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Tài liệu Báo cáo khoa học: Interaction between very-KIND Ras guanine exchange factor and microtubule-associated protein 2, and its role in dendrite growth – structure and function of the second kinase noncatalytic C-lobe domain docx

Interaction between very-KIND Ras guanine exchange
factor and microtubule-associated protein 2, and its role
in dendrite growth – structure and function of the second
kinase noncatalytic C-lobe domain
Jinhong Huang
1,
*, Asako Furuya
1
, Kanehiro Hayashi
1–3
and Teiichi Furuichi
1,2,4
1 Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Saitama, Japan
2 JST, CREST, Kawaguchi, Saitama, Japan
3 Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan
4 Faculty of Science and Technology, Tokyo University of Science, Chiba, Japan
Keywords
dendrite growth; KIND domain; MAP2;
protein–protein interaction; RasGEF
Correspondence
T. Furuichi, Laboratory for Molecular

Neurogenesis, RIKEN Brain Science
Institute, 2-1 Hirosawa, Wako 351-0198,
Japan
Fax: +81 48 467 6079
Tel: +81 48 467 5906
E-mail: tfuruichi@brain.riken.jp
*Present address
Discovery & Development Laboratory I,
Hanno Research Center, Taiho
Pharmaceutical Co., Ltd, Saitama, Japan
(Received 5 January 2011, revised 19
February 2011, accepted 28 February
2011)
doi:10.1111/j.1742-4658.2011.08085.x
The kinase noncatalytic C-lobe domain (KIND) is a putative protein–protein
interaction module. Four KIND-containing proteins, Spir-2 (actin-nuclear
factor), PTPN13 (protein tyrosine phosphatase), FRMPD2 (scaffold protein)
and very-KIND (v-KIND) (brain-specific Ras guanine nucleotide exchange
factor), have been identified to date. Uniquely, v-KIND has two KINDs (i.e.
KIND1 and KIND2), whereas the other three proteins have only one. The
functional role of KIND, however, remains unclear. We previously demon-
strated that v-KIND interacts with the high-molecular weight microtubule-
associated protein 2 (MAP2), a dendritic microtubule-associated protein,
leading to negative regulation of neuronal dendrite growth. In the present
study, we analyzed the structure–function relationships of the v-KIND–
MAP2 interaction by generating a series of mutant constructs. The interac-
tion with endogenous MAP2 in mouse cerebellar granule cells was specific to
v-KIND KIND2, but not KIND1, and was not observed for the KINDs
from other KIND-containing proteins. The binding core modules critical for
the v-KIND–MAP2 interaction were defined within 32 residues of the mouse
v-KIND KIND2 and 43 residues of the mouse MAP2 central domain. Three
Leu residues at amino acid positions 461, 474 and 477 in the MAP2-binding
core module of KIND2 contributed to the interaction. The MAP2-binding
core module itself promoted dendrite branching as a dominant-negative regu-
lator of v-KIND in hippocampal neurons. The results reported in the present
study demonstrate the structural and functional determinant underlying the
v-KIND–MAP2 interaction that controls dendrite arborization patterns.
Structured digital abstract
l
vKIND-KIND2 binds to Map2 by pull down (View interaction)
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Map2 physically interacts with vKIND-KIND2 by pull down (View interaction 1, 2, 3, 4, 5)
l
Map2 physically interacts with vKIND by pull down (View interaction)
l
Map2 physically i nteracts with vKIND-KIND2 by anti bai t co immunoprecipitation (View i nteraction)
l
vKIND-KIND2 physically interacts with Map2 by pull down (View interaction)
Abbreviations
CD, central domain; DIV, day in vitro; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; HMW, high-molecular-
weight; KIND, kinase noncatalytic C-lobe domain; KIND1, first kinase noncatalytic C-lobe domain; KIND2, second kinase noncatalytic C-lobe
domain; MAP2, microtubule-associated protein 2; GEF, guanine exchange factor; v-KIND, very-KIND.
FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1651
Introduction
Protein–protein interactions play important roles in
the molecular recognition and functional modulation
between proteins in many signal transduction path-
ways [1,2]. The kinase noncatalytic C-lobe domain
(KIND) was determined to be a putative signaling
domain based on bioinformatic analysis of the N-ter-
minal sequence of the Drosophila protein Spir, an
actin-nucleation factor [3]. The KIND domain shows
homology to the C-terminal protein kinase catalytic
fold (C-lobe), although it lacks the sequence similarity
critical for kinase activity [3]. Four proteins containing
KIND domains have so far been identified in mam-
mals: Spir [4,5], nonreceptor-type protein tyrosine
phosphatase 13 (PTPN13, or PTP-BL ⁄ PTP-BAS) [6,7],
FERM and PDZ-domain-containing 2 (FRMPD2) [8]
and Ras guanine exchange factor (RasGEF) very-
KIND (v-KIND, or kinase noncatalytic C-lobe
domain containing 1) [9]. The KIND domain in these
proteins is localized to the N-terminal region, and their
specific functional domains are located in the C-termi-
nal region. The C-lobe of protein kinases mediates the
interaction with activators, substrates and regulatory
subunits, implying that the KIND domain, an atypical
noncatalytic C-lobe, is involved in the interaction with
signaling proteins [3,5,10]. However, the structural and
functional properties of the KIND domains remain
largely unknown.
Within the KIND protein family, v-KIND is unique
because it possesses two tandem-repeated KIND
domains, KIND1 and KIND2, in the N-terminal
region. Recently, a heterozygous, nonsynonymous
somatic single nucleotide variation of human v-KIND
(KNDC1), in which Leu799 is changed to a Phe resi-
due, was reported in acute myeloid leukemia genomes
[11]. We previously identified v-KIND with a character-
istic spatiotemporal expression pattern during the post-
natal development of mouse brain: it shows low- or
moderate-level expression in the cerebrum, hippocam-
pus and thalamus in the first week after birth, whereas
its highest expression level occurs in cerebellar granule
cells of the internal granular layer by postnatal week 2
and thereafter [12]. We showed that v-KIND overex-
pression suppresses and v-KIND knockdown promotes
dendrite growth of cultured cerebellar granule cells
and hippocampal neurons, suggesting that v-KIND
acts as a signaling molecule in controlling or limiting
dendrite growth of neurons during development [12].
We also suggested that the protein–protein interaction
between v-KIND and the high-molecular-weight
(HMW) form, but not the low-molecular-weight form,
of microtubule-associated protein 2 (MAP2) via
KIND2 is critical for this signaling pathway [12].
HMW-MAP2 (referred to hereafter as MAP2) is
known to modulate polymerization, stability and rear-
rangement of microtubules in neuronal dendrites
[13–15] and is associated with some neurological and
psychiatric disorders [16,17]. However, the structure–
function relationship of the interaction between v-KIND
and MAP2, as well as its biological significance, remains
unclear.
In the present study, we determined the structural
and functional properties of the protein–protein inter-
action between v-KIND and MAP2. We defined the
binding core regions for the v-KIND–MAP2 interac-
tion and showed that the MAP2 binding core is not
only critical for targeting of v-KIND to neuronal den-
drites, but also is indispensable for the function of
v-KIND in negatively controlling dendrite growth and
branching.
Results
The KIND2 domain of v-KIND has a unique ability
to localize to dendrites via MAP2 binding,
which is absent in the KINDs from other
KIND-containing proteins
To examine the dendrite localization signal domains in
v-KIND, we first investigated the subcellular localiza-
tion of eight different Flag epitope-tagged v-KIND
derivatives with domain deletions (as shown in Fig. 1A)
in primary cultured mouse cerebellar granule cells,
coexpressed with enhanced green fluorescent protein
(EGFP) to visualize the protrusion patterns of transfect-
ed neurons. As shown in Fig. 1B, the full-length
v-KIND was specifically localized to dendrites and
soma, although not to axons. The expression of three
other KIND2-containing constructs (DKIND1, DRasN
and DGEF) was restricted to dendrites and soma
(Fig. 1B). On the other hand, two KIND2 domain-
lacking constructs, DKIND2 (Fig. 1B) and DKIND 1 + 2
(Fig. 1B), were widely distributed throughout the cells,
including the axons. Notably, the KIND2 domain has a
specific ability to localize to dendrites by itself, whereas
the KIND1 domain alone has lost this ability (Fig. 1B).
The results obtained indicate that the KIND2 domain
is necessary and sufficient for the targeting of v-KIND
to dendrites of neurons.
To investigate the MAP2-binding characteristics of
the KIND domain protein family, we generated four
glutathione S-transferase (GST)-fused proteins of
KIND domains (v-KIND KIND1, v-KIND KIND2,
Core modules for the very-KIND-MAP2 interaction J. Huang et al.
1652 FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS
Spir-2 KIND and PTPN13 KIND) (Fig. 2B) and ana-
lyzed their ability to interact with MAP2 protein
(Fig. 2A). Only GST-fused v-KIND KIND2 was able
to pull down MAP2 from mouse cerebellar lysates
(Fig. 2A, left), as well as purified MAP2 protein sam-
ples (Fig. 2A, right). Taken together, these data indi-
cate that the direct interaction with the dendritic
MAP2 protein is a unique feature of v-KIND KIND2,
among the four KIND domains tested.
Residues 702–745 in the central domain of MAP2
contain the v-KIND binding core module
MAP2 consists of three main structural domains: the
cAMP-dependent protein kinase regulatory subunit
RII binding domain, the central domain (CD) and
the microtubule-binding domain (Fig. 3A). To deter-
mine the v-KIND-binding region in MAP2, we first
divided MAP2 into five regions (Fig. 3A) and ana-
lyzed the bacterially expressed proteins of these con-
structs (Fig. S1A) for binding to v-KIND in
cerebellar lysates by a pull-down assay. Only the
GST-fused CD2 region (residues 600–1099) could pull
down endogenous v-KIND protein (Fig. 3B). These
results indicate that the region spanning amino acids
600–1099 of MAP2 (i.e. around the middle part of
the CD) interacts with the endogenous v-KIND in
mouse cerebellum.
To verify whether the CD2 region of MAP2 specifi-
cally binds to KIND2 in v-KIND, we screened the
v-KIND
Δ
KIND1
Δ
KIND2
Δ
KIND1+2
Δ
RasN
Δ
GEF
KIND1
KIND2
KIND1 KIND2 GEFRasNCC
1
37–217 456–620 1238–1364 1461–1734 1742
α
-FlagEGFP +
α
-Flag
Δ
KIND2-Flag
v-KIND-Flag
dendrite
axon
Δ
KIND1-Flag
Δ
KIND1+2-Flag
KIND1-Flag
Δ
RasN-Flag
α
-FlagEGFP +
α
-Flag
KIND2-Flag
Δ
GEF-Flag
A
B
Fig. 1. Domain structure of the MAP2-asso-
ciated RasGEF v-KIND and its dendritic tar-
geting via KIND2 domain. (A) Structures of
the v-KIND. KIND1, KIND2, coiled-coil (CC),
RasN and RasGEF domains. Flag-tagged
v-KIND derivatives: v-KIND, full-length
v-KIND; DKIND1, deletion of KIND1;
DKIND2, deletion of KIND2; DKIND1 + 2,
deletion of both KIND1 and KIND2; DRasN,
deletion of RasN; DGEF, deletion of
RasGEF; KIND1, KIND1 domain; KIND2,
KIND2 domain. (B) KIND2 domain anchors
v-KIND to dendrites. Flag-tagged v-KIND,
DKIND1, DKIND2, DKIND1 + 2, DRasN,
DGEF, KIND1 or KIND2 together with EGFP
were transfected into primary cultures of
mouse cerebellar granule cells at DIV1.
Transfected cells fixed at DIV14 were
immunostained with anti-Flag serum. Flag
immunoreactivity (red) and EGFP fluores-
cence (green) were observed by confocal
microscopy. Open and closed arrowheads
indicate dendrites and axons, respectively.
Scale bar = 50 lm.
J. Huang et al. Core modules for the very-KIND-MAP2 interaction
FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1653
GST-CD2 binding ability of six Flag-tagged v-KIND
derivatives (Fig. S2A). The pull-down assay with GST-
CD2, followed by immunoblotting with anti-Flag
serum, showed that four KIND2-containing constructs
(v-KIND, DKIND1, DRasN and DRasGEF) inter-
acted with GST-CD2, whereas two constructs lacking
KIND2 (D KIND2 and DKIND1 + 2) failed to bind
to GST-CD2 (Fig. S2A). This indicates that the
KIND2 domain of v-KIND is involved in the interac-
tion with the CD2 region of MAP2. Furthermore,
a similar pull-down assay with GST-CD2 to screen
four KIND domain constructs (Spir-2 KIND, PTPN13
KIND, v-KIND KIND1 and v-KIND KIND2)
showed that only v-KIND KIND2 could bind to
GST-CD2 (Fig. S2B). Taken together, these results
reveal the specific protein–protein interaction between
the v-KIND KIND2 domain and the middle CD
region of MAP2.
To narrow down the region responsible for v-KIND
binding within the MAP2 CD2 region, we first subdi-
vided the CD2 region (amino acids 600–1099)
into three subregions: CD2-1 (amino acids 600–767),
CD2-2 (amino acids 768–934) and CD2-3 (amino acids
935–1099) (Fig. 3C). Next, we analyzed the binding
ability of bacterially expressed proteins of these con-
structs (Fig. S1B) to endogenous v-KIND protein from
cerebellar lysates. As a result, only GST-CD2-1 pulled
down v-KIND (Fig. 3D). To identify the sequence crit-
ical for v-KIND binding within the CD2-1 (amino
acids 600–767) region, we next generated a series of
GST-fused deletions of the CD2-1 region (Fig. 3E)
and examined the interactions between bacterially
expressed proteins of these subregions (Fig. S1C) and
endogenous cerebellar v-KIND protein. All four C-ter-
minal subregions (CD2-1-1 to CD2-1-4) failed to pull
down v-KIND (Fig. 3F, upper), whereas three of the
four N-terminal subregions (CD2-1-6, CD2-1-7 and
CD2-1-8, but not CD2-1-5) could pull down v-KIND
(Fig. 3F, middle). Finally, we generated three GST-
fused C-terminal truncations of the CD2-1-6 subregion
(Fig. 3E, lower). The pull-down assay with bacterially
expressed proteins of these truncated constructs
(Fig. S1C) showed that GST-CD2-1-6-2 and
GST-CD2-1-6-3, but not GST-CD2-1-6-1, bound to
cerebellar v-KIND (Fig. 3F, lower). This indicates that
residues 702–744 of the smallest construct CD2-1-6-2
contain the core sequence for v-KIND binding.
To evaluate the v-KIND KIND2-binding specificity
of residues 702–744 of MAP2, we performed a pull-
down assay of combinations of EGFP-fused KIND1
or KIND2 of v-KIND with GST-fused CD2-1-6-1 or
CD2-1-6-2 of MAP2. We successfully detected a pull
down for the combination of EGFP-KIND2 and
GST-CD2-1-6-2, but not for other combinations
(Fig. S2C), suggesting that the core sequence critical
for the specific v-KIND KIND2 binding resides within
residues 702–744 of MAP2.
Residues 456–487 in the KIND2 domain of v-KIND
contain the core MAP2 binding module
To identify the core MAP2-binding site within the
KIND2 domain (amino acids 456–620), we analyzed
five EGFP-fused KIND2 domain derivatives, EGFP-
KIND2-1 to -KIND2-5 (Fig. 4A), by a pull-down
assay with GST-CD2 (MAP2 v-KIND binding core).
Input (cerebellum)
GST
v-KIND KIND1
v-KIND KIND2
Spir-2 KIND
PTPN13 KIND
Input (MAP2)
GST
v-KIND KIND1
v-KIND KIND2
Spir-2 KIND
PTPN13 KIND
PD from cerebellar lysates
PD from purified MAP2
IB:
α
-MAP2
kDa
50
37
25
CBB
A
B
Fig. 2. Of all members of the KIND family
of proteins, only KIND2 binds to MAP2.
Pull-down assay (PD) of the endogenous
MAP2 from cerebellar lysates of P21 mice
(A, left) and purified MAP2a ⁄ b (A, right) by
GST-fused KIND domains (v-KIND KIND1,
v-KIND KIND2, Spire-2 KIND and PTPN13
KIND) shown in (B), followed by immuno-
blotting with anti-MAP2 serum.
Core modules for the very-KIND-MAP2 interaction J. Huang et al.
1654 FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS
Immunoblotting with anti-EGFP serum showed that
three C-terminal truncations (KIND2-1, KIND2-2 and
KIND2-3), but not two N-terminal truncations
(KIND2-4 and -KIND2-5), were pulled down by
GST-CD2 (Fig. 4B). This suggests that the core
sequence critical for the specific MAP2 binding resides
within the 32 residues (amino acids 456–487) of the
v-KIND KIND2 domain.
To examine whether the MAP2 binding core site of
v-KIND binds to intact MAP2 protein, we coex-
pressed EGFP-fused KIND2-1 and full-length MAP2
in COS7 cells and analyzed their interaction by
Input
Mouse IgG
-MAP2
GST
RII
CD1
CD2
CD3
MBD
IP PD
IB: -v-KIND
250
150
IB: -v-KIND
Input
GST
CD2-1
CD2-2
CD2-3
CD2
PD
250
150
Input
GST
CD2-1-6-1
CD2-1-6-2
CD2-1-6-3
CD2-1
PD
IB: -v-KIND
250
150
Input
GST
CD2-1-5
CD2-1-6
CD2-1-7
CD2-1-8
CD2-1
PD
250
Input
GST
CD2-1-1
CD2-1-2
CD2-1-3
CD2-1-4
CD2-1
PD
250
150
MAP2
MBD
RII
CD1
CD2
CD3
1–147
148–599
600–1099
1100–1518
1519–1829
767
934
CD2-1
CD2-2
CD2-3
600
CD2
1099
Inter-
action
600 767
CD2-1
Inter-
action
CD2-1-1
634
CD2-1-2
667
CD2-1-3
701
CD2-1-4
734
CD2-1-5
735
CD2-1-6
702
CD2-1-7
668
CD2-1-8
635
CD2-1-6-1
734702
CD2-1-6-2
744
CD2-1-6-3
755
CDRII
MBD
Inter-
action
AB
CD
EF
Fig. 3. MAP2 interacts with v-KIND via residues 702–744 within the MAP2 center region of the CD. (A) MAP2a was subdivided into five
fragments [the cAMP-dependent protein kinase regulatory subunit RII binding domain (RII), the central domain (CD)1-3 and the microtubule-
binding domain (MBD)] and corresponding GST fusion proteins were generated. (B), Pull-down assay (PD) of v-KIND from mouse cerebellar
lysates (input) by bacterially expressed GST-fused MAP2 derivatives, followed by immunoblotting (IB) with anti-v-KIND serum. Immunopre-
cipitation assay (IP) of v-KIND from cerebellar lysates (input) by anti-MAP2 serum and normal mouse IgG as a positive and negative control,
respectively. (C) Division of the middle CD2 region (amino acids 600–1099) of the MAP2 CD into three subregions: CD2-1 (amino acids 600–
767), CD2-2 (amino acids 768–934) and CD2-3 (amino acids 935–1099). (D) Pull-down assay (PD) of v-KIND from cerebellar lysates (input) by
GST-fused CD2 and its subregions (CD2-1, CD2-2 or CD2-3), followed by immunoblotting (IB) with anti-v-KIND serum. (E) The series of GST-
fused MAP2 CD2-1 (amino acids 600–767) derivatives: CD2-1-1 (amino acids 600–634), CD2-1-2 (amino acids 600–667), CD2-1-3 (amino
acids 600–701), CD2-1-4 (amino acids 600–734), CD2-1-5 (amino acids 735–767), CD2-1-6 (amino acids 702–767), CD2-1-7 (amino acids 668–
767), CD2-1-8 (amino acids 635–767), CD2-1-6-1 (amino acids 702–735), CD2-1-6-2 (amino acids 702–744) and CD2-1-6-3 (amino acids 702–
755). (F) Pull-down assay of v-KIND from cerebellar lysates (input) by the GST-fused CD2 derivatives shown in (E), followed by immunoblot-
ting with anti-v-KIND serum.
J. Huang et al. Core modules for the very-KIND-MAP2 interaction
FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1655
a co-immunoprecipitation assay (Fig. 4C). Using anti-
MAP2 sera, we found that EGFP-KIND2-1 was
co-immunoprecipitated with MAP2 in the cell lysates.
Taken together, these data indicate that the MAP2-
binding core site of v-KIND is sufficient for the
v-KIND-MAP2 interaction within cells.
The MAP2-binding core module of v-KIND is
involved in targeting to neuronal dendrites and
dendrite growth
We next investigated the MAP2 binding core of
v-KIND in terms of its ability to target to dendrites
and to control dendrite morphology. We transfected
three HA-epitope-tagged v-KIND derivatives (v-KIND,
KIND2 domain and KIND 2-1 MAP2-binding core
region) into primary cultured hippocampal neurons at
7 days in vitro (DIV), fixed at DIV21, and analyzed
the dendrite localization of the expressed proteins as
well as the dendrite morphology by immunodetection
with anti-HA serum (Fig. 5A). Hippocampal neurons
were used because the dendrite morphology was much
more significant and easy to observe than that in cere-
bellar granule cells. The KIND2-1 core region was
localized to dendrites, as was the endogenous v-KIND
(Fig. S3), indicating that the MAP2-binding core con-
fers the dendritic targeting of v-KIND. Overexpression
of v-KIND decreased dendritic branching as reported
previously [12], whereas overexpression of KIND2 pro-
moted dendrite extension, compared to that of EGFP
alone (Fig. 5A). Interestingly, overexpression of the
KIND 2-1 domain resulted in a complex dendrite mor-
phology (Fig. 5A). Sholl analysis of the dendrite
PD: GST-CD2
IB:
-GFP
Input
IB:
-GFP
GST-CD2
(CBB stain)
EGFP
EGFP-KIND2-1
EGFP-KIND2-2
EGFP-KIND2-3
EGFP-KIND2-4
EGFP-KIND2-5
EGFP-KIND2
EGFP-KIND1
KIND2-1
487
KIND2-2
521
KIND2-3
555
KIND2-5
589
KIND2-4
488
456 620
KIND2
v-KIND
+
+
+


Interaction
EGFP
EGFP-
KIND2-1
Input
IB:
-GFP
IP:
-MAP2
IB:
-GFP
IP:
-MAP2
IB:
-MAP2
A
B
C
Fig. 4. Residues 456–487 within the v-KIND KIND2 domain contain
the MAP2-binding core site. (A) Division of the KIND2 domain
(456–620) into five subregions: KIND2-1 (amino acids 456–487),
KIND2-2 (amino acids 456–521), KIND2-3 (amino acids 456–555),
KIND2-4 (amino acids 488–620) and KIND2-5 (amino acids
589–620). These KIND2 subregions were fused with EGFP at the
N-termini and expressed in COS7 cells. (B) Pull-down assay (PD)
of EGFP-fused KIND2 derivatives (middle) by GST-fused CD2
(bottom), followed by immunoblotting (IB) with anti-GFP serum
(top). KIND2 and KIND1 constructs were used as positive and
negative controls, respectively. (C) Immunoprecipitation assay (IP)
of COS7 cells co-expressing MAP2 full-length (middle) and
GFP-fused KIND2-1 (bottom) with anti-MAP2 serum, followed by
immunoblotting with anti-GFP serum (top).
EGFP v-KINDEGFP v KIND
KIND2 KIND2-1
16
18
EGFP
***
6
8
10
12
14
EGFP
v-KIND
KIND2
KIND2-1
0
2
4
20 50 80 110 140 170 200 230 260 290 320 350
Distance from soma (µm)
Total number of intersections
A
B
Fig. 5. KIND 2-1 targets the dendrite and its overexpression sup-
pressed dendrite growth. (A) Flag-tagged v-KIND, v-KIND KIND2
(amino acids 456–620) or KIND2-1 (amino acids 456–487) together
with EGFP were cotransfected into primary cultured mouse hippo-
campal neurons at DIV7. Cells fixed at DIV21 were immunostained
with anti-HA serum. HA immunofluorescence and its colocalization
with GFP fluorescence were observed by confocal microscopy.
Scale bar = 100 lm. (B) Sholl analysis of the dendrite complexity of
individual neurons. Data were obtained from three independent
transfection experiments (for each experiment, n = 15 per con-
struct). The results are the mean ± SEM. ***P<0.001 for v-KIND
versus KIND2-1.
Core modules for the very-KIND-MAP2 interaction J. Huang et al.
1656 FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS
branching patterns showed that the number of den-
drites within 130 lm from the soma was decreased in
neurons overexpressing v-KIND, but increased in neu-
rons overexpressing either KIND2 or KIND2-1, com-
pared to neurons expressing EGFP (Fig. 5B). Neurons
overexpressing KIND2-1 had an increased number of
dendrite branches of < 70 lm compared to neurons
overexpressing KIND2. Interestingly, the number of
dendrites > 300 lm was slightly increased by overex-
pressing v-KIND compared to the overexpression of
EGFP, KIND2 or KIND2-1 (Fig. 5B). These results
suggest that KIND2 and KIND2-1, overexpressed
in neurons, act as dominant-negative regulators of
v-KIND and suppress the negative regulation of den-
drite growth by endogenous v-KIND.
Three conserved Leu residues are critical for the
v-KIND and MAP2 interaction
We examined the 32 residues of the MAP2-binding
core (amino acids 456–487) of mouse v-KIND by com-
paring them with those of human and Gallus v-KIND,
as well as the corresponding residues of mouse
v-KIND KIND1, Spire-2 KIND and PTPN13 KIND
(Fig. 6A). The amino acid sequences of the core
regions of mouse, human and Gallus v-KIND shared
65.6% homology, and 17 identical and four function-
ally similar residues, including seven conserved Leu
residues, were identified. By contrast, the correspond-
ing sequence in the three other KIND domains from
mouse Spir-2, v-KIND and PTPN13 shared 46.9%
homology and had only two or three conserved Leu
residues out of seven residues. It is notable that four
Leu residues (amino acids 461, 474, 477 and 482) and
one Thr residue (amino acid 487) were well conserved
in the KIND2 of v-KIND in all species analyzed,
although not in the other KINDs. To investigate the
possible involvement of these conserved Leu residues
in the interaction between v-KIND and MAP2, we
generated EGFP-tagged KIND2 mutants with an Ala
substitution at Leu461, 474, 477, 482 or 485 (L461A,
L474A, L477A, L482A or L485A) and conducted a
pull-down assay with the v-KIND-binding core mod-
ule CD2-1-6-2 of MAP2 (Fig. 6B). KIND2 L461A,
L474A and L477A mutants failed to bind to CD2-1-6-
2 of MAP2, whereas the L482A and L485A mutants
did bind to this module. These results suggest that the
Leu461, 474 and 477 residues conserved in the KIND2
domain alone are indispensable for the v-KIND–
MAP2 interaction.
The Leu474 residue of the KIND2 MAP2-binding
core module is important for dendrite growth of
hippocampal neurons
To clarify the biological significance of the v-KIND
MAP2-binding core module, we generated full-length v-
KIND containing the L474A substitution mutation and
analyzed its role in dendrite growth by cotransfection
with EGFP in hippocampal neurons (Fig. 7). When
compared with neurons transfected with wild-type
v-KIND, which decreased the number of dendrites, neu-
rons transfected with the L474A mutant had more com-
plex dendritic arborization (Fig. 7A). Sholl analysis
showed that the L474A mutant-transfected neurons had
an increased number of dendrites within 150 lm from
the soma compared to wild-type v-KIND-transfected
neurons (Fig. 7B). In addition, L474A-transfected neu-
rons had more dendritic branches at 90–140 lm from
the soma compared to neurons transfected with EGFP
alone, although the two transfectants had similar num-
bers of proximal dendrites < 80 lm and distal dendrites
> 150 lm from the soma. In addition, the maximal
length of each dendrite in v-KIND-overexpressed neu-
rons was significantly greater than that of EGFP- or
L474A-overexpressed neurons (Fig. 7C). These results
suggest that the Leu474 residue contributed to the func-
tional interaction between v-KIND and MAP2 in the
regulation of neuronal dendrite growth.
A
B
Fig. 6. Conserved Leu residues are important for the interaction
between v-KIND and MAP2. (A) Alignment of the KIND2-1 region in
human, mouse and Gallus (top) and the corresponding mouse
v-KIND KIND1, Spir-2 KIND and PTPN13 KIND domains (bottom).
The residues conserved across all species in the KIND2 domain
and in any other KIND domain are highlighted in gray, whereas
those conserved only in the KIND2 domain are highlighted in black.
The Leu residues at positions 461, 474, 477 and 482 were substi-
tuted by Ala. (B) Pull-down assay (PD) of EGFP-fused KIND2,
L461A, L474A, L477A, L482A or L485A mutant of v-KIND KIND2
by the v-KIND-binding core module CD2-1-6-2 of MAP2. EGFP
alone was used as a control. Asterisks in (A) show the Leu
residues essential for binding.
J. Huang et al. Core modules for the very-KIND-MAP2 interaction
FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1657
Discussion
The present study revealed the structural and func-
tional determinants of the protein–protein interaction
between v-KIND and MAP2 (i.e. the MAP2-binding
core module in v-KIND and the v-KIND-binding core
module in MAP2) across a range of amino acid resi-
dues, and provided evidence that these novel protein–
protein interaction core modules play a pivotal role in
regulating dendrite growth and branching of cerebellar
granule cells and hippocampal neurons.
Among the KIND domains in four KIND-containing
proteins (Spir-2, PTPN13, FRMPD2 and v-KIND),
only the KIND2 domain of v-KIND specifically bound
to MAP2. It is noteworthy that the v-KIND MAP2-
binding core polypeptides of 32 residues expressed in
hippocampal neurons were very effective in promoting
dendrite branching, which is opposite to the effect of the
overexpression of v-KIND, but similar to the effect of
knockdown of v-KIND [12], thereby suggesting that the
32 amino acids core polypeptide acts as a dominant-neg-
ative molecule by competing with endogenous v-KIND
for MAP2 binding. However, v-KIND-overexpressing
neurons showed decreased dendrite branches, but
formed slightly longer dendrites than the control
neurons and KIND2- or binding core-overexpressing
neurons. A previous in vitro study indicated that the
RasGEF activity of v-KIND induces the phosphoryla-
tion of MAP2 by JNK1 and ⁄ or ERK via the activation
of the Ras–Raf–MAP kinase pathway [12]. These results
appear to be in agreement with those of previous studies
showing that the phosphorylation of MAP2 by JNK1
(such as downstream of v-KIND overexpression [12])
enhances the activity of MAP2 to bind to microtubules
and promote their assembly [18], and that the inhibition
of JNK1 (such as downstream of v-KIND knockdown
[12]) increases the number of dendrite branches, but
decreases the mean dendrite length [19]. Taken together,
these results indicate that the KIND2 domain regulates
dendrite complexity via targeting of v-KIND RasGEF
(an activator of the Ras pathway) to MAP2 associated
with the dendritic microtubule cytoskeleton.
The present study indicates that the conserved amino
acid sequence of the MAP2 binding core module in the
mouse, human and Gallus v-KIND is indispensable for
the v-KIND–MAP2 interaction. The Ala substitution
for Leu at amino acids 461, 474 or 477, which are con-
served among the mouse, human and Gallus KIND2 but
not in the mouse Spir-2 and PTPN13 KIND, as well as
the mouse v-KIND KIND1, impaired the interaction
with MAP2. In addition, neurons transfected with
v-KIND bearing the L474A mutation induced more
complex dendritic arborization patterns than those of
neurons transfected with wild-type v-KIND, which
exhibited a decrease in total number of dendrites and an
increased mean length of dendrites. These findings dem-
onstrate the structural and functional importance of the
Leu474 residue in the v-KIND–MAP2 interaction-medi-
ated regulation of dendrite growth.
The interaction with v-KIND is specific to HMW-
MAP2, but not to LMW-MAP2, which lacks the CD
[14]. Although the functional property of the CD, the
EGFP
v-KIND
L474A
20 50 80 110 140 170 200 230 260 290 320 350
14
12
10
8
6
4
2
0
***
Distance from soma (µm)
Total number of intersections
EGFP v-KIND L474A
0
100
200
300
400
EGFP v-KIND L474A
Average length of dendrite (µm)
*** *
A
BC
Fig. 7. Contribution of the Leu474 residue
in the MAP2-binding core of v-KIND to den-
drite growth and branching in hippocampal
neurons. (A) HA-tagged v-KIND or the
L474A mutant was cotransfected with
EGFP into primary-cultured mouse hippo-
campal neurons at DIV7. Transfection with
EGFP alone was used as a control. Cells
fixed at DIV21 were immunostained with
anti-HA serum. Cell morphology images
with EGFP fluorescence were obtained by
confocal microscopy. Scale bar = 100 lm.
(B) Sholl analysis of dendrite complexity of
individual neurons. Data were obtained from
three independent transfection experiments
(for each experiment, n = 15 per construct).
The results are the mean ± SEM. *P < 0.05
and ***P < 0.001 for v-KIND versus L474A.
(C) Mean dendrite length in individual
neurons. The results are the mean ± SEM.
*P < 0.05 and ***P < 0.001.
Core modules for the very-KIND-MAP2 interaction J. Huang et al.
1658 FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS
largest domain of MAP2 (1372 amino acids, account-
ing for 70% of the total size) is not yet fully under-
stood, the data obtained in the present study indicate
that the 43 residues (amino acids 702–744 in mice) that
reside in the middle region of the CD act as the
v-KIND binding core. The v-KIND-binding core mod-
ule of MAP2 is also well conserved among human,
mouse and Gallus, and contains six conserved Leu resi-
dues (Fig. S4). Thus, it would be interesting to deter-
mine whether a hydrophobic interaction between the
Leu residues from v-KIND and MAP2 contributes to
the interaction between the two proteins.
In conclusion, the present study has clarified the
structural and functional importance of the v-KIND
and MAP2 interaction core modules in the regulation
of dendrite growth and branching in hippocampal neu-
rons and cerebellar granule cells. Further studies of
these newly-identified protein–protein interaction core
modules, including tertiary structural analyses, will
shed light on the molecular mechanism by which the
v-KIND–MAP2 interaction regulates the dendrite
arborization patterns that are critical for shaping neu-
ronal circuits, and also may provide a clue to the
understanding of some MAP2-associated neurodegen-
erative and psychiatric disorders [16,17].
Materials and methods
Animals
Mice (ICR) were purchased from Nihon SLC (Hamamatsu,
Japan) and used in accordance with protocols approved by
the Animal Care and Use Committee of RIKEN.
Plasmid construction and expression
Plasmid construction and expression in Escherichia coli or
African green monkey kidney cell line COS7 cells were per-
formed essentially as described previously [12]. Mouse
v-KIND cDNA [12] and its derivatives were cloned into a
mammalian cell expression vector, pCAG, with Flag or HA
tags at the C-terminal ends. The fragments of the KIND1
and KIND2 domains were generated by PCR and inserted
into pEGFP-C1 (Clontech Laboratories, Inc., Mountain
View, CA, USA) and pGEX-4T-2 (GE Healthcare UK Ltd,
Little Chalfont, UK) vectors for EGFP and GST fusion con-
structs, respectively. HMW MAP2 cDNA and its derivatives
generated by PCR were cloned into pGEX-4T-2. The KIND
domains of Spir-2 and PTPN13 were generated by RT-PCR
and inserted into pEGFP-C1 and pGEX-4T-2. The N- and
C-terminal regions of the v-KIND KIND2 and the MAP2
CD were generated by PCR and cloned into appropriate
expression vectors. Site-directed mutagenesis for substitution
of conserved Leu residues with Ala in the KIND2 domain of
v-KIND was performed using the QuikChange XL Site
Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA).
Pull-down assay
GST-fusion proteins were prepared and used for the pull-
down assays, as described previously [12]. Briefly, E. coli
expressing GST fusion proteins were lysed in ice-chilled lysis
buffer (50 mm Tris-HCl pH 7.4, 25% sucrose, 1% Triton
X-100 and 5 mm MgCl
2
). Then, 10 lgofE. coli lysates
containing GST fusion protein was coupled to glutathione-
sepharose (GE Healthcare UK Ltd) by rotating for 1 h at
4 °C. After washing with lysis buffer, the GST fusion protein
coupled with sepharose was mixed with 1 mg of protein
lysates prepared from mouse cerebella. After rotating for 1 h
at 4 °C, the GST fusion protein complex was washed with
lysis buffer and subjected to immunoblot analysis.
Immunoprecipitation
Immunoprecipitation was performed as described previ-
ously [20]. Briefly, COS7 cells or mouse cerebella were lysed
and homogenized in ice-chilled lysis buffer (50 mm Hepes,
150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm
MgCl
2
,1mm EGTA, 100 mm NaF, 1 mm Na
3
VO
4
,
10 lgÆmL
)1
aprotinin, 10 lgÆmL
)1
leupeptin and 1 m m
phenylmethanesulfonyl fluoride). After centrifugation at
1000 g for 10 min at 4 °C, the supernatants were mixed
with the antibody and incubated on ice for 1 h, followed by
rotation with protein A-sepharose or protein G-sepharose
(GE Healthcare UK Ltd) at 4 °C. Proteins immunoprecipi-
tated with the antibody–protein A- or G-sepharose com-
plexes were washed with lysis buffer and subjected to
immunoblot analysis.
Immunoblotting
SDS ⁄ PAGE and immunoblotting were performed essen-
tially as described previously [12]. The anti-(rabbit
v-KIND) serum [12] was used at 0.5 lgÆmL
)1
. Antibodies
against MAP2a ⁄ b (catalog number: AP20; Sigma-Aldrich,
St Louis, MO, USA), MAP2 (catalog number: M1406;
Sigma-Aldrich), EGFP (catalog number: SC-9996; Santa
Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Flag
(catalog number: 3165; Sigma-Aldrich) and HA (catalog
number: 1867423; Roche Diagnostics, Basel, Switzerland)
were used at dilutions of 1 : 1000 for immunoblotting,
1 : 200 for immunocytochemistry and 1 lgÆmL
)1
for co-
immunoprecipitation.
Primary cultures, transfection and imaging of
hippocampal and cerebellar neurons
Hippocampal and cerebellar dissociated primary cultures
were prepared from ICR mice (Nippon SLC, Hamamatsu,
J. Huang et al. Core modules for the very-KIND-MAP2 interaction
FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1659
Japan) at embryonic day 16 and postnatal day 0, respec-
tively. Cells were transfected with Flag- or HA-tagged full-
length v-KIND or mutant constructs together with the
EGFP vector as a cell morphology marker, essentially as
described previously [12]. Briefly, hippocampal neurons
(1 · 10
5
cellsÆcm
)2
) at DIV7 were transfected using Lipofec-
tamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) and
cultured in Opti-MEM (Invitrogen). Cerebellar neurons
(5 · 10
5
cellsÆcm
)2
) at DIV1 were transfected by the Ca
2+
-
phosphate-mediated method using a CellPhect Transfection
kit (GE Healthcare UK Ltd) and were cultured in serum-
free Eagle’s minimum essential medium (Nissui Pharmaceu-
tical Co., Ltd, Tokyo, Japan). Transfected cells were visual-
ized by EGFP fluorescence and immunocytochemical
staining with anti-Flag or anti-HA sera. Cell images were
acquired by confocal microscopy (LSM510; Carl Zeiss,
Inc., Oberkochen, Germany).
Morphometric analysis of dendritic arborization
patterns
To quantify dendrite growth and branching, 15 neurons
transfected with each construct were randomly chosen for
each experiment, and EGFP fluorescent images of their
dendrites were analyzed with neurolucida software (MBF
Bioscience, Williston, VT, USA). Image data were statisti-
cally quantified by repeated-measures analysis of variance
with Bonferroni post-hoc analysis.
Acknowledgements
We thank Dr N. Cowan (NYU Medical Center) and
Dr J. Miyazaki (Osaka University) for their kind gifts
of the MAP2 cDNA and the pCAG expression vector,
respectively. The present study was supported by
grants from the Japanese Ministry of Education,
Culture, Sports, Science and Technology; the Japan
Society for the Promotion of Science; and the Japan
Science and Technology Agency.
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Supporting information
The following supplementary material is available:
Fig. S1. The CBB-stained gel images of GST-fused
proteins tested.
Fig. S2. Pull-down assay of Flag-tagged v-KIND dele-
tion derivatives and EGFP-fused KIND domains by
GST-fused MAP2 CD2 region.
Fig. S3. Localization of HA-tagged v-KIND MAP2-
binding core (KIND2-1-HA) in dendrites of cultured
hippocampal neurons.
Fig. S4. Domain structure of MAP2 and the alignment
of v-KIND-binding core (BD) region of MAP2 CD2
domain (702–744 aa) in human, mouse and Gallus
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
J. Huang et al. Core modules for the very-KIND-MAP2 interaction
FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1661

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