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J Biol Chem, Vol. 275, Issue 9, 6395-6403, March 3, 2000
From the Department of Neuroscience, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
Communication between membranes and the actin
cytoskeleton is an important aspect of neuronal function. Regulators of
actin cytoskeletal dynamics include the Rho-like small GTP-binding
proteins and their exchange factors. Kalirin is a brain-specific
protein, first identified through its interaction with
peptidylglycine- Communication between membranes and the cytoskeleton involves
small GTP-binding proteins of the Rho family and their regulators (1,
2). Many Dbl family members function as guanine-nucleotide exchange factors (3, 4), catalyzing the exchange of bound GDP with GTP
on small GTP-binding proteins of the Rho family. Rho family proteins
also regulate gene expression (5-7) and a growing number of other
processes in cells (8).
Kalirin, a Dbl family member, was first identified as an interactor
with the cytoplasmic COOH-terminal domain of membrane peptidylglycine- Along with its DH and PH domains, Kalirin-8 has nine spectrin-like
repeats, an Src homology 3 domain, and two putative PEST sequences
(Fig. 1). A human protein, DUO, identified through its interaction with
Huntingtin-associated protein 1 (14), is 98% identical with rat
Kalirin-8 over the overlapping region but differs from Kalirin-8 at
both its NH2- and COOH-terminal ends. Kalirin and DUO are
closely related to human TRIO, an interactor with LAR, a transmembrane
protein-tyrosine phosphatase thought to be a regulator of cell-matrix
interactions (15). TRIO has a structure similar to that of Kalirin but
is larger and contains additional functional domains (Fig. 1). UNC-73,
a Caenorhabditis elegans homolog of Kalirin and TRIO, is a
regulator of the cytoskeleton during cell and growth cone migration and
is required for axon guidance (16). Two forms of UNC-73 have been
identified; UNC-73B has a domain structure similar to Kalirin-8, while
UNC-73A contains an additional DH and PH domain (Fig.
1).
Little is known about the natural forms of Dbl family proteins and
their functions in neurons, since the majority of studies have utilized
transfected cells, and endogenous proteins have been difficult to
detect. We first searched for a form of rat Kalirin equivalent to human
DUO. We demonstrate that a 7-kb transcript encodes a rat protein
(Kalirin-7) equivalent to human DUO. Using a general Kalirin antiserum,
we identify a family of endogenous Kalirin proteins in adult rat brain.
Using an antiserum specific to the COOH terminus of Kalirin-7, we show
that Kalirin-7 is highly enriched in the postsynaptic density fraction.
We use cultured neurons to localize Kalirin-7 to spine-like structures
and show that other forms of Kalirin are localized in the cell soma and neurites. We explore the ability of Kalirin-7 and its DH-PH domain to
induce formation of lamellipodia and membrane ruffling by transient expression in NIH 3T3 fibroblasts and demonstrate that the DH-PH domain
is an active guanine-nucleotide exchange factor for Rac1. Kalirin-7 may
tether membrane proteins to the actin cytoskeleton, potentially
providing a link between postsynaptic membrane signaling and
cytoskeletal rearrangements involved in neurite growth and synaptic function.
Materials
Antibodies--
PDZ domain monoclonal antibody was from Upstate
Biotechnology, Inc. (Lake Placid, NY); BiP polyclonal antibody was from
Affinity Bioreagents; p38 (synaptophysin) monoclonal antibody was from Roche Molecular Biochemicals; Rac-1 and Munc18-1 (nSec1) monoclonal antibodies were from Transduction Laboratories; and rat TGN38 antibody
was raised in our laboratory (17). Neuron-specific tubulin monoclonal
antibody was from Babco; actin polyclonal antibody was a gift from Dr.
Douglas Murphy (Department of Cell Biology and Anatomy, Johns Hopkins University).
Plasmids and DNA--
Rac1-N17 plasmid was a gift from Dr.
Silvio Gutkind (Laboratory of Cellular Development and Oncology, NIDR,
National Institutes of Health). Ost expression plasmid was generously
provided by Dr. Toru Miki (Laboratory of Cellular and Molecular
Biology, NCI, National Institutes of Health); TRIO expression plasmid
was a gift from Dr. Michel Streuli (Division of Tumor Immunology, Dana Farber Cancer Institute and Department of Pathology, Harvard Medical School). The plasmid encoding constitutively active Rac1 (Rac1-Q61L) (5) was a gift from Dr. Anirvan Ghosh (Johns Hopkins). Oligonucleotides were synthesized at the Johns Hopkins Molecular Biology Core.
Cloning of Kalirin-7 cDNA
Rat cerebral cortex cDNA was generated by reverse
transcription of poly(A)+ mRNA with an
oligo-(dT)15 primer (Promega). A 900-bp PCR product was
generated by amplification with a Kalirin-8 sense primer (nt 4071-4092) and an antisense primer
5'-GGTCTAGACGGCTAACCCGGTCAGCT-3' (rat nt 4978-4960)-based
Homo sapiens EST T74341, which included an XbaI
site (underlined). The PCR product was subcloned and sequenced. A
cDNA encoding full-length Kalirin-7 was constructed by replacing the 3'-end of Kalirin-8 (with the P-CIP10a 5'-end (2)) with the 3'-end
of Kalirin-7. The PCR product was digested with NsiI (nt
4316) and XbaI, and the 662-bp fragment was isolated for
subcloning into pBS.Kalirin-8 digested with the same two enzymes to
generate pBS.Kalirin-7. The mammalian expression vector
pSCEP.Myc-Kalirin-7 was constructed in a similar manner: a 0.7-kb
NsiI (nt 4316)-NotI (3'-MCS) fragment isolated
from pBS.Kalirin-7 was subcloned into pSCEP.Myc-Kalirin-8 cut with the
same two enzymes. Another mammalian expression vector,
pEAK10.His-Myc-Kalirin-7, was constructed by excising the entire insert
from pSCEP.Myc-Kalirin-7 with NcoI and NotI and
inserting it into pEAK10.His (Edge Biosystems) digested with
BspLU11I and NotI. The DNA fragment encoding the
DH-PH domain of Kalirin-8 (amino acids 1231-1575) was generated by PCR
amplification of Kalirin-8 (nt 3715-4749) with sense primer
5'-GCTCTAGACATATGGCCATGGGGTTCAGACCGGGAGGTCA and
antisense primer
5'-GGGAAAAGCGGCCGCTTATTTGGCTGGTGTTTTTGG, bearing the restriction sites for NcoI (underlined),
and NotI (double underlined). pEAK10.His-Myc-DH-PH was
constructed by inserting the NcoI-NotI fragment
of the DH-PH PCR product into pEAK10.His-Myc cut with Bsp LU11 I and
NotI. All constructs were verified by sequencing.
RT-PCR to Identify the Kalirin-7 5'-Ends
PCR sense primers were as follows: nt 1-27 of P-CIP10a,
5'-GCCCGGTCTCTCAGGGCAGCCATAATG-3'; nt 327-352 of P-CIP10b,
5'-CGCAGACGCCCTCCCCCAGTCATGAA-3'; nt 98-123 of human DUO,
5'-GGGATGACGGACCGCTTCTGGGACCA-3'. Antisense primer was KAL 7-stop,
nucleotides 4951-4925 of P-CIP10a (5'-AGTCCCAGGCTGCGCGCTAAACGTAAG-3'). Reverse transcription was performed with Superscript II (Life Technologies, Inc.), and the resultant cDNA was amplified using the
High Fidelity PCR System (Roche Molecular Biochemicals). Samples were
denatured for 15 s at 94 °C, annealed for 30 s at
62 °C, and extended for 4 min at 68 °C for a total of 30 cycles.
Extension times were increased 20 s per cycle for the last 20 cycles.
Northern Analysis
A DUO-specific probe for use in Northern blot analysis was
generated by RT-PCR. Total or poly(A)+ cerebral cortical
RNA was reverse transcribed using oligo(dT). Amplification was carried
out with Kalirin-7-specific sense (5'-GTGACAAGGATGGCAACCTT-3' (nt
4865-4884)) and antisense (5'-GGAAACATGTTGCCCTCTGA-3' (nt 4987-4967))
primers (nt numbers are for DUO). This 122-bp fragment was used to
probe a Northern blot of rat cerebral cortex poly(A)+ mRNA.
Antisera
Rabbit polyclonal antisera JH2580-2582 were generated against
Kalirin spectrin-like repeats 4-7 (rat Kalirin-8-(517-976)) (4).
Affinity purification of these antisera did not alter the pattern
observed on Western blots. Rabbit polyclonal antisera JH2958 and JH2959
were generated at Covance (Denver, PA) by immunizing rabbits with the
synthetic peptide comprising the COOH-terminal 19 residues of rat
Kalirin-7 (1625GNLVPRWHLGPGDPFSTYV1644)
cross-linked to keyhole limpet hemocyanin with glutaraldehyde (2 mg of
peptide, 5 mg of hemocyanin). This peptide was synthesized by Dr. Henry
Keutmann (Endocrine Unit, Massachusetts General Hospital). Following
ammonium sulfate precipitation, JH2959 was affinity-purified using the
rat Kalirin-7 COOH-terminal peptide linked to Affi-Gel 10. Affinity-purified antibody was eluted with 0.2 M glycine
HCl, 0.1 M NaCl, 0.1% Triton X-100, pH 2.3, immediately
neutralized with Tris-HCl, dialyzed into 100 mM sodium
phosphate buffer, pH 7.4, and used at a dilution of 1:100.
Tissue Preparations
Parietal cortex and liver were dissected from adult female
Harlan Sprague Dawley rats (Harlan), and tissues were homogenized in 10 volumes of 20 mM Tris-HCl (pH 7.5), containing
phenylmethylsulfonyl fluoride (0.3 mg/ml), and a protease inhibitor
mixture (lima bean trypsin inhibitor (50 µg/ml), leupeptin (2 µg/ml), benzamidine (16 µg/ml), and pepstatin (2 µg/ml)) with
five strokes of a glass/Teflon homogenizer. Large debris and unbroken
cells were removed by centrifugation at 5,000 × g for
5 min. The supernatant was centrifuged in a Beckman TL100
ultracentrifuge at 450,000 × gmax for 15 min to obtain crude soluble and crude particulate fractions.
Subcellular Fractionations
Extracts of rat brain cortex and olfactory bulb were
fractionated in isotonic buffer according to the method of Huttner
et al. (18). An equal percentage of each fraction was
analyzed by SDS-PAGE and Western blotting. Postsynaptic densities were purified from brain cortex and olfactory bulb from 10 female adult Holtzman rats (Harlan) using the protocol described by Carlin et al. (19) with the exception that the
homogenization buffer A contained 4 mM HEPES, pH 7.5.
Transient Transfections
NIH3T3 cells were cultured in Dulbecco's modified Eagle's
medium/F-12 containing 10% fetal bovine serum (Hyclone) and 10% NuSerum (Collaborative Research). Briefly, cells grown on glass slides
for 3 days to 40-60% confluence were transfected with 1 µg of
plasmid DNA/4-cm2 (pEAK10.His-Myc-DH-PH,
pEAK10.His-Myc-Kalirin-7, or a plasmid carrying preproneuropeptide Y
(20)) and 4 µl of lipofectamine (Life Technologies, Inc.) in 1 ml of
complete serum-free medium for 5 h, after which they were fed with
growth medium. After 1 day, the medium was replaced with Dulbecco's
modified Eagle's medium/F-12 containing no serum for 16 h. Cells
were fixed in 4% formaldehyde in phosphate-buffered saline (50 mM NaPi, pH 7.5, 150 mM NaCl) for
30 min at room temperature, permeabilized, and stained as described
previously (17). Cell morphology was evaluated by analyzing
approximately 30 transfected cells and counting the fraction of cells
displaying a particular phenotype. Protein expression levels in
transfected cells were compared by quantifying the intensities of
immunofluorescence upon detection with Myc antibody, using the Scion
image software.
Primary Cultures of Rat Cortical and Olfactory Bulb Neurons
Olfactory bulbs dissected from postnatal rat brains were
dissociated by treatment with porcine pancreas trypsin (Sigma) (3 mg/ml) and Benzonase (EM Science) (4 mg/ml) for 20 min at 37 °C. Cells were plated onto polylysine-coated plastic slides and grown in
Dulbecco's modified Eagle's medium/F-12 containing 10% fetal bovine
and 10% NuSerum. Plating medium was replaced with serum-free Neurobasal medium (Life Technologies, Inc.) containing 1% N-2 supplement (Life Technologies, Inc.), 0.5 mM
L-glutamine (Sigma), and 25 µM
L-glutamic acid (Sigma) after 24 h at 37 °C (21). After 4-8 days in culture, cells were fixed with 4% formaldehyde for
30 min and stained as described above. Rat cerebral hemispheres dissected from embryonic day 17 or 18 rat embryos were dissociated by
treatment with papain (Sigma) (200 units) for 40 min at 37 °C. Cells
were plated onto polylysine- and laminin-coated glass coverslips and
grown in Neurobasal medium (Life Technologies, Inc.) containing 2%
B-27 supplement (Life Technologies, Inc.), 2.1 mM
L-glutamine (Sigma), and 5 µg/ml gentamycin. After 7 days in culture, cells were fixed with 4% formaldehyde for 30 min and stained as described above.
GDP/GTP Exchange Factor Assay
GST-Rac1 was expressed in Escherichia coli and
purified on glutathione affinity resin (Amersham Pharmacia Biotech).
For expression of DH-PH, pEAK-Rapid cells (Edge Biosystems) were
transiently transfected with pEAK10.His-Myc.DH-PH using Lipofectamine
(Life Technologies, Inc.). The DH-PH protein was enriched using the Talon affinity resin (Qiagen) following the manufacturer's
instructions. The GDP/GTP exchange assay was performed according to
Debant et al. (15) and Steven et al. (16).
Cloning of Kalirin-7--
Human DUO (14) and rat Kalirin-8 are
>98% identical in their 1612-amino acid region of overlap, differing
at only 22 positions (Fig.
2A). This degree of identity
strongly suggests that DUO and Kalirin are encoded by equivalent human
and rat genes, respectively. Human DUO and rat Kalirin-8 differ at
their NH2- and COOH-terminal ends. Human DUO has a
22-residue N terminus preceding the homologous region, while two
different rat Kalirin N termini (10a, 4 residues; 10b, 24 residues)
were identified (10). The three NH2-terminal sequences
share no sequence similarity. Recombinant Kalirin-8 included the
4-residue N terminus, because transcripts with this start site were
more efficiently expressed following in vitro transcription/translation. At its COOH terminus, DUO contains 20 residues not present in Kalirin-8, while Kalirin-8 extends 283 residues
beyond the 3'-divergence point (Fig. 2A).
To find out if rats express a form of Kalirin equivalent to human DUO,
we performed RT-PCR on rat cerebral cortex poly(A)+ RNA
(Fig. 2B). A cDNA fragment of the predicted size was
amplified with a sense primer common to human DUO and rat Kalirin-8 and an antisense primer specific to human DUO (based on human EST T74341).
EST T74341 is identical to Kalirin and DUO up to their 3'-divergence
point, after which it is identical to DUO. The polypeptide encoded by
the PCR product was identical to Kalirin-8 until the 3'-divergence
point, after which the 20 COOH-terminal residues were identical to the
corresponding region of human DUO and were followed by a stop codon.
The 5'-ends of Kalirin-8 and DUO diverge at exactly the same point at
which the two previously identified Kalirin 5'-ends (10a and 10b)
diverged. To determine which of the three potential Kalirin 5'-ends
encode Kalirin mRNAs with the DUO-like 3'-end, we amplified
cDNA with sense primers specific for each of the three 5'-ends
(10a, 10b, DUO) and a DUO-specific 3' antisense primer. Products of the
predicted size (5 kb) were obtained with all three primer pairs,
indicating that all three 5'-ends were present in mRNAs that
include a DUO-type 3'-end. Southern analysis using a cDNA probe to
Kalirin-spectrin domains 1-3 (nt 580-1531) was used to confirm that
the amplified products correspond to Kalirin (Fig. 2C).
Northern blot analysis was performed to determine the sizes of the
Kalirin mRNAs including various regions of Kalirin (Fig. 2D). A probe specific for the DUO-type 3'-end detected 5.0- and 7.0-kb Kalirin transcripts and no larger forms. Hybridization with
a probe corresponding to the first three spectrin-like repeats of
Kalirin (spectrin 1-3) revealed that the 7-kb transcripts, as well as
several longer transcripts, contained this region, while the 5-kb
transcript did not. A probe specific for the 10b 5'-end also identified
7-kb transcripts (Fig. 2D). Based on the PCR data shown in
Fig. 2C, 7-kb Kalirin transcripts could also include the
DUO- and 10a 5'-ends. Since the functional significance of the
different 5'-ends is not yet apparent, the three Kalirin proteins
encoded by the 7-kb mRNA will be referred to collectively as
Kalirin-7 (Fig. 2E).
A full-length Kalirin-7 cDNA was constructed by joining the 5'-end
of Kalirin-8, bearing the 10a 5'-end (10), with the DUO-type 3'-fragment. The open reading frame of Kalirin-7 encodes a protein of
1644 residues (190 kDa). The Kalirin-7 protein includes the 10a
sequence, nine spectrin-like repeats, a DH domain, and a PH domain
(Fig. 2E). It lacks the Src homology 3 domain present in Kalirin-8 and contains instead a unique 20-residue COOH terminus. The
final COOH-terminal 3 residues correspond to the recognition motif for
proteins with PDZ domains ((S/T)XV), suggesting that Kalirin-7 and Kalirin-5, unlike larger Kalirin proteins, could interact
with such proteins.
Kalirin Antisera Detect Multiple Proteins in Rat Brain--
Two
sets of antisera were used to detect endogenous Kalirin-related
proteins. Kalirin-spectrin antisera JH2581 and JH2582 were raised
against recombinant spectrin-like repeats 4 to 7 of Kalirin (13). We
tested the cross-reactivity of the Kalirin-spectrin antibodies with the
homologous fragments of TRIO and Ost. The Kalirin-spectrin antisera had
little (JH2581) or no (JH2582) cross-reactivity with the corresponding
regions of TRIO and Ost (Fig.
3A). We used a 19-residue
synthetic peptide corresponding to the unique COOH terminus of
Kalirin-7 to generate Kalirin-7-specific antisera (JH2958 and
JH2959).
The two Kalirin-spectrin antisera and a Kalirin-7 antiserum were used
to detect Kalirin proteins in crude soluble and particulate fractions
of rat brain cortex homogenate (Fig. 3B). Both
Kalirin-spectrin antisera detected a family of proteins in the soluble
and particulate fractions. The bands recognized by both
Kalirin-spectrin antisera and not present in the liver, a tissue that
does not express detectable levels of Kalirin mRNA by Northern
blotting (10), were assumed to represent forms of Kalirin: 115, 190, 370, 420, and 470 kDa (Fig. 3B). The 150-kDa protein is
considered nonspecific because it is not detected by both
Kalirin-spectrin antisera and corresponds to a cross-reactive protein
in liver. The 190-kDa Kalirin protein was enriched in the particulate fraction.
The Kalirin-7 antibodies detected a major 190-kDa protein and a minor
115-kDa protein (Fig. 3B). Proteins of the same size were
detected by both Kalirin-spectrin antisera. The 190-kDa protein detected by the Kalirin-7 antiserum is the mass predicted for Kalirin-7, and the 115-kDa protein is small enough to be encoded by the
Kalirin-5 transcripts. Staining by the Kalirin-7 antiserum was
eliminated by inclusion of excess antigen (Fig. 3C). Since Kalirin-7 is enriched in the particulate fraction (Fig. 3B),
the abundance of Kalirin-7 in extracts varies with the extraction conditions used. In particulate fractions of adult rat cerebral cortex,
Kalirin-7 was one of the most abundant Kalirin proteins. To our
surprise, we found it difficult to detect a protein with the mass of
Kalirin-8 (217 kDa), suggesting that Kalirin-8 is not an abundant isoform.
To confirm the identity of this 190-kDa protein as Kalirin-7, we
performed an immunodepletion experiment (Fig. 3D). Since we
used a crude soluble fraction of the rat brain to reduce background problems during immunoprecipitation, Kalirin-7 was not enriched in the
starting material (IN-Cortex-S). Kalirin-7 antibody was incubated with
the sample, and antibody-bound proteins were collected with protein
A-agarose beads. Incubation with the Kalirin-7 antibody specifically
depleted the 190-kDa Kalirin protein from the brain homogenate, while
the other Kalirin-related proteins remained in the flow-through
fraction (F.T.). Preincubation of the Kalirin-7 antiserum
with antigenic peptide or use of preimmune serum prevented immunoprecipitation of the 190-kDa protein, demonstrating that binding
to the antibody was specific. Further work is in progress to uncover
the identity of the other Kalirin forms; the 115-kDa protein
specifically bound to the Kalirin-7 antibody could represent the
protein encoded by the Kalirin-5 transcript.
Subcellular Distribution of Kalirin Proteins in Rat
Brain--
Based on Western blot analysis of cerebral cortex
homogenates, Kalirin-7 is a major form of Kalirin in the rat brain and
is recovered primarily from particulate fractions. To further
investigate its localization, we used differential centrifugation to
prepare fractions enriched in different organelles (Fig.
4A) (18); antibodies to
specific marker proteins were used to verify the identity of each
subcellular compartment. We used a method optimized for the isolation
of synaptosomes, pinched off presynaptic boutons with their enclosed
synaptic vesicles and cytosol (18); the apposed postsynaptic membrane
remains strongly attached (18, 19). An equal proportion of each
subcellular fraction was analyzed.
As expected, using the Kalirin-spectrin antibody, multiple Kalirin
proteins were identified in the soluble, cytosolic fraction (fraction
S3). Fraction P3, enriched in internal membranous organelles such as
the endoplasmic reticulum and Golgi apparatus (as indicated by BiP and
TGN38 markers, respectively), contained more Kalirin-7 than did the
cytosolic fraction (S3); the other forms of Kalirin were equally
abundant in P3 and S3. The crude synaptosomal pellet (P2) was subjected
to hypotonic lysis and then centrifuged to pellet a fraction enriched
in synaptosomal plasma membranes and attached postsynaptic membranes
(LP1). The resultant supernatant was separated into fractions enriched
in synaptosomal cytosol (LS2) and synaptic vesicles (LP2). A cytosolic
marker (GAD-65), was recovered from S3 and LS2; very little Kalirin was
recovered in LS2. In contrast, the synaptosomal plasma membrane
fraction (LP1) was substantially enriched in Kalirin-7. The synaptic
vesicle fraction (as shown by the synaptophysin/p38 marker) did not
contain detectable levels of any Kalirin proteins. A significant
proportion of the total Kalirin-7 was recovered in the synaptosomal
membrane (LP1) fraction, while the higher molecular weight Kalirin
proteins were evenly distributed among the cytosolic (S3), P3, and
LP1 fractions.
Kalirin-7 Is Highly Enriched in the Postsynaptic Density
Fraction--
Since a significant amount of the Kalirin-7 fractionated
with the synaptosomal membrane fraction (LP1), we explored the
possibility that Kalirin-7 was concentrated at postsynaptic densities.
The postsynaptic density fraction prepared by the method of Carlin et al. (19) is enriched in postsynaptic densities
(electron-dense structures located on the postsynaptic sides of
neuronal synapses) along with the apposed presynaptic membranes. These
structures are enriched in receptors and ion channels along with
cytoskeletal proteins and scaffolding proteins thought to anchor
receptors and signaling molecules (22).
We generated a synaptosomal fraction (P2) by sucrose gradient
centrifugation (19, 23). The proteins of the postsynaptic density are
resistant to detergent extraction (19, 23). We compared the behavior of
Kalirin-7 to that of two PSD markers ((PSD95 (23) and Chapsyn-110 (24))
and a presynaptic membrane-associated protein, Munc18-1 (25). Equal
amounts of protein from each fraction were examined. Kalirin-7 was
highly enriched in the fraction that remained insoluble after one or
two rounds of Triton extraction (1-Triton, P and
2-Triton, P (Fig.
5)). The higher molecular weight Kalirin
proteins were progressively depleted from the Triton-insoluble pellets
by Triton extraction. Kalirin-7 was enriched in the postsynaptic density fraction ("2-Triton"-P). PSD-95 and Chapsyn-110
co-fractionated with Kalirin-7 throughout the Triton extractions.
Munc18-1, a peripheral membrane protein (25), was progressively
depleted from the Triton-insoluble pellets.
Extraction of the 1-Triton pellet with Sarcosyl
(N-lauroylsarcosinate), a much stronger detergent, leaves
behind a "postsynaptic density core," which contains a more
restricted set of proteins (22). After extraction with Sarcosyl, a
smaller but still significant fraction of the Kalirin-7 remains in the
"postsynaptic density core" (Sarcosyl-P) (19, 23). Chapsyn-110 and
PSD95 are more prevalent in the "postsynaptic density core" than
Kalirin-7.
Kalirin Immunostaining in Cultured Neurons--
To date, few
endogenous Dbl-family members have been localized at a subcellular
level in their native tissues. To examine the cellular and subcellular
localization of Kalirin proteins, we prepared primary cultures of
cortical and olfactory bulb neurons. To determine whether Kalirin were
expressed in neurons and/or glia, we stained cultures simultaneously
with a polyclonal Kalirin-spectrin antibody and a monoclonal antibody
for neuron-specific tubulin (Fig. 6).
Staining of the same cells with both the Kalirin-spectrin and
neuron-specific tubulin antibodies showed that Kalirin proteins were
expressed in neurons (Fig. 6) in both types of culture. All neuron-specific tubulin-positive cells also expressed Kalirin, and all
cells that stained with Kalirin also expressed neuron-specific tubulin.
Western blot analysis indicated that only the higher molecular weight
forms of Kalirin were expressed in the olfactory bulb at this
developmental stage; Kalirin-7 was undetectable (Fig. 7A). In contrast, a
significant amount of 190-kDa Kalirin-7 was expressed in the cerebral
cortex at this developmental stage. Thus, by using the Kalirin-spectrin
antibody to visualize Kalirin proteins in olfactory bulb neurons, we
localized the higher molecular weight forms of Kalirin, not Kalirin-7
(Fig. 7B). The higher molecular weight Kalirin proteins were
most prevalent in the cell soma (s), often concentrated in
the perinuclear region, but they were also detectable in processes
(p) (Fig. 7B). To detect the higher molecular weight forms of Kalirin in cortical neurons, we stained 2-day-old cultures, which express very low levels of Kalirin-7 (Fig. 7, C and D). The staining patterns observed with the
Kalirin-spectrin antibody in cultured cortical neurons resembled the
patterns observed in cultured olfactory bulb neurons.
Because the DH domain of Kalirin binds specifically to
nucleotide-depleted Rac1 (9) and is highly similar to the first DH
domain of TRIO and UNC-73, both nucleotide exchange factors for Rac1,
we stained neurons simultaneously with Kalirin-spectrin and Rac1
antibodies (Fig. 7, B-D). Rac1 was especially enriched in
neuronal processes and the cortical (subplasmalemmal) region. The high
molecular weight Kalirin proteins in olfactory bulb neurons were
detected in these same regions but were more prevalent in the cell soma
(Fig. 7B). In cortical neurons, Kalirin staining coincides
with Rac1 in the subplasmalemmal regions of the soma (s) and
in some of the processes (p) (Fig. 7, C and
D). In long processes (p), Rac1 was significantly
enriched in the distal portion, where Kalirin was also enriched (Fig.
7D).
With time, cortical cultures express Kalirin-7 along with the high
molecular weight Kalirin isoforms (Fig. 7A). Thus, by
staining older cortical cultures with the Kalirin-7 antibody, we asked whether Kalirin-7 exhibited a distinct pattern of localization (Fig.
7E). This allowed us to compare the staining with the
Kalirin-spectrin and Kalirin-7 antibodies. Kalirin-7 was visualized as
beads along the sides of neurites and the cell soma (Fig.
7E). This staining pattern is typical of synaptic proteins
and is dramatically different from that of the higher molecular weight
forms of Kalirin (Fig. 7, C and D). Little
Kalirin-7 was detected in the cell body or the cytoplasm of neurites.
Kalirin-7 was undetectable in 1-day-old cortical cultures. Rac1 was
detectable in the processes and at the plasma membrane, in areas
immediately adjacent to Kalirin-7. Signals from both Kalirin antisera
in both types of culture were blocked by preincubation with the
appropriate antigen (not shown), demonstrating that the signal was specific.
Transient Expression of Kalirin-7 and DH-PH Domain of Kalirin in
NIH 3T3 Fibroblasts--
To determine whether Kalirin affects the
cytoskeletal organization of NIH 3T3 fibroblasts in a manner typical of
factors that activate Rho-like small GTP-binding proteins, we
transiently transfected these cells with plasmids encoding full-length
Kalirin-7 or its isolated DH/PH domain (DH-PH) (Fig.
8). This system has allowed sensitive and
specific detection of the activation of particular Rho-like proteins,
through analysis of rearrangements in the actin cytoskeleton and
effects on cellular morphology that are specific for each Rho subfamily
member (8).
As a control, we transfected fibroblasts with a plasmid encoding
preproneuropeptide Y (NPY), a soluble, secreted protein that should not
affect cellular morphology and the actin cytoskeleton (Fig.
8A). NPY was localized to the Golgi area and punctate
structures in processes. NPY-expressing cells were polygonal with
concave edges and were indistinguishable from nontransfected cells. The actin cytoskeleton was not different from nontransfected cells. Thus,
neither this method of transfection nor expression of a soluble,
secreted protein affected the cytoskeleton and cell morphology.
Fibroblasts expressing full-length Kalirin-7 exhibited a markedly
different cellular morphology from that of nontransfected cells (Fig.
8B). Kalirin-7 expressing cells were mostly flat, with
massive generation of lamellipodia (95% of transfected cells). Kalirin-7-expressing cells also had fewer stress fibers, and actin staining was generally punctate. Kalirin-7 was distributed throughout the entire cytoplasm, with more intense staining in the perinuclear region. In some cases, we observed Kalirin-7 enrichment at the edges of
protruding lamellipodia. Filamentous actin was also enriched at the
edges of the lamellipodia.
To determine whether induction of these changes in cell morphology and
cytoskeleton were mediated by the DH-PH domain of Kalirin-7, it was
transiently expressed in fibroblasts (Fig. 8C). All cells transiently expressing the DH-PH domain of Kalirin were flat, and a
significantly larger fraction of the cells expressing the DH-PH domain
had a circular or near circular shape (58%) than the cells expressing
Kalirin-7 (9.5%) (Fig. 8E). Cells expressing the DH-PH
domain also displayed ruffles much more massively (61%) than cells
expressing the full-length protein (9.5%). Both DH-PH and filamentous
actin were co-localized in ruffles. Filamentous actin again exhibited
punctate staining. Based on visualizing Kalirin-7 and DH-PH with a Myc
antibody, the proteins were expressed at similar levels (staining
intensity for Kalirin-7 was 80% that of DH-PH).
To determine whether these changes in cell morphology and cytoskeletal
organization resembled those caused by activation of Rac1, we expressed
constitutively active Rac1 (Rac1-Q61L) (5) in NIH3T3 cells (Fig.
8D). These cells also exhibited massive lamellipodia
formation, a phenotype similar to that induced by Kalirin-7 or its
DH-PH domain. The punctate localization of filamentous actin observed
in cells expressing Kalirin-7 or DH-PH was not observed in cells
expressing Rac1-Q61L.
In Vitro GDP/GTP Exchange Activity of the DH-PH Domain of
Kalirin--
The DH-PH domain of Kalirin is most closely homologous to
the DH1-PH1 domains of TRIO (15) and UNC-73 (16); these proteins are
both exchange factors for Rac1. Moreover, Kalirin-8 was previously shown to bind nucleotide-depleted Rac1, but not nucleotide-depleted RhoA or Cdc42 (10). Hence, we tested whether the DH-PH domain of
Kalirin could activate in vitro the release of bound GDP
from Rac1 (Fig. 9). The His.Myc-tagged
DH-PH domain of Kalirin-7 was expressed transiently and enriched by
binding to a metal chelate resin. While in the presence of buffer only,
the release of bound [3H]GDP from Rac1 was slow; in the
presence of the DH-PH domain of Kalirin-7, the release of
[3H]GDP was significantly accelerated.
Based on analysis of both RNA and protein, the Kalirin gene
encodes a variety of proteins in the rat central nervous system (Figs.
2 and 3). The Kalirin-spectrin antibodies detected proteins ranging in
size from 115 to 470 kDa in homogenates of adult rat brain. Kalirin
mRNAs ranging in size from 5 to over 11 kb were detected on
Northern blots (Fig. 2D). The probability that these proteins are splice variants is supported by the existence of at least
three different Kalirin NH2-terminal sequences (10a, 10b,
and DUO) and at least two different COOH-terminal sequences (Kalirin-7
and -8); the higher molecular weight forms of Kalirin may include
COOH-terminal domains similar to those of TRIO and UNC-73A (Fig.
1).
In this study, we focused on characterizing one of the most abundant
Kalirin transcripts, Kalirin-7. Kalirin-7 is the rat equivalent of
human DUO and differs from the previously characterized Kalirin
transcript, Kalirin-8, at the 3'-end. While the COOH-terminal region of
Kalirin-8 includes an Src homology 3 domain and several PEST sequences,
the COOH-terminal region of Kalirin-7 lacks these domains and has
instead a 20-amino acid segment whose sequence could allow interactions
with PDZ domain-containing proteins. Although heterogeneity is observed
at the 5'-end of Kalirin-7, with three different 5'-sequences expressed
with the Kalirin-7 3'-end (Fig. 2, C and D), the
functional significance of these differences is not clear.
Using antisera to the spectrin-like repeat region of Kalirin, we were
able to detect the naturally occurring forms of Kalirin in rat brain.
Few Dbl family members have been characterized in their native tissues.
Although more work is required to determine the identity of each of the
forms of Kalirin, concomitant use of the Kalirin-spectrin and Kalirin-7
antisera clearly indicates that the 190-kDa Kalirin-7 protein and the
higher molecular weight Kalirin isoforms occupy different subcellular
locations. This conclusion is supported both by subcellular
fractionation of rat brain and immunofluorescence localization of
Kalirin in cultured neurons. The distinctive subcellular distribution
of Kalirin-7 suggests that its functions are distinct from those of the
higher molecular weight forms of Kalirin.
Biochemically, Kalirin-7 is enriched in the postsynaptic density
fraction, while the higher molecular weight Kalirin proteins are
equally distributed between the cytosol and membranous organelles (Fig.
5). By immunostaining, Kalirin-7 is enriched in neurites in structures
with the properties of spines, while the Kalirin-spectrin antiserum
visualizes a substantial amount of Kalirin in the cell soma. Depletion
of Munc18-1, a protein associated with the presynaptic membrane by
strong protein-protein interactions, from the postsynaptic density
fraction suggests that Kalirin-7 is enriched on the postsynaptic side.
The COOH terminus of Kalirin-7 (-STYV) matches the recognition sequence
for proteins with PDZ domains (Thr/Ser-X-Val) (26, 27). Many
of the proteins identified in the postsynaptic density fraction
(e.g. PSD-95 and Chapsyn-110; markers shown in Fig. 5) have
PDZ domains. Kalirin-7 localization to the PSD fraction may require
interactions of its unique COOH terminus with PSD-localized PDZ domain proteins.
We show that expression of Kalirin-7 or its DH-PH domain in 3T3 cells
alters cell morphology and the actin cytoskeleton in a manner similar
to that of activated Rac1, with disruption of stress fibers and
formation of lamellipodia and ruffles (1, 2). The first DH domain of
TRIO and the DH domain of UNC-73B are highly similar to Kalirin, and
both activate Rac1 in vitro and in transfected cells.
In vitro, the DH-PH domain of Kalirin is indeed a GDP/GTP
exchange factor for Rac1. Since Kalirin-7 can induce Rac1-like
cytoskeletal rearrangements in 3T3 cells, Kalirin-7 may fulfill a
similar function at the sites of its localization in vivo in neurons.
Kalirin-7 is the first guanine nucleotide exchange factor localized to
the postsynaptic density. Rho-like proteins have not yet been
implicated in synaptic function, but actin filaments are abundant in
postsynaptic densities and dendritic spines (29). Cytoskeletal
rearrangements are believed to be important in receptor clustering
(30), dendritic remodeling (31, 32), generation of spines (33), and
organization of postsynaptic density structures (34). In the
postsynaptic density, Kalirin-7 may activate Rac1, which in turn could
regulate rearrangements of the actin cytoskeleton. Thus, Kalirin may
participate in the generation and dynamics of the postsynaptic density.
Based on immunostaining and subcellular fractionation, the higher
molecular weight forms of Kalirin are found in both the cell soma and
in neurites. Kalirin may subserve different functions in these
different locations. Overexpression of Kalirin-8 in AtT-20 cells
affected the biosynthetic and endocytic trafficking of a membrane
protein localized to the trans-Golgi network and immature secretory
granules. Kalirin proteins localized to the cell soma may play a role
in the trafficking of both newly synthesized and endocytosed membrane
proteins. In cultured cortical neurons, the higher molecular weight
forms of Kalirin were abundant in neuritic processes. A role for
Kalirin in neurite growth is suggested by the fact that, when stably
expressed in AtT-20 cells, Kalirin-8 induced formation of longer and
more branched neurites (9). Small GTP-binding proteins of the Rho
subfamily have been implicated in dendritic growth and remodeling in
cortical neurons (35) and axonal development in cerebellar Purkinje
cells (33). In general, Rac1 appears to mediate neurite extension
(36-38). In C. elegans, UNC-73 is involved in axon growth
(16). Kalirin forms are homologous to UNC-73 and may fulfill a similar
function in mammalian neurons.
Dr. Martin Schiller (Dept. Pathology and
Anesthesiology, Johns Hopkins University) generously provided the NIH
3T3 cells. Dr. Michel Streuli (Division of Tumor Immunology,
Dana-Farber Cancer Institute) kindly provided a cDNA fragment of
human TRIO. The Rac1-N17 plasmid was a gift from Dr. Silvio Gutkind
(Laboratory of Cellular Development and Oncology, NIDR, National
Institutes of Health), and the Rac1-QL plasmid was a gift from Dr.
Anirvan Ghosh (Johns Hopkins University School of Medicine). Dr. Toru Miki (Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health) generously provided the Ost expression plasmid. We thank Dr. Douglas Murphy (Department of Cell Biology and Anatomy, Johns Hopkins University) for the actin polyclonal antibody and Dr.
Henry Keutmann (Endocrine Unit, Massachusetts General Hospital) for the
synthesis of the antigenic peptide. We thank Lixian Jin and Marie Bell
for general laboratory assistance. We thank Dr. Bo Xiao and Dr. Kevin
O'Donovan for reading the manuscript.
*
This work was supported by National Institutes of Health
Grants DA-00266 and DK-32948.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
DH, Dbl homology;
PH, pleckstrin homology;
PEST, Pro, Glu, Ser, Thr-rich
protease-sensitive region (39);
kb, kilobase pair(s);
bp, base pair(s);
nt, nucleotide(s);
EST, expressed sequence tag;
PCR, polymerase chain
reaction;
PAGE, polyacrylamide gel electrophoresis;
NPY, preproneuropeptide Y.
An Isoform of Kalirin, a Brain-specific GDP/GTP Exchange Factor,
Is Enriched in the Postsynaptic Density Fraction*
ABSTRACT
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DISCUSSION
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-amidating monooxygenase. In this study, we cloned
rat Kalirin-7, a 7-kilobase mRNA form of Kalirin. Kalirin-7
contains nine spectrin-like repeats, a Dbl homology domain, and a
pleckstrin homology domain. We found that the majority of Kalirin-7
protein is associated with synaptosomal membranes, but a fraction is
cytosolic. We also detected higher molecular weight Kalirin proteins.
In rat cerebral cortex, Kalirin-7 is highly enriched in the
postsynaptic density fraction. In primary cultures of neurons,
Kalirin-7 is detected in spine-like structures, while other forms of
Kalirin are visualized in the cell soma and throughout the neurites.
Kalirin-7 and its Dbl homology-pleckstrin homology domain induce
formation of lamellipodia and membrane ruffling, when transiently
expressed in fibroblasts, indicative of Rac1 activation. Using Rac1,
the Dbl homology-pleckstrin homology domain catalyzed the in
vitro exchange of bound GDP with GTP. Kalirin-7 is the first
guanine-nucleotide exchange factor identified in the postsynaptic
density, where it is positioned optimally to regulate signal
transduction pathways connecting membrane proteins and the actin cytoskeleton.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amidating monooxygenase, an important
neuropeptide-processing enzyme (9, 10). Northern blot analysis
identified several forms of Kalirin mRNA, with expression
restricted to the central nervous system and highest levels in cerebral
cortex and hippocampus. The Dbl homology
(DH)1 domain of Kalirin, with
its adjacent pleckstrin homology (PH) domain, is conserved among all
Dbl family members (Fig. 1). Several Dbl family members, including Ost
(11) and Tiam (12), are expressed in the brain, but only Kalirin is
restricted to the central nervous system. AtT-20 corticotrope tumor
cells overexpressing one form of Kalirin (Kalirin-8) and
peptidylglycine--amidating monooxygenase developed longer and more
branched neuritic processes (10). Both AtT-20 and Chinese hamster ovary
cells overexpressing Kalirin-8 displayed rearrangements of the actin
cytoskeleton (13).
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Fig. 1.
Domain structures and alignment of Kalirin-8
and related proteins. Domains indicated are DH, PH, PEST,
fibronectin III-like (FnIII), and immunoglobulin-like
(Ig). The region of the spectrin domains of Kalirin used to
immunize rabbits is indicated at the top
(Kalirin-spectrin). For clarity, the 1899-residue form of
rat Kalirin described earlier (10) is referred as Kalirin-8. UNC-73 A
and B are alternatively spliced transcripts of a C. elegans
gene involved in axon guidance and neuronal cell migration (16). TRIO
is an interactor of transmembrane protein-tyrosine phosphatase LAR
(15). Ost is an oncoprotein cloned from an osteosarcoma cDNA
library, which acts as a guanine-nucleotide exchange factor for RhoA
and Cdc42 and binds activated Rac1 (11). Dbl is an oncoprotein cloned
from a diffuse B-cell lymphoma library, with guanine-nucleotide
exchange factor activity for RhoA and Cdc42 (40).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Fig. 2.
Cloning of rat Kalirin-7 cDNA.
A, the relationship of rat Kalirin-8, human DUO, and human
EST T74341 is shown with the position of the PCR primers. Two 5'-ends
for Kalirin were identified (10a and 10b); the coding regions are
indicated. The 5'-end of DUO is a third distinct 5'-end. B,
construction of full-length rat Kalirin-7 cDNA.
Oligo(dT)15 was used to generate cDNA from rat cerebral
cortex mRNA by reverse transcription. A sense primer (S)
derived from the DH domain of Kalirin-8 and an antisense primer
(As) based on human EST T74341 were used to amplify a
cDNA fragment whose sequence encoded the rat equivalent of human
DUO. The Kalirin-7 specific fragment was joined with the 5' region of
Kalirin-8 to generate the full-length rat Kalirin-7; the 5'-end of this
mRNA is 10a. Dotted lines represent
3'-untranslated region. C, the Kalirin-7 mRNAs contain
all three potential 5'-ends. Amplification of oligo(dT)-primed cDNA
was performed with sense primers specific for the three different
5'-ends (10a, 10b, and DUO), and the products were analyzed by Southern
blotting with a Kalirin-spectrin probe. D, Northern blot
analysis with different Kalirin probes. A probe specific for the 3'-end
of Kalirin-7 detected mRNAs of 5.0 and 7.0 kb, while the
Kalirin-spectrin repeat 1-3 probe detected mRNAs of 7.0 kb and
larger. The 10b probe detected the 7-kb mRNA. E, domain
structure of Kalirin-7 and amino acid sequence specific to Kalirin-7.
The position of the peptide used to generate the Kalirin-7-specific
antisera is indicated by the bar.
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Fig. 3.
Antibodies generated against various regions
of Kalirin detect multiple proteins in rat brain. A,
specificity of Kalirin-spectrin antisera. Kalirin (residues 517-976),
TRIO (residues 423-859), and Ost (residues 6-439) Gal4-fusion
proteins were expressed in yeast cells. Detection with anti-Gal4
antibody showed that similar amounts of each fusion protein were
applied to the gel. The indicated antisera were used to detect Gal4
fusion proteins. B, Kalirin antisera detected multiple
proteins in rat cerebral cortex homogenates. Crude soluble
(Cortex-S) and particulate fractions (Cortex-P)
(50 µg of protein from each) were analyzed by Western blotting. Liver
homogenate was used as negative control. A cross-reactive nonspecific
protein is marked by an asterisk. C, blocking of
Kalirin antisera. Antisera were preincubated with antigen (+) and then
used for Western blotting of rat cerebral cortex homogenate crude
soluble fraction (Cortex-S, 50 µg of protein) at the same
dilution (1:1000) as nonblocked antisera ( 
). D,
immunodepletion of Kalirin-7. Rat cerebral cortex crude soluble
fraction (Cortex-S) (100 mg) was incubated with Kalirin-7
antiserum (JH2958), Kalirin-7 antiserum prebound to antigenic peptide
(Blocked, 10 µg of peptide/µl of serum), or preimmune
serum. Flow-through and bound proteins were separated by SDS-PAGE and
detected by Western blotting with Kalirin-spectrin antiserum
(JH2581).
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Fig. 4.
Subcellular distribution of Kalirin in rat
cerebral cortex. Left, isotonic centrifugal
fractionation of rat cerebral cortex homogenate was performed to
generate subcellular fractions. Right, an equal percentage
of each fraction was separated by SDS-PAGE. Kalirin proteins were
detected with Kalirin-spectrin antiserum (JH2582). Fractions are as
follows. S3, cytosol; P3, Golgi complex and
endoplasmic reticulum (ER); LS2, cytosol from the
lysed synaptosomal fraction; LP1, synaptosomal plasma
membrane; LP2, small synaptic vesicles. Subcellular
compartments were identified by detection of resident proteins with the
corresponding antibody. These markers are BiP (for endoplasmic
reticulum), TGN38 (for the Golgi complex), p38 (synaptophysin) (for
small synaptic vesicles (SSV)), and GAD65 (for cytosol and
synaptosomal cytosol).
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Fig. 5.
Kalirin-7 is highly enriched in the
postsynaptic density fraction. Rat cerebral cortex post-nuclear
supernatant (S1) was centrifuged at 100,000 × g for 15 min (100K) to obtain crude soluble
(S2) and particulate (P2) fractions. Fraction P2
was then separated on a discontinuous sucrose gradient. The fraction
containing the purified synaptosomes was collected and extracted in
0.5% Triton X-100 to yield the 1-Triton pellet (P) and
supernatant (S). The 1-Triton pellet was split in half and
either re-extracted in 0.5% Triton (2-Triton) or 3%
Sarcosyl to yield pellet and supernatant. All lanes contain
5 µg of protein. Kalirin was detected with Kalirin-spectrin antiserum
(JH2581). A PDZ-family specific antibody was used to detect PSD95 and
Chapsyn 110, and Munc18-1 antibody was used to detect Munc18-1.
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Fig. 6.
Kalirin is expressed in neurons.
7-Day-old cultures of primary olfactory bulb (A) or cortical
(B) neurons were visualized simultaneously with the
Kalirin-spectrin antibody and a neuron-specific tubulin
(nst) antibody. Cell cultures and immunostaining procedures
were as described under "Experimental Procedures." Phase images are
shown for comparison.
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Fig. 7.
Forms of Kalirin different from Kalirin-7 are
detected in soma and neurites, while Kalirin-7 is localized to
synapse-enriched regions. A, SDS-PAGE. Equal amounts of
protein (25 µg) from homogenates of olfactory bulb and cerebral
cortex from 7-day-old rat pups were analyzed by SDS-PAGE and Western
blotting with the Kalirin-spectrin antibody. While the higher molecular
weight forms of Kalirin are present in both tissues, Kalirin-7 (190 kDa) and the 115-kDa form are detected only in the cerebral cortex.
B, 7-day-old cultures of primary olfactory bulb neurons were
visualized simultaneously with Kalirin-spectrin and Rac1 antibodies.
s, soma; p, processes. C and
D, 2-day-old cultures of primary cortical neurons were
visualized simultaneously with Kalirin-spectrin and Rac1 antibodies.
The arrows indicate co-localization of Kalirin with Rac1 in
neurites (p) and in the subplasma membrane region of the
soma (s). E, 7-day-old cultures of primary
cortical neurons were visualized simultaneously with affinity-purified
Kalirin-7 and Rac1 antibodies. The arrows indicate
localization of Kalirin-7 to neurites, with intense punctate staining
that could represent dendritic spines. Rac1 was concentrated in the
subplasma membrane region of the cell body and throughout
neurites.
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Fig. 8.
Intact Kalirin-7 or the Kalirin DH-PH domain
induces alterations in the actin cytoskeleton (formation of
lamellipodia and membrane ruffles) in fibroblasts. NIH 3T3
fibroblasts transiently expressing proneuropeptide-Y,
His-Myc-Kalirin-7, His-Myc-DH-PH, or Rac1-Q61L were serum-starved for
16 h, fixed, and visualized with NPY antibody, Kalirin-spectrin
antibody (Kalirin-7), Myc monoclonal antibody (DH-PH, Kalirin-7), or
Rac1 monoclonal antibody. Filamentous actin was detected with
fluorescein isothiocyanate-phalloidin. A, transient
expression of a control secreted protein (neuropeptide-Y) does not
affect the cytoskeleton or cellular morphology. Note the presence of
punctate staining (ps) for NPY. B, fibroblasts
transiently expressing Kalirin-7 are flat and exhibit massive
lamellipodia (L) formation. Kalirin-7 is sometimes
co-localized with filamentous actin at the edges of lamellipodia
(line arrows). C, fibroblasts
expressing the Kalirin DH-PH domain are rounded and exhibit
lamellipodia and abundant membrane ruffles (r) on the cell
surface (open arrows). Ruffles are enriched in
both Kalirin-7 and filamentous actin (line
arrows). D, fibroblasts expressing constitutively
activated Rac1 (Rac1-Q61L) form massive lamellipodia. E,
plot of fraction of the transfected cells displaying lamellipodia,
ruffles, and a circular shape. For quantitation, the structures
indicated in B and C were considered as
reference.
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Fig. 9.
The DH-PH domain of Kalirin is a GDP/GTP
exchange factor for Rac1. Two µg of bacterially expressed
purified GST-Rac1 preloaded with [3H]GDP was incubated
alone or with 14 µg of partially purified DH-PH, expressed in
pEAK-Rapid cells, as described under "Experimental Procedures."
Each assay was performed in duplicate, and the data points represent
averages from three separate assays. Error bars
represent S.D.
DISCUSSION
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: The Johns Hopkins
University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-6937; Fax: 410-955-0681; E-mail: beipper@jhmi.edu.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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