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: [email protected]
*Present address
Discovery & Development Laboratory I,
Hanno Research Center, Taiho
Pharmaceutical Co., Ltd, Saitama, Japan

l
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-

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-

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
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-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

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

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

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

(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 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

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

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 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

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

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

)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

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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
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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