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Distinct Roles for the Two Rho GDP/GTP Exchange Factor Domains of Kalirin in Regulation of Neurite Growth and Neuronal Morphology

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Distinct Roles for the Two Rho GDP/GTP Exchange Factor Domains of Kalirin in Regulation of Neurite Growth and Neuronal Morphology The Journal of Neuroscience, November 1, 2001, 21(21):8426–8434 Distinct Roles for the Two Rho GDP/GTP Exchange Factor Domains of Kalirin in Regulation of Neurite Growth and Neuronal Morphology Peter Penzes,1 Richard C. Johnson,1 Vikram Kambampati,1 Richard E. Mains,2 and Betty A. Eipper2 1Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and 2Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 06030 The actin cytoskeleton, essential for neuronal development, is GEF domain alone induces axonal over-elongation and abun- regulated in part by small GTP binding proteins of the Rho dant filopodial neurites, mediated by RhoA. Coordination of the subfamily. Kalirin-9, with two Rho subfamily-specific GDP/GTP actions of the individual GEF domains through their presence in exchange factor (GEF) domains, localizes to neurites and Kalirin-9, with its Sec14p, spectrin, and Src homology domain growth cones of primary cortical neurons. Kalirin-9 overexpres- 3 motifs, is essential for regulating neurite extension and neu- sion in cultured cortical neurons induces longer neurites and ronal morphology. altered neuronal morphology. Expression of the first GEF do- main alone results in drastically shortened axons and excessive Key words: Rac1; RhoA; Trio; spectrin; cytoskeleton; growth growth cones, mediated by Rac1. Expression of the second cone The actin cytoskeleton plays an essential role in morphological domains, an Src homology domain 3, a second tandem DHPH changes during neuronal development and plasticity. Rho domain, a region of Ig-like repeats, and a serine–threonine GTPases are key regulators of the actin cytoskeleton (Hall, 1998), kinase-like domain (see Fig. 1a). Kalirin-9 and Kalirin-12, the and there is ample evidence involving Rho GTPases in growth of major isoforms expressed during embryonic development (Han- neurites and axon guidance (Luo et al., 1994;Threadgill et al., sel et al., 2001), both contain two DH domains, whereas Kalirin-7, 1997; Li et al., 2000). The immediate activators of Rho-like the major adult isoform, contains only one (Johnson et al., 2000; GTPases are GDP/GTP exchange factors (GEFs). All GEFs for Penzes et al., 2000). Rho subfamily members contain a catalytic Dbl-homology (DH) Studies in Drosophila and C. elegans indicate that a protein domain, followed immediately by a pleckstrin homology (PH) homologous to Kalirin-9 (dTrio and UNC-73, respectively) is an domain (Whitehead et al., 1997). Neuronal GEFs include pro- essential player in multiple axon guidance pathways (Steven et al., teins with two DHPH domains, such as Kalirin (Alam et al., 1997; 1998; Awasaki et al., 2000; Bateman et al., 2000; Liebl et al., 2000; Johnson et al., 2000; Penzes et al., 2000), Trio (Debant et al., Newsome et al., 2000) (Fig. 1a). Mutational studies indicated an 1996), Caenorhabditis elegans UNC-73 (Steven et al., 1998), and essential role for the first DHPH domain, with no clear role for Drosophila Trio (Awasaki et al., 2000; Bateman et al., 2000; Liebl the second DHPH domain (Steven et al., 1998; Bateman et al., et al., 2000; Newsome et al., 2000) (see Fig. 1a). 2000; Liebl et al., 2000; Newsome et al., 2000). In this study, we Tremendous progress has been made in uncovering extracel- sought to understand the importance of having multiple DHPH lular signals and the receptors that regulate neurite outgrowth domains included in a single protein capable of interacting simul- and growth cone guidance (Tessier-Lavigne and Goodman, taneously with several proteins and with membrane lipids. 1996). As reflected in the ability of cyclic nucleotide levels to switch guidance cues from attractive to repulsive, the rapid ex- MATERIALS AND METHODS tension and retraction of processes associated with neurite growth DNA and antibodies. Rac1-N17 and RhoA-N19 plasmids were gifts from requires careful coordination (Song and Poo, 1999). We won- Dr. Silvio Gutkind (National Institute of Dental Research, National dered whether this task involved neuronal proteins containing Institutes of Health, Bethesda, MD), whereas glutathione S-transferase multiple GEF domains. (GST)-RhoA, GST-Rac1, and GST-Cdc42 plasmids and constitutively active Rac1 (Rac1-Q61L) and RhoA (RhoA-Q63L) plasmids were gifts Kalirin, a Dbl family member with multiple isoforms, is highly from Dr. Richard Cerione (Cornell University, Ithaca, NY). Rabbit expressed in adult rat CNS (Alam et al., 1997). The longest polyclonal antisera against regions of Kalirin were affinity purified Kalirin protein, Kalirin-12, contains a Sec14-like putative lipid (Johnson et al., 2000; Penzes et al., 2000). Monoclonal antisera to binding domain, nine spectrin-like repeats, tandem DH and PH neuronal-specific tubulin (Babco, Richmond, CA), Rac1 (Upstate Bio- technology, Lake Placid, NY), and RhoA (Santa Cruz Biotechnology, Santa Cruz, CA) were purchased. FITC-phalloidin was from Sigma (St. Received June 19, 2001; revised Aug. 1, 2001; accepted Aug. 7, 2001. Louis, MO). pEGFP-N2 vector was from Clontech (Palo Alto, CA). C3 This work was supported by National Institutes of Health Grants DA-00266 and transferase expression vector was from Dr. Anirvan Ghosh (Johns Hop- DK-32948. We thank Dr. L. Redmond for her help with neuronal transfections. We thank Drs. A. Ghosh, A. Kolodkin, and C. Hopf for their useful advice and for kins University, Baltimore, MD). critically reading this manuscript. Cell cultures and transient transfections. NIH3T3 cells were cultured Correspondence should be addressed to Betty A. Eipper, Department of Neuro- in DMEM–F-12 containing 10% fetal bovine serum (HyClone, Logan, science, University of Connecticut Health Center, 263 Farmington Avenue, Farm- UT) and 10% NuSerum (Collaborative Research, Bedford, MA). For ington, CT 06030. E-mail: [email protected]. immunocytochemistry, cells grown on glass chamber slides for3dto Copyright © 2001 Society for Neuroscience 0270-6474/01/218426-09$15.00/0 40–60% confluence were transfected with 1 ␮g of plasmid DNA per 4 Penzes et al. • Kalirin Regulates Neurite Growth J. Neurosci., November 1, 2001, 21(21):8426–8434 8427 cm 2 of pEAK10.His-Myc-DHPH1, pEAK10.His-Myc-DHPH2, or pEAK10.His-Myc-Kal-9 and 4 ␮l of Lipofectamine (Life Technolo- gies, Gaithersburg, MD) in 1 ml of complete serum-free medium for 5 hr, after which they were washed and fed with growth medium. After 1 d, medium was replaced with DMEM–F-12 for 16 hr. Cells fixed in 3.7% formaldehyde in PBS (50 mM NaPi, pH7.5, and 150 mM NaCl) for 30 min at room temperature were permeabilized and stained. Neuronal cultures and transfections. Rat embryo cerebral cortex (em- bryonic day 18) digested with papain for 30 min at 37°C was dissociated by pipetting. Neurons were plated in chamber slides precoated with poly-L-lysine in MEM containing 2 mM glutamine and 5% heat- inactivated horse serum (Life Technologies), penicillin, and streptomy- cin.After2dat37°Cin5%CO2, cultures were transfected using the calcium phosphate method for 15 min (Penzes et al., 2001). Cells in one chamber of a two-chamber slide were transfected with 1 ␮g of pEGFP-N2 and 1 ␮g of a pEAK10.His-Myc Kalirin-encoding vector. Vectors encod- ing Rho GTPases (2–3 ␮g) were transfected along with pEGFP-N2 (1 ␮g) and, where indicated, a Kalirin-encoding vector (1 ␮g).After2dat 37°C, neurons were fixed in 3.7% formaldehyde for 20 min, permeabil- ized, and blocked in PBS containing 2% normal goat serum and 0.1% Triton X-100 for 1 hr. Antibody was added in PBS with 2% normal goat serum for 2 hr at room temperature. Transfected myc-tagged Kalirin constructs were visualized by immunostaining with monoclonal or poly- clonal myc antibodies. Morphological features were quantified with the help of an independent observer using the IPLab software (Scanalytics Inc., Fairfax, VA). Pictures of randomly selected fields were taken at low magnification, and the length of the longest process on each individual neuron in the field was measured after it was traced using the computer program; at least 30 neurons from two to three independent transfections were analyzed. In addition, transfected neurons were evaluated for the presence of growth cones on the cell soma and filopodial structures along neurites. A neuron was scored as positive if it had one or more growth cones and abundant filopodial structures. Rac1 GTPase activation assays. Activation of Rac1 was measured with the Rac1/Cdc42 activation assay kit (Upstate Biotechnology). Briefly, cells that had been transfected 1 d earlier were serum-starved overnight, and cell lysates (ϳ0.5 mg of protein) were incubated for 1 hr at 4°C with 10 ␮l of GST-PAK1-PBD resin per sample. Bound proteins were eluted and analyzed by SDS-PAGE and Western blotting with Rac1 and Cdc42 monoclonal antibodies. Figure 1. Expression of Kalirin. a, Kalirin isoforms and related proteins. DHPH binding to Rho GTPases. The GST fusion proteins of Rho Bars indicate antigens used to generate antibodies. b, Expression of ␮ GTPases purified from Escherichia coli were depleted of bound nucleo- Kalirin in rat cerebral cortex (25 g of protein) during postnatal devel- tide by incubation in 10 mM EDTA. pEAK-rapid human embryonic opment, detected with the Kal-spec antibody (1:1000). c, Expression of ␮ kidney 293 (HEK-293) cells expressing myc-DHPH1 or myc DHPH2 Kalirin isoforms in 4-d-old cultures of dissociated cortical neurons (25 g were extracted in binding buffer (20 mM Tris-HCl, pH 7.5, and 50 mM of protein), detected with the indicated Kalirin antibodies. Kal-spec was NaCl containing 1% Triton X-100). For each binding reaction, 5 ␮gof used at 1:1000; Kalirin-9, Kalirin-12, and Kalirin-7 affinity-purified anti- GST-GTPase bound to 25 ␮l of glutathione-Sepharose beads was mixed bodies were used at 1:100. Kalirin-7 cannot be detected at this age.
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