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 Huang1,*, Asako Furuya1, Kanehiro Hayashi1–3 and Teiichi Furuichi1,2,4 Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Saitama, Japan JST, CREST, Kawaguchi, Saitama, Japan Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan 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 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 demonstrated that v-KIND interacts with the high-molecular weight microtubuleassociated protein (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 interaction 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 regulator 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) 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 interacts with vKIND-KIND2 by anti bait coimmunoprecipitation (View interaction) 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-molecularweight; 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 Core modules for the very-KIND-MAP2 interaction J Huang et al Introduction Protein–protein interactions play important roles in the molecular recognition and functional modulation between proteins in many signal transduction pathways [1,2] The kinase noncatalytic C-lobe domain (KIND) was determined to be a putative signaling domain based on bioinformatic analysis of the N-terminal 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 mammals: Spir [4,5], nonreceptor-type protein tyrosine phosphatase 13 (PTPN13, or PTP-BL ⁄ PTP-BAS) [6,7], FERM and PDZ-domain-containing (FRMPD2) [8] and Ras guanine exchange factor (RasGEF) veryKIND (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-terminal 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 residue, was reported in acute myeloid leukemia genomes [11] We previously identified v-KIND with a characteristic spatiotemporal expression pattern during the postnatal development of mouse brain: it shows low- or moderate-level expression in the cerebrum, hippocampus 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 and thereafter [12] We showed that v-KIND overexpression 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 (MAP2) via 1652 KIND2 is critical for this signaling pathway [12] HMW-MAP2 (referred to hereafter as MAP2) is known to modulate polymerization, stability and rearrangement 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 interaction between v-KIND and MAP2 We defined the binding core regions for the v-KIND–MAP2 interaction and showed that the MAP2 binding core is not only critical for targeting of v-KIND to neuronal dendrites, 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 localization 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 transfected 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 domainlacking constructs, DKIND2 (Fig 1B) and DKIND1 + (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, FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS J Huang et al Core modules for the very-KIND-MAP2 interaction A 37–217 456–620 KIND1 KIND2 v-KIND ΔKIND1 ΔKIND2 ΔKIND1+2 ΔRasN ΔGEF KIND1 KIND2 ΔKIND1-Flag RasN ΔKIND2-Flag 1461–1734 1742 GEF ΔKIND1+2-Flag dendrite axon ΔGEF-Flag KIND1-Flag KIND2-Flag α-Flag ΔRasN-Flag EGFP + α-Flag Fig Domain structure of the MAP2-associated RasGEF v-KIND and its dendritic targeting 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 fluorescence (green) were observed by confocal microscopy Open and closed arrowheads indicate dendrites and axons, respectively Scale bar = 50 lm EGFP + α-Flag α-Flag B v-KIND-Flag 1238–1364 CC Spir-2 KIND and PTPN13 KIND) (Fig 2B) and analyzed 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 samples (Fig 2A, right) Taken together, these data indicate 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 determine the v-KIND-binding region in MAP2, we first divided MAP2 into five regions (Fig 3A) and analyzed the bacterially expressed proteins of these constructs (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 specifically binds to KIND2 in v-KIND, we screened the FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1653 J Huang et al PTPN13 KIND Spir-2 KIND v-KIND KIND2 v-KIND KIND1 GST PD from purified MAP2 Input (MAP2) PTPN13 KIND Spir-2 KIND v-KIND KIND2 v-KIND KIND1 PD from cerebellar lysates GST A Input (cerebellum) Core modules for the very-KIND-MAP2 interaction IB: α-MAP2 B kDa 50 CBB 37 25 GST-CD2 binding ability of six Flag-tagged v-KIND derivatives (Fig S2A) The pull-down assay with GSTCD2, followed by immunoblotting with anti-Flag serum, showed that four KIND2-containing constructs (v-KIND, DKIND1, DRasN and DRasGEF) interacted with GST-CD2, whereas two constructs lacking KIND2 (DKIND2 and DKIND1 + 2) failed to bind to GST-CD2 (Fig S2A) This indicates that the KIND2 domain of v-KIND is involved in the interaction 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 subdivided 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 constructs (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 critical 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 1654 Fig 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 immunoblotting with anti-MAP2 serum endogenous cerebellar v-KIND protein All four C-terminal 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 GSTfused 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 pulldown 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, EGFPKIND2-1 to -KIND2-5 (Fig 4A), by a pull-down assay with GST-CD2 (MAP2 v-KIND binding core) FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS J Huang et al Core modules for the very-KIND-MAP2 interaction MAP2 RII CD1 CD2 CD3 MBD RII InterMBD action CD 1–147 148–599 600–1099 1100–1518 1519–1829 B IP PD Input Mouse IgG -MAP2 GST RII CD1 CD2 CD3 MBD A 250 150 IB: -v-KIND CD2 CD2-1 CD2-2 CD2-3 D action PD Input GST CD2-1 CD2-2 CD2-3 CD2 1099 Inter- 600 250 767 150 934 IB: -v-KIND E F CD2-1 CD2-1-1 634 CD2-1-2 667 CD2-1-3 701 CD2-1-4 CD2-1-5 735 CD2-1-6 702 CD2-1-7 668 CD2-1-8 635 CD2-1-6-1 702 CD2-1-6-2 CD2-1-6-3 Interaction PD Input GST CD2-1-1 CD2-1-2 CD2-1-3 CD2-1-4 CD2-1 767 250 150 PD 734 Input GST CD2-1-5 CD2-1-6 CD2-1-7 CD2-1-8 CD2-1 600 734 744 755 250 PD Input GST CD2-1-6-1 CD2-1-6-2 CD2-1-6-3 CD2-1 C 250 150 IB: -v-KIND Fig 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 microtubulebinding 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 Immunoprecipitation 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 GSTfused 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 immunoblotting with anti-v-KIND serum 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 coexpressed EGFP-fused KIND2-1 and full-length MAP2 in COS7 cells and analyzed their interaction by FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1655 Core modules for the very-KIND-MAP2 interaction v-KIND KIND2 KIND2-1 KIND2-2 KIND2-3 KIND2-4 KIND2-5 A 620 521 555 488 589 EGFP EGFP-KIND2-1 EGFP-KIND2-2 EGFP-KIND2-3 EGFP-KIND2-4 EGFP-KIND2-5 EGFP-KIND2 EGFP-KIND1 v KIND v-KIND KIND2 KIND2-1 + + + – – 487 B EGFP C IP: -MAP2 IB: -GFP PD: GST-CD2 IB: -GFP Input IB: -GFP IP: -MAP2 IB: -MAP2 Input IB: -GFP GST-CD2 (CBB stain) Fig 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) a co-immunoprecipitation assay (Fig 4C) Using antiMAP2 sera, we found that EGFP-KIND2-1 was co-immunoprecipitated with MAP2 in the cell lysates Taken together, these data indicate that the MAP2binding 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 days in vitro (DIV), fixed at DIV21, and analyzed the dendrite localization of the expressed proteins as 1656 EGFP Interaction Total number of intersections 456 EGFPKIND2-1 A J Huang et al B 18 16 14 12 10 *** 20 50 80 EGFP v-KIND KIND2 KIND2-1 110 140 170 200 230 260 290 320 350 Distance from soma (µm) Fig KIND 2-1 targets the dendrite and its overexpression suppressed 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 hippocampal 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 construct) The results are the mean ± SEM ***P < 0.001 for v-KIND versus KIND2-1 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 cerebellar 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 confers the dendritic targeting of v-KIND Overexpression of v-KIND decreased dendritic branching as reported previously [12], whereas overexpression of KIND2 promoted dendrite extension, compared to that of EGFP alone (Fig 5A) Interestingly, overexpression of the KIND 2-1 domain resulted in a complex dendrite morphology (Fig 5A) Sholl analysis of the dendrite FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS J Huang et al branching patterns showed that the number of dendrites within 130 lm from the soma was decreased in neurons overexpressing v-KIND, but increased in neurons overexpressing either KIND2 or KIND2-1, compared 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 overexpressing 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 dendrite 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 comparing 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 A B Fig 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 substituted 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 Core modules for the very-KIND-MAP2 interaction 65.6% homology, and 17 identical and four functionally similar residues, including seven conserved Leu residues, were identified By contrast, the corresponding 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 module CD2-1-6-2 of MAP2 (Fig 6B) KIND2 L461A, L474A and L477A mutants failed to bind to CD2-1-62 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 vKIND 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, neurons transfected with the L474A mutant had more complex 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 neurons had more dendritic branches at 90–140 lm from the soma compared to neurons transfected with EGFP alone, although the two transfectants had similar numbers 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 neurons was significantly greater than that of EGFP- or L474A-overexpressed neurons (Fig 7C) These results suggest that the Leu474 residue contributed to the functional interaction between v-KIND and MAP2 in the regulation of neuronal dendrite growth FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1657 Core modules for the very-KIND-MAP2 interaction J Huang et al A EGFP v-KIND L474A C 14 12 *** 10 EGFP v-KIND L474A 20 50 80 110 140 170 200 230 260 290 320 350 Average length of dendrite (µm) Total number of intersections B 400 *** 300 200 100 EGFP Distance from soma (µm) Discussion The present study revealed the structural and functional 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 residues, 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 MAP2binding 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-negative 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 phosphorylation 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 1658 * v-KIND L474A Fig Contribution of the Leu474 residue in the MAP2-binding core of v-KIND to dendrite growth and branching in hippocampal neurons (A) HA-tagged v-KIND or the L474A mutant was cotransfected with EGFP into primary-cultured mouse hippocampal 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 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 conserved 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 demonstrate the structural and functional importance of the Leu474 residue in the v-KIND–MAP2 interaction-mediated regulation of dendrite growth The interaction with v-KIND is specific to HMWMAP2, but not to LMW-MAP2, which lacks the CD [14] Although the functional property of the CD, the FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS J Huang et al largest domain of MAP2 (1372 amino acids, accounting for 70% of the total size) is not yet fully understood, 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 module of MAP2 is also well conserved among human, mouse and Gallus, and contains six conserved Leu residues (Fig S4) Thus, it would be interesting to determine 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 neurons 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 neuronal circuits, and also may provide a clue to the understanding of some MAP2-associated neurodegenerative and psychiatric disorders [16,17] Core modules for the very-KIND-MAP2 interaction 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 pulldown 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 mm MgCl2) Then, 10 lg of E coli lysates containing GST fusion protein was coupled to glutathionesepharose (GE Healthcare UK Ltd) by rotating for h at °C After washing with lysis buffer, the GST fusion protein coupled with sepharose was mixed with mg of protein lysates prepared from mouse cerebella After rotating for h at °C, the GST fusion protein complex was washed with lysis buffer and subjected to immunoblot analysis Immunoprecipitation 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 Immunoprecipitation was performed as described previously [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 MgCl2, mm EGTA, 100 mm NaF, mm Na3VO4, 10 lgỈmL)1 aprotinin, 10 lgỈmL)1 leupeptin and mm phenylmethanesulfonyl fluoride) After centrifugation at 1000 g for 10 at °C, the supernatants were mixed with the antibody and incubated on ice for h, followed by rotation with protein A-sepharose or protein G-sepharose (GE Healthcare UK Ltd) at °C Proteins immunoprecipitated with the antibody–protein A- or G-sepharose complexes were washed with lysis buffer and subjected to immunoblot analysis Plasmid construction and expression Immunoblotting Plasmid construction and expression in Escherichia coli or African green monkey kidney cell line COS7 cells were performed 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 constructs, 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 SDS ⁄ PAGE and immunoblotting were performed essentially as described previously [12] The anti-(rabbit v-KIND) serum [12] was used at 0.5 lgỈmL)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 : 1000 for immunoblotting, : 200 for immunocytochemistry and lgỈmL)1 for coimmunoprecipitation Materials and methods Animals Primary cultures, transfection and imaging of hippocampal and cerebellar neurons Hippocampal and cerebellar dissociated primary cultures were prepared from ICR mice (Nippon SLC, Hamamatsu, FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1659 Core modules for the very-KIND-MAP2 interaction J Huang et al Japan) at embryonic day 16 and postnatal day 0, respectively Cells were transfected with Flag- or HA-tagged fulllength v-KIND or mutant constructs together with the EGFP vector as a cell morphology marker, essentially as described previously [12] Briefly, hippocampal neurons (1 · 105 cellsỈcm)2) at DIV7 were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) and cultured in Opti-MEM (Invitrogen) Cerebellar neurons (5 · 105 cellsỈcm)2) at DIV1 were transfected by the Ca2+phosphate-mediated method using a CellPhect Transfection kit (GE Healthcare UK Ltd) and were cultured in serumfree Eagle’s minimum essential medium (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) Transfected cells were visualized 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 statistically quantified by repeated-measures analysis of variance with Bonferroni post-hoc analysis 10 Acknowledgements 12 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 References 13 14 15 Pawson T & Nash P (2000) Protein–protein interactions define specificity in signal transduction Genes Dev 14, 1027–1047 Pawson T & Nash P (2003) Assembly of cell regulatory systems through protein interaction domains Science 300, 445–452 Ciccarelli FD, Bork P & Kerkhoff E (2003) The KIND module: a putative signalling domain evolved from the C lobe of the protein kinase fold Trends Biochem Sci 28, 349–352 Otto IM, Raabe T, Rennefahrt UE, Bork P, Rapp UR & Kerkhoff E (2000) The p150-Spir protein provides a 1660 11 16 17 18 link between c-Jun N-terminal kinase 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cerebellar granule cell axons Mol Biol Cell 17, 3187–3196 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 deletion derivatives and EGFP-fused KIND domains by GST-fused MAP2 CD2 region Core modules for the very-KIND-MAP2 interaction Fig S3 Localization of HA-tagged v-KIND MAP2binding 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 FEBS Journal 278 (2011) 1651–1661 ª 2011 The Authors Journal compilation ª 2011 FEBS 1661 ... PDZ -domain- containing (FRMPD2) [8] and Ras guanine exchange factor (RasGEF) veryKIND (v-KIND, or kinase noncatalytic C-lobe domain containing 1) [9] The KIND domain in these proteins is localized to the. .. determined the structural and functional properties of the protein? ? ?protein interaction between v-KIND and MAP2 We defined the binding core regions for the v-KIND–MAP2 interaction and showed that the. .. 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,