immunoprecipitated with IQGAP1

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(Figure 2A left). Reciprocally, IQGAP1 was co-immunoprecipitated with VCP

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(Figure 2A right). The interaction between IQGAP1 and VCP was reconfirmed

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using rat brain lysate (Supplementary Figure 1). These results suggest that

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IQGAP1 interacts with VCP under physiological conditions.

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It has been reported that VCP contains two AAA-ATPase domains. To

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examine which AAA-ATPase domain is responsible for the interaction with

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IQGAP1, we constructed two deletion mutants, N-half and C-half of VCP, which

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contain AAA-ATPase domain 1 and AAA-ATPase domain 2, respectively (Figure

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2B). When HEK cells were co-transfected with VCP-N-half and either IQGAP1

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N-half or C-half, VCP-N-half was co-precipitated with IQGAP1 C-half and

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IQGAP1 N-half. The band intensity of VCP-N-half precipitated with IQGAP1

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C-half was relatively higher than that precipitated by IQGAP1-N-half (Figure 2C).

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This result suggests that VCP interacts with IQGAP1 not only in NR region but

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also C-half region. VCP-C-half bound to neither IQGAP1 N-half nor C-half,

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indicating that IQGAP1 binds to VCP N-half mainly via its C-half region.

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Three isoforms of IQGAP have been identified. Few studies showing

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functional deference between IQGAP isoforms were reported. To investigate the

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isoform specificity of the interaction, an immunoprecipitation assay was

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performed in HEK293T cells expressing myc-VCP with either GFP-IQGAP1, 2,

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or 3. Not only IQGAP1, but also IQGAP2 and IQGAP3, were immunoprecipitated

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by VCP (Figure 2D), suggesting that all of the IQGAP isoforms interact with VCP.

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3.3 Localization of IQGAP1 and VCP in cultured hippocampal neurons Localization of IQGAP1 and VCP in primary cultured neurons was

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examined. Immunoreactivity of IQGAP1 in cultured hippocampal neurons (DIV4)

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was observed at the growth corn, as previously reported [19]. Under this

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condition, VCP localized inside of the growth corn were partially co-localized

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with IQGAP1 (Figure 3A, Supplementary Figure 2A). Co-localization of IQGAP1

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and VCP was also observed in the axon shaft, cell body, and dendrites. In

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neurons at DIV14, IQGAP1 co-localized with VCP in the dendritic shaft (Figure

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3B, Supplementary Figure 2B).

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3.4 Interaction of IQGAP1 with disease-related mutant VCP VCP mutation was observed in patients with neurodegenerative

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disorders such as inclusion body myopathy with Paget’s disease of bone and

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frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS) [20].

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It has been reported that IBMPFD mutations of VCP are clustered in the

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N-terminal region and AAA1 domain [21]. Accordingly, we next examined

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whether disease-related mutation of VCP affects its interaction with IQGAP1 by

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an immunoprecipitation assay. We generated two VCP mutants, R155H and

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A232E. R155H is the most common VCP-associated mutation, and A232E is

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linked to a severe clinical phenotype [20, 22]. Interaction of IQGAP1-C-half with

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both VCP-N-half R155H and A232E was significantly decreased compared with

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WT VCP (F(2,6) = 13.27, p<0.01, Figure 4A-B).

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Discussion IQGAP1 is also involved in spine morphogenesis through regulation

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actin cytoskeleton [9, 15]. Neurons from IQGAP1 knockout mice show

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decreases in the number of dendritic spines [15]. In the present study, we

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identified VCP as a novel IQGAP1-interacting protein, and showed that VCP

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interacted with IQGAP1 in physiological conditions and partially co-localized with

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IQGAP1 in neurons. In stage 3 neurons with a single axon and several minor

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processes, VCP was observed at the tip of the growth corn where IQGAP1 is

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localized. In mature neurons, however, VCP was localized at dendrite where

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IQGAP1 is also localized. A previous our study has demonstrated that IQGAP2

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and 3, but not IQGAP1, were involved in neurite outgrowth at stage 2-3 [19].

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Recent study has shown that VCP regulates spine formation and protein

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synthesis in neurons [23, 24]. Accordingly, we speculate that IQGAP1 may

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regulate spine dynamics through VCP.

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Posttranslational

modification

of

IQGAP1

affects

its

function.

Phosphorylation of IQGAP1 by GSK3β results in a reduced interaction with

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CLASP2, which leads to a polarization defect [25]. One of the major functions of

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VCP is to act as a molecular chaperon, in which polyubiquitinated proteins lead

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to proteasome degradation. VCP binds some cofactor proteins such as E3

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ligase, p37, and p47 [26]. Thus, depending on the cofactor to form the complex,

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VCP may promote degradation of polyubiquitinated substrates or unfolding

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aggregated proteins. Although it was unclear whether IQGAP1 forms a complex

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with VCP and its cofactors, preliminary proteomic analysis using the NCBI

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database showed that the VCP/p47 complex-interacting protein, p135

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(VCIP135), is a candidate for IQGAP1-interacting proteins (data not shown).

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Since VCIP135 is involved in the protein deubiquitination process [27], VCP may

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regulate the polyubiquitination state of IQGAP1 by cooperating with VCIP135. It

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is not known whether IQGAP1 undergoes ubiquitination under physiological

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conditions.

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VCP has been identified as causative gene in IBMPFD, a

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neurodegenerative disorder with cognition defects [20]. A point mutation of VCP

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was found in familiar IBMPFD and ALS. Single amino acid mutations were

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clustered in the conserved N-terminal region of VCP. VCP interacted with

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IQGAP1 in its N-half region, suggesting that disease-related mutations alter the

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interaction with IQGAP1. In fact, we showed that two disease-related mutations

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of VCP decreased the interaction with IQGAP1. Although the number of VCP

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mutations in which we examined their effects on the interaction with IQGAP1

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was limited, we speculate that the interaction of other VCP mutants with IQGAP1

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may be decreased compared with wild type. Therefore, different interacting

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properties of VCP to IQGAP1 may be associated with VCP mutation-related

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diseases.

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In conclusion, this study provides evidence that IQGAP1, and also

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IQGAP2 and 3, interact with VCP. Disease-related mutations in VCP, R155H,

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and A232E, decrease the interaction with IQGAP1, suggesting the involvement

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of IQGAP1 in IBMPFD. Further study is required to clarify the pathophysiological

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significance of the interaction between IQGAP1 and VCP in neurodegenerative

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disorders.

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Author contributions

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N.I., T.N., and K.Y. designed the study; N.I. performed the experiments and

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analyzed the data; T.N., T.W., A. I., K.K., and K.Y. analyzed and interpreted the

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data; N.I., T.N., and K.Y. wrote the manuscript. All of the authors discussed the

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results and agreed on the content of this manuscript.

6 Competing financial interests

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The authors declare no competing financial interests.

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9 Acknowledgement

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This work was supported by the Japan Society for the Promotion of Science

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(JSPS) KAKENHI (Grant-in Aid for Scientific Research (S) 20227006 to K.K.,

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Grant-in Aid for Scientific Research (B) 17H04031 to K.Y., 17H04252 to

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T.Nabeshima, Grant-in Aid for Challenging Exploratory Research 16K15201 to

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K.Y., Grant-in Aid for Young Scientists (B) 16K21080 to N.I., 22790279 to T. W.)

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

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(MEXT), the Astellas Foundation for Research on Metabolic Disorders, and

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partially supported by the Strategic Research Program for Brain Sciences from

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Japan Agency for Medical Research and Development, AMED.

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We thank all members of the Kaibuchi and Yamada labs for their helpful

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discussions and support of this work.

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The Journal of biological chemistry, 271 (1996) 21732-21737. [14] S. Kuroda, M. Fukata, K. Kobayashi, M. Nakafuku, N. Nomura, A. Iwamatsu, K. Kaibuchi, Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1, The Journal of biological chemistry, 271 (1996) 23363-23367. [15] C. Gao, S.F. Frausto, A.L. Guedea, N.C. Tronson, V. Jovasevic, K. Leaderbrand, K.A. Corcoran, Y.F. Guzman, G.T. Swanson, J. Radulovic, IQGAP1 regulates NR2A signaling, spine density, and cognitive processes, The Journal of neuroscience : the official journal of the Society for Neuroscience, 31 (2011) 8533-8542. [16] N. Itoh, M. Nakayama, T. Nishimura, S. Fujisue, T. Nishioka, T. Watanabe, K. Kaibuchi, Identification of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3-kinase) as Par3 partners by proteomic analysis, Cytoskeleton (Hoboken, N.J.), 67 (2010) 297-308. [17] T. Nakai, T. Nagai, M. Tanaka, N. Itoh, N. Asai, A. Enomoto, M. Asai, S. Yamada, A.B. Saifullah, M. Sokabe, M. Takahashi, K. Yamada, Girdin phosphorylation is crucial for synaptic plasticity and memory: a potential role in the interaction of BDNF/TrkB/Akt signaling with NMDA receptor, The Journal of neuroscience : the official journal of the Society for Neuroscience, 34 (2014) 14995-15008. [18] J. Liu, J.J. Guidry, D.K. Worthylake, Conserved sequence repeats of IQGAP1 mediate binding to Ezrin, Journal of proteome research, 13 (2014) 1156-1166. [19] S. Wang, T. Watanabe, J. Noritake, M. Fukata, T. Yoshimura, N. Itoh, T. Harada, M. Nakagawa, Y. Matsuura, N. Arimura, K. Kaibuchi, IQGAP3, a novel effector of Rac1 and Cdc42, regulates neurite outgrowth, Journal of cell science, 120 (2007) 567-577. [20] G.D. Watts, J. Wymer, M.J. Kovach, S.G. Mehta, S. Mumm, D. Darvish, A. Pestronk, M.P. Whyte, V.E. Kimonis, Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein, Nature genetics, 36 (2004) 377-381. [21] R. Schroder, G.D. Watts, S.G. Mehta, B.O. Evert, P. Broich, K. Fliessbach, K. Pauls, V.H. Hans, V. Kimonis, D.R. Thal, Mutant valosin-containing protein causes a novel type of frontotemporal dementia, Annals of neurology, 57 (2005) 457-461. [22] V.E. Kimonis, S.G. Mehta, E.C. Fulchiero, D. Thomasova, M. Pasquali, K. Boycott, E.G. Neilan, A. Kartashov, M.S. Forman, S. Tucker, K. Kimonis, S. Mumm, M.P. Whyte, C.D. Smith, G.D. Watts, Clinical studies in familial VCP myopathy associated with Paget disease of bone and frontotemporal dementia, American journal of medical genetics. Part A, 146a (2008) 745-757. [23] H.F. Wang, Y.T. Shih, C.Y. Chen, H.W. Chao, M.J. Lee, Y.P. Hsueh, Valosin-containing protein and neurofibromin interact to regulate dendritic spine density, The Journal of clinical investigation, 121 (2011) 4820-4837. [24] Y.T. Shih, Y.P. Hsueh, VCP and ATL1 regulate endoplasmic reticulum

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and protein synthesis for dendritic spine formation, Nature communications, 7 (2016) 11020. [25] T. Watanabe, J. Noritake, M. Kakeno, T. Matsui, T. Harada, S. Wang, N. Itoh, K. Sato, K. Matsuzawa, A. Iwamatsu, N. Galjart, K. Kaibuchi, Phosphorylation of CLASP2 by GSK-3beta regulates its interaction with IQGAP1, EB1 and microtubules, Journal of cell science, 122 (2009) 2969-2979. [26] H. Meyer, M. Bug, S. Bremer, Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system, Nature cell biology, 14 (2012) 117-123. [27] Y. Wang, A. Satoh, G. Warren, H.H. Meyer, VCIP135 acts as a deubiquitinating enzyme during p97-p47-mediated reassembly of mitotic Golgi fragments, The Journal of cell biology, 164 (2004) 973-978.

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Figure legends

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Figure 1 Screening of novel IQGAP1-interacting proteins by a proteomic

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approach

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(A) Schematic representation of IQGAP1. CHD: calponin-binding domain, IQ

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repeats: IQGAP-specific repeat motif, WW: double tryptophan motif, IQ motif:

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calmodulin-binding domain, GRD: Ras-GAP-related domain, RasGAP C:

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RasGAP C-terminus.

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(B) Silver staining of affinity-purified proteins. Eluents from affinity column

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chromatography were subjected into SDS-PAGE followed by silver staining.

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Arrow and arrowhead indicate identified protein and GST protein, respectively.

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(C) Western blotting analysis of eluents. Eluents from affinity column

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chromatography were subjected into SDS-PAGE. Proteins were reacted with an

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anti-VCP antibody.

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Figure 2 Interaction of IQGAP1 with VCP

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(A) Immunoprecipitation from rat brain lysates. Rat brain lysates were incubated

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with an anti-IQGAP1 antibody or an anti-VCP antibody. The same amount of

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rabbit or mouse IgG was used as a control antibody. Coimmunoprecipitates

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were detected by western blotting using the indicated antibodies.

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(B) Schematic representatives of VCP. AAA1: AAA-ATPase domain 1, AAA2:

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AAA-ATPase domain 2.

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(C) Determination of the region in IQGAP1 and VCP that is responsible for the

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interaction. HEK cell lysates containing the indicated proteins were precipitated

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with an anti-GFP antibody. The coprecipitated myc-VCP deletion mutant was

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detected by an anti-myc antibody. Asterisks indicate specific band for IgG.

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(D) Immunoprecipitation of IQGAP isoforms with VCP. HEK cell lysates

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containing myc-VCP and GFP-IQGAPs were precipitated with an anti-GFP

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antibody. Coprecipitated myc-VCP was detected with an anti-myc antibody.

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Figure 3 Localization of IQGAP1 with VCP in primary cultured neurons.

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(A) Immunostaining of IQGAP1 and VCP. Neurons (DIV4) were immunostained

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with the indicated antibodies. Scale bar: 20 µm.

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(B) Immunostaining of IQGAP1 with VCP in neurons. Neurons (DIV14) were

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immunostained with the indicated antibodies. Scale bar: 20 µm.

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Figure 4 Interaction of IQGAP1 with disease-related VCP mutants

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(A) Immunoprecipitation from HEK293T cells expressing deletion mutants of

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IQGAP1 and VCP mutants. HEK cell lysates containing the indicated proteins

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were incubated with an anti-GFP antibody. Coprecipitated myc-VCP proteins

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were detected with an anti-myc antibody.

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(B) Quantitative analysis of the interaction of IQGAP1 with VCP. n=3. *p< 0.05.

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A

B

CHD

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IQ motif

GRD

RasGAP-C

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IQ repeats

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GST

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GST-IQGAP1-NR

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Chalf

Mr (kDa) 220 160 120 100 90 80 70 60 50

GST GST-IQGAP1 (216-683 AA)

Figure. 1

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Figure. 2 A

B

IQGAP1

VCP N-half

Blot

IP sample

Blot myc *

Mr (kDa)

mock

C-half

N-half

GFP

C-half

input myc-VCP

GFP-IQGAP1

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GFP-IQGAP1 GFP

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GFP

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GFP-IQGAP1

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GFP

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GFP

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Blot

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Mr (kDa) 250

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GFP-IQGAP3

IP: GFP

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GFP-IQGAP2

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GFP-IQGAP1

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mock

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IQGAP1

GFP-IQGAP2

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GFP-IQGAP1

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IgG

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Figure. 3 A

Merged

VCP

Magnified

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IQGAP1

Merged

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VCP

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IQGAP1

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Fig. 4

A GFP

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GFP-IQGAP1-C-half

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R155H

WT

A232E

R155H

WT

myc-VCP-N-half

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myc

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Relative Ratio

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Highlights ・ VCP is a novel IQGAP1 interacting protein. ・ IQGAP1 interacts with N-terminal region of VCP.

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・ IQGAP1 is co-localized with VCP in cultured hippocampus neuron.

・ Some disease-related mutations of VCP decrease its interaction with

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IQGAP1.