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Molecular and Cellular Biology, October 1998, p. 5838-5851, Vol. 18, No. 10
Department of Microbiology and Cancer Center,
University of Virginia Health Science Center, Charlottesville,
Virginia 22908
Received 23 April 1998/Returned for modification 21 May
1998/Accepted 18 June 1998
Cortactin is an actin-binding protein that contains several
potential signaling motifs including a Src homology 3 (SH3) domain at
the distal C terminus. Translocation of cortactin to specific cortical
actin structures and hyperphosphorylation of cortactin on tyrosine have
been associated with the cortical cytoskeleton reorganization induced
by a variety of cellular stimuli. The function of cortactin in these
processes is largely unknown in part due to the lack of information
about cellular binding partners for cortactin. Here we report the
identification of a novel cortactin-binding protein of approximately
180 kDa by yeast two-hybrid interaction screening. The interaction of
cortactin with this 180-kDa protein was confirmed by both in vitro and
in vivo methods, and the SH3 domain of cortactin was found to direct
this interaction. Since this protein represents the first reported
natural ligand for the cortactin SH3 domain, we designated it CortBP1
for cortactin-binding protein 1. CortBP1 contains two recognizable
sequence motifs within its C-terminal region, including a consensus
sequence for cortactin SH3 domain-binding peptides and a sterile alpha
motif. Northern and Western blot analysis indicated that CortBP1 is
expressed predominately in brain tissue. Immunofluorescence studies
revealed colocalization of CortBP1 with cortactin and cortical actin
filaments in lamellipodia and membrane ruffles in fibroblasts
expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin
was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during
neurite outgrowth.
Cells undergo rearrangement of the
cortical cytoskeleton, a submembranous actin filament (F-actin)-based
network, during a variety of cellular processes including
differentiation, proliferation, migration, and oncogenic transformation
(9, 10, 43, 67). The cortical cytoskeleton not only controls
cell morphology but is also involved in transmitting signals between
the plasma membrane and intracellular compartments (7, 8,
38). A large body of evidence indicates that small GTP-binding
proteins, tyrosine kinases, and serine/threonine kinases play a pivotal
role in regulating the dynamic structure of the cortical cytoskeleton
(30, 33). The molecular mechanisms by which these enzymes
regulate cortical actin polymerization and reorganization are currently
unclear. Identification of actin-associated targets of these enzymes is important for unveiling signaling pathways correlated with the cortical F-actin remodeling.
Cortactin, an F-actin-binding substrate for the nonreceptor tyrosine
kinase pp60src (39, 78), is
distinguished by the presence of several potential signaling sequence
motifs (63, 77, 79). The N-terminal half of the protein
contains six and a half tandem repeats of a 37-amino-acid sequence. The
repeat region is required and sufficient for efficient association with
F-actin as assessed by in vitro cosedimentation assays (78).
The role of this region in mediating the interaction with F-actin has
been further confirmed by the blockage of the cosedimentation of
cortactin with F-actin by a cortactin-specific monoclonal antibody
(MAb) whose epitope is located in the repeats (78). Since
this region does not show significant sequence similarities with other
actin-binding proteins, cortactin represents a distinctive family of
F-actin-binding proteins. The C-terminal half of cortactin consists of
a predicted Studies of tyrosine phosphorylation and intracellular distribution of
cortactin have suggested involvement of cortactin in signaling pathways
induced by oncogenic transformation and cell surface activation. In a
number of nontransformed adherent cells grown in culture, cortactin
localizes to poorly defined punctate structures in the cytoplasm and
within F-actin-based lamellipodia and membrane ruffles at the cell
cortex (73, 78). Lamellipodia and membrane ruffles are
veil-like membrane structures that have been implicated in guiding the
migration of motile fibroblasts and of the neuronal growth cones
(59). Cortactin is normally phosphorylated on serine and
threonine, but it becomes heavily phosphorylated on tyrosine in
oncogenic pp60src-transformed fibroblasts
(39, 77). Src-induced transformation causes accumulation of
cortactin and F-actin in podosomes (77), which are labile
cell substratum adhesion sites in the transformed cells
(71). Overexpression of cortactin and redistribution of cortactin into podosome-like structures (56, 61, 63) have been observed in human carcinoma cells with amplification of the chromosome 11q13 region in which the human cortactin gene is located (61). Since podosomes contain a substratum-degrading
proteolytic activity (12) and amplification of the 11q13
region is correlated with poor tumor prognosis (50, 62),
cortactin is suggested to be involved in upregulating an invasive and
metastatic behavior associated with tumors with 11q13 amplification
(63). In addition, a transient increase in
tyrosyl-phosphorylated cortactin is induced by a variety of types of
cell surface activation, including treatment of fibroblasts with growth
factors (23, 79), activation of platelets with thrombin
(76), activation of intercellular adhesion molecule 1 in
brain endothelial cells (25), and invasion of epithelial
cells by Shigella flexneri (19). All these
cellular processes involve dramatic changes in the cortical
cytoskeleton, and translocation of cortactin to specific cortical
cytoskeleton structures has been observed in response to some of these
external stimuli. For example, during S. flexneri-mediated
cell invasion, which requires assembly of F-actin at the bacterial
entry sites (14), cortactin coaccumulates with F-actin in
the membrane ruffles at the sites of bacterial entry and in the
periphery of the early phagosomes (19).
Given its multidomain structure and regulated subcellular localization,
cortactin has been suggested to recruit other signaling molecules to
the cortical cytoskeleton (25, 53, 76, 78). In this study,
we have identified a novel cortactin-binding protein of approximately
180 kDa, which is designated CortBP1 for cortactin-binding protein 1. A
consensus sequence for cortactin SH3 domain-binding peptides
(66) is present in the CortBP1 sequences, and the cortactin SH3 domain is required and sufficient for the interaction with CortBP1.
The C terminus of CortBP1 contains a sterile alpha motif (SAM), which
has been found in a number of signaling proteins involved in
developmental regulation (60). CortBP1 is expressed predominately in brain tissue as determined by both Northern and Western blot analysis. Immunofluorescence studies revealed
colocalization of CortBP1 with cortactin and cortical F-actin in
lamellipodia and membrane ruffles in fibroblasts overexpressing
CortBP1. Colocalization of endogenous CortBP1 and cortactin was
observed in growth cones in differentiating hippocampal neurons. These
data place CortBP1 and cortactin in the same subcellular compartment
and implicate a possible involvement of CortBP1 and cortactin in the
dynamic actin reorganization during neuronal growth cone extension.
DNA constructs.
To generate the construct pPC97-SR5,
expressing the cortactin SH3 domain fused to the GAL4 DNA-binding
domain (GAL4 BD), the mouse cortactin cDNA clone mp85.L7
(51) was amplified by PCR with primers 1597 (5'-GTCCCCCGGGCATCACAGCCATCGCC-3') and 1786 (5'-CCGGAATTCTGGGTGGCAGCCCTACT-3'). The resultant PCR
product was inserted as an SmaI/EcoRI fragment
into pPC97 (13). The expression construct for GAL4
BD-cortactinSH3W525K was generated by subcloning the
BglII/NotI fragment (amino acids 506 to 546) of
pFlag-cortactinW525K (described below) into
BglII/NotI-digested pPC97-SR5.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of a Novel Cortactin SH3 Domain-Binding Protein
and Its Localization to Growth Cones of Cultured Neurons
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-helix of 50 to 60 residues, a region enriched in
proline, serine, and threonine, and a Src homology 3 (SH3) domain that
has been found in numerous signaling proteins (15). The
cortactin SH3 domain has significant sequence and topological
similarity with the SH3 domains of the cortactin-related protein HS1
(76%; discussed below), the yeast actin-binding protein 1 (53%
[24]), and the c-Abl tyrosine kinase substrate Abi2
(53% [17]). All SH3 domains characterized so far
mediate protein-protein interactions via recognition of polyproline
motifs with a left-handed helical conformation (49). The SH3
domain-mediated interactions are implicated in regulation of enzyme
activities, targeting of proteins to specific subcellular compartments,
and coupling of signaling pathways (15). Little is known
about the function of the cortactin SH3 domain except for its selective
interaction with peptides containing a consensus sequence of +PP
PXKP
(66). It is interesting to note that a cortactin-related
protein termed HS1, which is expressed exclusively in hematopoietic
cells (40), contains three and a half copies of a
37-amino-acid motif in the N-terminal region and an SH3 domain at the
distal C terminus. Each unit of the repeats and the SH3 domain of HS1
display approximately 70% sequence identity to analogous regions in
cortactin, whereas sequence similarity in other regions is limited
(40, 41). Proliferative responses and clonal deletion of B
cells and T cells upon antigen receptor cross-linking are significantly
impaired in mice lacking HS1, implicating important roles of HS1 in
antigen receptor-initiated signaling processes (70).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
) (Invitrogen). The full-length
CortBP1 cDNA, pcDNA3.62, was then made by subcloning the 2.4-kb
EcoRV/BamHI fragment of pcDNA3.2 into
EcoRV/BamHI-digested pcDNA3.6 construct.
Yeast two-hybrid analysis, cDNA cloning, and sequence
analysis.
Transformation of yeast strain Y190 (34) was
performed by the lithium acetate method (29), and
-galactosidase activity in individual transformants was measured by
the filter lift assay (4). Initially, five variants of
cortactin fused to GAL4 BD were generated as baits. However, Y190 cells
harboring each of four constructs which encoded full-length proteins or
the C-terminal regions (amino acids 329 to 546, 396 to 546, or 473 to
546) exhibited readily detectable -galactosidase activity. A
construct, pPC97-SR5, lacking the 17-amino-acid sequence
(473-GHYQAEDDTYDGYESDL-489) common to the four
transactivating constructs did not induce any -galactosidase
activity and was used to screen a rat brain library. Yeast
transformants containing pPC97-SR5 were subsequently transformed with a
rat hippocampus cDNA library in pPC86 (13) and selected for
growth on Leu
Trp His plates
with 25 mM 3-aminotriazole. Three independent positive clones that
showed detectable -galactosidase activity within 12 h of
analysis were identified from 5 × 105 yeast
transformants and purified by three rounds of colony purification. Loss
of pPC97-SR5 from purified positive colonies was induced by
Trp selection, and positive library plasmids were
isolated. To verify the interaction, purified positive plasmids were
retransformed into Y190 cells together with pPC97, pPC97-SR5, or
pPC97-SR5W525K, and resultant transformants were analyzed for
-galactosidase activity.
gt11 rat
brain cDNA library (Clontech) was screened with an
[-32P]CTP-labeled 0.87-kb
SalI/BglII fragment of insert 2H (see Fig. 2A).
Subsequently, a 5' 0.88-kb EcoRI-digested fragment derived from clone 10.a (see Fig. 2A) was labeled with
[-32P]CTP and used as probe to screen a
5'-plus-stretch rat hippocampus library in gt11 (Clontech) until
clones (clones 6.b and 7.b [Fig. 2A]) containing 5' in-frame stop
codons were isolated. Throughout the complete coding region, at least
two independent clones of the same region were sequenced on both
strands. DNA sequence was obtained by the dideoxy chain termination
method. Both DNA and amino acid sequences were analyzed with Genetics
Computer Group sequence analysis software.
Northern blot analysis.
A 0.87-kb
SalI-BglII fragment of insert 2H, a 1.25-kb
EcoRI-SacI fragment of the mouse cortactin cDNA
clone mp85.L7 (51), and a -actin cDNA probe (Clontech)
were labeled with [-32P]CTP by primer extension with
random hexamernucleotide primer (Pharmacia Biotech). A multiple-tissue
Northern blot (Clontech), each lane of which contains approximately 2 µg of poly(A)+ RNA from specific adult mouse tissues, was
incubated with each probe (2 × 108 to 5 × 108 cpm/µg) overnight at 42°C. The filter was then
washed twice (5 min) in washing buffer 1 (2× SSC [1× SSC is 0.15 M
NaCl plus 0.015 M sodium citrate], 0.1% sodium dodecyl sulfate
[SDS]) at room temperature, twice (5 min) in washing buffer 2 (0.2×
SSC, 0.1% SDS) at room temperature, and then twice (15 min) in
prewarmed washing buffer 2 at 42°C. After each hybridization, the
probe was stripped by incubating the filter in boiled 0.5% SDS for 10 min and then in 2× SSC for 5 min at room temperature.
GST fusion proteins and in vitro binding analysis. GST fusion proteins were expressed and purified as described elsewhere (65). Briefly, 5 ml of clarified lysates from Escherichia coli BL21 expressing GST fusion proteins in NETN (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 50 µg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, 0.05 U of aprotinin per ml) was incubated with 200 µl of 50% glutathione-Sepharose bead slurry (Pharmacia Biotech) at 4°C for 1 h with constant rocking. The beads were then washed three times with 1 ml of NETN. To make antigen for CortBP1-specific antiserum, purified GST-CortBP1cte proteins were eluted by incubating the beads with glutathione elution buffer (20 mM reduced glutathione, 120 mM NaCl, 100 mM Tris-HCl, pH 8.0) at room temperature for 10 min with constant rocking. The eluates were then dialyzed against phosphate-buffered saline (PBS) and concentrated to approximately 1 mg/ml.
For in vitro binding experiments, immobilized GST fusion proteins were mixed with 1 ml of 1-mg/ml cell lysates in modified radioimmunoprecipitation assay buffer (RIPA) (50 mM HEPES [pH 7.2], 150 mM NaCl, 2 mM EDTA, 0.5% sodium deoxycholate, 1% Nonidet P-40, 50 µg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride, 0.05 U of aprotinin per ml, 1 mM sodium vanadate, 40 mM sodium fluoride) at 4°C for 2 to 3 h. After being washed once with 1 ml of modified RIPA and three times with 1 ml of HNTG (20 mM HEPES [pH 7.0], 150 mM NaCl, 0.1% Triton X-100, 10% glycerol), precipitated proteins were subjected to Western blot analysis with the cortactin-specific MAb 4F11.Antibodies and immunoprecipitation analysis. Rabbit polyclonal antiserum (anti-CortBP1) was raised against purified GST-CortBP1cte (amino acids 941 to 1252). Both anti-CortBP1 and preimmune serum were protein G purified. The specificity of anti-CortBP1 against CortBP1 was confirmed as follows: 500 µg of lysates from NIH 3T3 cells overexpressing Myc epitope-tagged CortBP1cte was immunoprecipitated with anti-CortBP1, preimmune serum, or the Myc-specific MAb 9E10. Precipitated proteins were resolved on SDS-12% polyacrylamide gel electrophoresis (PAGE) gels and subjected to Western blot analysis with anti-CortBP1. Anti-CortBP1 recognized a species present in the immunocomplex of anti-CortBP1 but not in preimmune immunocomplex that comigrated with the recombinant protein present in the immunocomplex of MAb 9E10. Direct Western blot analysis of 100 µg of rat brain lysate revealed a major polypeptide of approximately 180 to 200 kDa (see Fig. 8A) and a minor peptide species of approximately 75 kDa (data not shown).
The cortactin-specific polyclonal antibody, anti-Cterm, was generated by purifying rabbit antiserum raised against a GST-cortactin fusion protein (GST.p80 [78]) by using a cyanogen bromide-Sepharose 4B column (Pharmacia) coupled with a GST fusion protein of the C-terminal half of mouse cortactin (amino acids 329 to 546 [73]). The cortactin-specific MAb 4F11 has been previously described (39). Mouse MAbs specific for NCK (anti-NCK), for the Myc epitope (9E10), and for the Flag epitope (M5) were purchased from Transduction Laboratories, Santa Cruz Biotechnology, and Sigma, respectively. For immunoprecipitating CortBP1, 5 to 10 µg of protein G-purified anti-CortBP1 or preimmune serum was incubated with 500 µl of modified RIPA containing 50 µl of protein A-Sepharose beads (Sigma) at 4°C for 1 h, followed by three washes with 1 ml of modified RIPA. Tissue extracts prepared by homogenizing adult male rat (Sprague-Dawley) tissues in modified RIPA containing 0.05% SDS or cell lysates were incubated with immobilized anti-CortBP1 or preimmune antiserum at 4°C for 2 to 3 h. After three washes with 1 ml of modified RIPA, immunoprecipitates were subjected to Western blot analysis with 1 µg of the MAb 4F11 per ml, 5 µg of anti-CortBP1 per ml, or 250 ng of the MAb anti-NCK per ml.Cell culture, transfection, and immunofluorescence microscopy. NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin-streptomycin. Both 10T1/2neo cells, which are a stable transfectant of C3H-10T1/2 murine fibroblasts with pSV2neo, and 5Hd47 cells (c-Src overexpressors), which are a stable transfectant of C3H-10T1/2 cells with pSV2neo and a plasmid encoding chicken c-Src (11), were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin-streptomycin, and G418. PC12 cells were cultured as described elsewhere (32). Primary cultures of hippocampal neurons prepared from embryonic day-18 rats (5) were generously provided by Gary Banker.
Cells were transiently transfected with SuperFect (Qiagen). Thirty-six hours posttransfection, NIH 3T3 cells transfected with expression constructs were lysed and subjected to further analysis. For detection of CortBP1 localization in fibroblasts, 10T1/2neo or 5Hd47 cells were seeded on glass coverslips, grown to approximately 60% confluence, and transfected with pRK5-CortBP1cte. Sixteen hours posttransfection, cells were fixed and processed for immunofluorescence analysis. For immunofluorescence experiments, cultured fibroblasts were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.5% Triton X-100 in PBS for 3 min. Cultured hippocampal neurons were fixed and permeabilized with methanol for 20 min. After three washes with PBS, cells were blocked with 20% goat serum-2% bovine serum albumin in PBS for 1 h and incubated with primary antibodies (2.0 to 5.0 µg of anti-CortBP1 per ml, 5 µg of preimmune serum per ml, 0.2 µg of 9E10 per ml, 1 to 2 µg of 4F11 per ml, or 1.0 µg of anti-Cterm per ml) in PBS containing 10% goat serum and 1% bovine serum albumin for 1 h. After three washes with PBS, cells were then incubated with 1.5 µg of fluorescein isothiocyanate- or Texas Red-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse [Jackson ImmunoResearch]) per ml for 45 min. To visualize F-actin, 0.4 U of Texas Red-conjugated phalloidin (Molecular Probes) per ml was coincubated with secondary antibodies. For the antigen blocking experiment, 5 µg of anti-CortBP1 per ml was preincubated with 5 µg of purified GST-CortBP1cte per ml for 2 h at room temperature prior to labeling. Cells were viewed with a Leitz DMR fluorescence microscope and photographed with a cooled charge-coupled device camera controlled by the ISee software program (Inovision). Images were processed on a Macintosh computer with Adobe Photoshop.Nucleotide sequence accession number. The nucleotide sequence of the CortBP1 cDNA (nucleotides 1 to 4636) has been given the GenBank accession no. AF060116.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Identification and molecular cloning of cortactin-binding protein 1. To better define the role of cortactin in cell signaling events, we utilized the yeast two-hybrid system (28) to isolate cDNA clones encoding cortactin-binding proteins. With GAL4 BD-cortactinSH3 as the bait (Fig. 1A), three unique positive clones were isolated from a rat hippocampus cDNA library. The clone pPC86-2H, which reproducibly exhibited the highest affinity for the bait (data not shown), was further characterized. The identified interaction was dependent upon the SH3 domain since the GAL4 BD alone or GAL4 BD-cortactinSH3W525K, in which a tryptophan residue highly conserved in SH3 domains (69) was replaced with a lysine residue, did not display any detectable interaction with the fusion protein encoded by pPC86-2H (Fig. 1B).
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-helices
linked by loops (58). SAM domains have been identified in a
variety of proteins (60) including EPH family receptor tyrosine kinases, yeast sterile proteins including the serine/threonine kinases Byr2p and Ste11p, the subunit of trimeric G protein Ste4p,
and the cytoskeleton-associated proteins Boi1p and Boi2p, as well as
several Drosophila proteins involved in developmental regulation. As the first reported natural ligand for the cortactin SH3
domain, this protein was designated CortBP1 for cortactin-binding protein 1.
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, aromatic residues. (D) Comparison of the CortBP1 ppI motif, the
consensus for cortactin SH3 domain-binding peptides, and the consensus
for class II ligands of SH3 domains. Predicted SH3-contacting residues
are capitalized. +, R or K; 3, 4, and 5. The predicted
termination codon (nucleotides 4181 to 4183) was present in two
additional clones (clone 15.a and clone 2.b [Fig. 2A]). The 3'-most
cDNA clone, clone 2.b, contained a 3' untranslated region of
approximately 2.7 kb. However, neither a polyadenylation signal
(75) nor a polyadenosine sequence was found at the 3' end of
clone 2.b, implicating an even longer 3' noncoding sequence.
The CortBP1 open reading frame, deduced from sequences of the partial
cDNA clones, encoded a 1,252-amino-acid protein with a calculated
molecular mass of 134 kDa (Fig. 2B). The deduced N-terminal 940 residues of CortBP1 5' cDNA did not show significant overall similarity
to any identified proteins in the existing databases. Amino acid
composition analysis revealed that the CortBP1 protein is considerably
rich in proline and serine residues (12 and 10%, respectively).
Tissue distribution analysis of the CortBP1 transcript. To investigate the tissue distribution of the CortBP1 expression, a CortBP1 cDNA fragment (nucleotides 3245 to 4111) derived from the clone 2H was used to probe a mouse multiple-tissue Northern blot. A major mRNA species of approximately 8 kb was detected exclusively in brain tissue, whereas a much less abundant species of approximately 9.5 kb was detected in brain, kidney, liver, and lung tissue (Fig. 3A). No hybridization to mRNAs from heart, spleen, skeletal muscle, and testis tissue was detected. Northern blot analysis using two other independent probes (from cDNA 2H and clone 10.a) that represented different regions of CortBP1 cDNA yielded identical results (data not shown).
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Expression of the CortBP1 protein. To characterize the endogenous CortBP1 protein, a rabbit polyclonal serum (anti-CortBP1) was raised against the C-terminal region (amino acids 941 to 1252) of CortBP1 (CortBP1cte) as described in Materials and Methods. CortBP1 expression in a number of adult rat tissues was then assessed by immunoprecipitation and Western blot analysis with anti-CortBP1. As shown in Fig. 4A, a protein of approximately 180 kDa was detected in brain lysates while preimmune serum showed no reactivity with this protein (data not shown). Interestingly, no immunoreactive bands were detected in the other tissues examined, including heart, spleen, lung, liver, skeletal muscle, kidney, and testis. Analysis of several cell lines revealed expression of the CortBP1 protein in the rat adrenal pheochromocytoma cell line PC12, whereas expression of p180 CortBP1 was undetectable in the rat fibroblast cell line RAT1 (Fig. 4B) or in mouse NIH 3T3 cells (Fig. 4C, lane 5).
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Characterization of the interaction between CortBP1 and cortactin. The identification of CortBP1 by two-hybrid screening indicated an interaction between the cortactin SH3 domain and the CortBP1cte containing the ppI motif. To further explore the interaction between CortBP1 and cortactin, we examined the ability of GST-CortBP1cte to associate with endogenous cortactin in NIH 3T3 cell lysates. GST, GST-CortBP1cte, or GST-FAKcte (amino acids 687 to 1054 [35]) fusion proteins were coupled to glutathione-Sepharose beads and incubated with NIH 3T3 cell lysates. GST-FAKcte was used as a control to specify the interaction since the FAKcte polypeptide contains two class II SH3 ligand motifs that have been shown to serve as the binding sites for the SH3 domains of p130cas and Graf (35, 37). The amount of cortactin bound to immobilized GST fusion proteins was then determined by Western blot analysis with the cortactin-specific MAb 4F11. As shown in Fig. 5A, cortactin coprecipitated with immobilized GST-CortBP1cte fusion proteins in a dose-dependent manner whereas little cortactin coprecipitated with beads coated with either GST alone or GST-FAKcte.
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Subcellular localization of ectopically expressed CortBP1 in fibroblasts. Previous studies have shown that a significant portion of cortactin colocalizes with F-actin-containing structures including lamellipodia and membrane ruffles in fibroblasts as well as in podosomes in Src-transformed fibroblasts (77, 78). To assess whether CortBP1 would colocalize with cortactin present in the cortical cytoskeleton, Myc epitope-tagged CortBP1cte (Fig. 6A) was expressed in 10T1/2 cells and 10T1/2 cells overexpressing c-Src (10T1/2-c-Src). Localization of CortBP1 was determined by immunofluorescence staining with either anti-CortBP1 or Myc epitope-specific MAb 9E10. In 10T1/2 cells overexpressing CortBP1, both antibodies revealed intense staining of CortBP1 within lamellipodia and membrane ruffles (Fig. 6B [a and b]). Accumulation of CortBP1 at the cell cortex was most prominent at the leading and trailing edges of motile cells (Fig. 6B and 7). These lamellipodia and membrane ruffles were enriched with F-actin as shown by costaining experiments with phalloidin (Fig. 6B [e and f]). Little background staining was detected in untransfected cells with either anti-CortBP1 (Fig. 7a and b) or MAb 9E10 (Fig. 6B [e and f]). Similar staining patterns of CortBP1 were also observed in two other fibroblast cell lines, REF52 and Swiss 3T3, overexpressing CortBP1cte (data not shown). When expressed in 10T1/2-c-Src cells, CortBP1 accumulated in dot-like structures in the peripheral extensions in some cells as well as throughout the cytoplasm in others (Fig. 6B [c and d]). As shown in Fig. 6B (g and h), these dot-like structures were enriched with F-actin and are likely membrane-substratum contact sites similar to podosomes (77).
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Analysis of CortBP1 and cortactin in differentiating hippocampal neurons. The intense costaining of CortBP1 and cortactin within the lamellipodia and membrane ruffles prompted our studies of the intracellular distribution of these two proteins in differentiating neurons which contain similar F-actin cortical structures in growth cones (45). To find neurons expressing both proteins, we first examined the expression levels of cortactin and CortBP1 in brain lysates isolated from rats at different developmental stages. Western blot analysis of brain lysates revealed that the expression levels of CortBP1, the major bands recognized by anti-CortBP1 (data not shown), increased during the course of embryonic development and remained at high levels during postnatal development (Fig. 8A). In contrast, the expression level of cortactin in rat brain was relatively constant at all of the nine developmental stages examined (Fig. 8A). Since expression of both CortBP1 and cortactin is readily detected in the embryonic 18-day rat brain lysates, in vitro-cultured neurons explanted from embryonic 18-day rat hippocampus were used to assess the subcellular localization of CortBP1 and cortactin by indirect immunofluorescence with the CortBP1-specific anti-CortBP1 and the cortactin-specific MAb 4F11. After 24 h in culture, a majority of cells displayed extended neurites with defined growth cone structures as previously described (31). At this stage, cortactin displayed intense staining within growth cones and weaker staining in the periphery of the cell body (Fig. 8B [a and b]). Little staining of cortactin was observed in neurite shafts. Interestingly, CortBP1 was observed within growth cones and the cell body (Fig. 8B [c and d]). Preincubation of anti-CortBP1 with purified antigen effectively blocked the staining in growth cones, leaving residual fluorescence within the cell body (Fig. 8B [g and h]). Staining of the cell body but not growth cones was seen with preimmune serum (Fig. 8B [e and f]). The staining patterns of cortactin and CortBP1 within growth cones are very similar in that they primarily localized at the central portion of growth cones. Coimmunostaining analysis further suggested the colocalization of CortBP1 and cortactin within growth cones (Fig. 9). These experiments clearly indicate that endogenous cortactin and CortBP1 reside in the same intracellular compartment in outgrowing neurites, supporting a direct interaction of these two proteins in vivo.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Previous studies have suggested that the actin-binding protein cortactin may mediate aspects of cell signaling associated with the cortical cytoskeleton. The present study provides evidence that cortactin forms an SH3 domain-dependent protein complex with a novel 180-kDa protein. This protein represents the first identified natural ligand for the cortactin SH3 domain and is thus referred to as CortBP1. Sequence analysis of CortBP1 revealed the presence of two readily identifiable sequence motifs in the C-terminal region: a sequence virtually identical to a consensus sequence defined for the cortactin SH3 domain-binding peptides (66) and a sequence similar to recently identified SAM domains (58). The expression pattern of CortBP1 is restricted, being expressed predominantly in brain tissue. The stable interaction of cortactin and CortBP1 was demonstrated by coimmunoprecipitation of cortactin with CortBP1 in PC12 cell lysates. In addition, CortBP1, overexpressed in fibroblasts, colocalizes with cortactin and cortical F-actin within lamellipodia and membrane ruffles in normal cells and within podosome-like structures in c-Src overexpressors. Colocalization of endogenous cortactin and CortBP1 was also observed in growth cones of differentiating rat hippocampal neurons. These data indicate that endogenous CortBP1 and cortactin reside within the same subcellular compartment and suggest a possible involvement of cortactin and CortBP1 in the dynamic actin reorganization during neuritogenesis.
Data obtained by yeast two-hybrid and in vitro interaction analysis
indicate that the SH3 domain of cortactin is responsible for mediating
the observed interaction with CortBP1. Mutation of the highly conserved
tryptophan residue to lysine in the cortactin SH3 domain efficiently
blocked the interaction of full-length cortactin with CortBP1, as
evidenced by the inability of GST-CortBP1 to pull down mutant cortactin
from cell lysates. These data, coupled with the observation that mutant
SH3 domain fusions fail to bind CortBP1 in the yeast two-hybrid assay,
indicate the dependence of the interaction with CortBP1 on the
structural integrity of the cortactin SH3 domain. The critical role of
the cortactin SH3 domain for interacting with CortBP1 is further
supported by the presence of the ppI motif in CortBP1, which displays a
precise match with the consensus sequence (Fig. 2D) for cortactin SH3 domain-binding peptides. The sequence of this consensus motif, identified by screening a biased X6PXXPX6
peptide library with GST-cortactin SH3 fusion proteins, is related to
class II ligands for SH3 domains (66). Interestingly, the
cortactin SH3 domain does not show detectable interaction with peptides
preferred by SH3 domains of other proteins including Src, Yes, Abl,
Crk, Grb2, and phospholipase C
(66). The apparent
specificity of the cortactin SH3 domain may derive from two unusual
residue selections in cortactin SH3 domain-binding peptides. First,
whereas an aliphatic residue is present at the 1 position (Fig. 2D)
in most class II ligands (27), a positively charged residue
(lysine or arginine) is present at the analogous position in cortactin
SH3 domain-binding peptides. Second, the cortactin SH3 domain-binding
peptides show a strict preference for lysine over arginine at the 5 position (Fig. 2D). We have examined the contribution of the CortBP1
ppI motif to interaction with cortactin by mutational analysis.
Mutation of proline 948 and proline 950 to alanine within the ppI motif
(e.g., ...KPA948VA950PKP...) reduced the
ability of CortBP1 to bind the cortactin SH3 domain in two-hybrid
interaction analysis and with endogenous cortactin in in vitro GST
pull-down experiments (data not shown). While these results underscore
the importance of the CortBP1 ppI motif in binding the cortactin SH3
domain, we cannot rule out the contribution of other CortBP1 sequences
in mediating the stable interactions with the cortactin SH3 domain.
The C-terminal 67 amino acids of CortBP1 display extensive similarity to SAM domains, recently identified protein modules present in diverse eukaryotic organisms from yeasts to humans (60). SAM-containing proteins have been implicated in developmental regulation and signal transduction (60). For example, SAM-containing proteins are essential for pheromone-induced sexual differentiation in yeast (Byr2p, Ste11p, and Ste4p [16]) and for regulating anterior-posterior patterning in Drosophila oocytes (polyhomeotic protein and Bicaudal-C [18, 46]). Two yeast SAM domain-containing proteins, Boi1p and Boi2p, are associated with cytoskeleton and play an important role in the maintenance of cell polarity during bud formation (6). While the molecular function of SAM domains remains largely unclear, recent studies of several SAM-containing proteins suggest a possible role of SAM domains in mediating protein-protein interactions (3, 48, 55, 64). Given the apparent association of CortBP1 with cortactin-F-actin complexes, it will be interesting to further investigate whether the CortBP1 SAM domain is responsible for recruiting other signaling proteins to the dynamic cortical cytoskeleton in growth cones.
In contrast to the cortactin gene, which is expressed in a wide variety of tissues, the expression pattern of the CortBP1 gene appears restricted. Northern blot analysis has shown that the major species of CortBP1 transcript (~8 kb) is present exclusively in brain tissue and a less abundant species (~9.5 kb) is present in brain, kidney, lung, and liver tissues. Whereas the CortBP1 protein is readily detected in brain tissues by Western blot analysis, we have failed to detect p180 CortBP1 in kidney, lung, and liver tissues. The difficulty of detecting the CortBP1 protein in these tissues may be due to the lower sensitivity of the Western blot analysis. The restricted expression pattern of CortBP1 suggests that the cortactin-CortBP1 interaction may have physiological significance in neurons and that in other cell types cortactin may interact with either CortBP1-like protein(s) or another tissue-specific binding partner(s).
Previous studies have shown that cortactin displays an intense staining within lamellipodia as well as a punctate staining within the cytoplasm in adherent cells cultured in the presence of serum (77, 78). Recent studies from our laboratory (73) have shown that the distribution of cortactin between the cortical cytoskeleton and cytoplasmic structures is regulated. Sequestration of cortactin to the cytoplasmic pool increases upon serum starvation. Translocation of cortactin into lamellipodia and membrane ruffles is dramatically enhanced by activation of Rac1, a small GTP-binding protein of the Rho family. The cortical cytoskeleton-targeting signal is located within the N-terminal half of cortactin, whereas sequences within the C-terminal half appear to be dispensable for cortical actin localization (74). Based on these data and the documented function of SH3 domains in recruiting their ligands to certain subcellular compartments (15), we propose that the cortactin SH3 domain plays a role in targeting its binding partner(s) to the cortical cytoskeleton. In support of this, we have shown here that a significant portion of overexpressed CortBP1 colocalizes with cortactin and F-actin in lamellipodia and membrane ruffles of cultured murine fibroblast cells. The colocalization of CortBP1 with cortactin and F-actin was also observed in podosome-like structures in 10T1/2 cells overexpressing c-Src. These observations strongly suggest that cortactin mediates formation of multiprotein complexes within the cortical cytoskeleton, by the concomitant interaction with cortical F-actin via its N-terminal repeats and with other cellular proteins via the SH3 domain at its C terminus. Thus, cortactin would serve as a cortical actin-specific docking protein for binding partners such as CortBP1.
We have further shown in this study that endogenous cortactin and CortBP1 colocalize within the cortical F-actin-containing subcellular compartment in primary cultures of differentiating rat neurons. Indirect immunofluorescence analysis suggests that both cortactin and CortBP1 are components of growth cones in cultured neurons isolated from embryonic 18-day rat hippocampus. These cells, when placed in culture, extend neurites mimicking their developmental morphology in vivo (22). The immunostaining of CortBP1 within growth cones appeared to be specific, since preimmune serum and antigen-blocked antibody failed to stain growth cones. The cortactin staining is essentially confined to growth cones, similar to the staining pattern of cortactin observed within growth cones of cultured Xenopus spinal cord neurons (57). Both CortBP1 staining and cortactin staining in growth cones of rat hippocampal neurons are primarily localized within the central portion of growth cones, very similar to the previously described staining pattern of F-actin in these cells (31). Additionally, costaining experiments support the colocalization of cortactin and F-actin in growth cones of the rat hippocampal neurons (data not shown). Previous studies have suggested that the F-actin remodeling within neuronal growth cones, in response to extracellular attraction or repulsion cues, plays an important role in regulating directional neurite extension (7, 45). Many proteins, including members of the Src family of tyrosine kinases (36, 47); actin-binding proteins such as profilin (26), gelsolin (68), myosin-V (72), and neurabin (52); and membrane-associated protein GAP-43 (2), have been identified as components of growth cones. Of note, the recently identified neural tissue-specific F-actin-binding protein neurabin has been implicated in neurite formation (52). The localization of cortactin and CortBP1 within growth cones, together with the brain-specific expression of CortBP1, suggests that the cortactin-CortBP1 interaction may also be involved in signaling pathways associated with the formation and migration of growth cones during neurite outgrowth.
A database search for CortBP1 homologs in other organisms reveals two human brain cDNA fragments in the expressed sequence tag database (accession no. m86079 and h41098 [1]) and one human DNA segment in the sequence-tagged site database (accession no. z51760 [21]). Both display high DNA sequence identity (81, 90, and 88%, respectively) to rat CortBP1 cDNA. The similarity between rat CortBP1 and the predicted open reading frames of these DNA sequences spans the N-terminal region (amino acids 294 to 405, 27 to 171, and 1 to 57, respectively). The existence of a putative human CortBP1 homolog is an indication of the conservation of CortBP1 function in other organisms and an involvement of CortBP1 in common biological processes in diverse organisms.
| |
ACKNOWLEDGMENTS |
|---|
We thank G. Banker and H. Asmussen for in vitro-cultured rat hippocampal neurons, S. J. Parsons for 10T1/2neo and 5Hd47 cells, A. A. Lanahan for pPC86 and pPC97 vectors and the rat hippocampus cDNA library in pPC86, A. Hall for the pRK5myc vector, J. S. Morrow for the pcDNA3Flag2AB vector, and M. T. Harte for the GST-FAKcte expression vector. We also thank M. E. Cox for helpful discussions.
This work was supported by grants CA29243 and CA40042 from the DHHS-NCI and grant 4491 from the Council for Tobacco Research, Inc., to J. T. Parsons; S. A. Weed is supported by NIH postdoctoral fellowship 1 F32 CA75695-01; W.-C. Xiong is supported by NIH NRSA fellowship NS09918.
| |
ADDENDUM IN PROOF |
|---|
CortBP1 contains a single PDZ domain (amino acids 38 to 133) that has sequence similarity to the PDZ domains of PSD95, Disc large-1, and Z0-1.
| |
FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Box 441, Health Science Center, University of Virginia, Charlottesville, VA 22908. Phone: (804) 924-5395. Fax: (804) 982-1071. E-mail: jtp{at}virginia.edu.
Present address: c/o CMHA (NF Division), St. John's, Newfoundland
A1C 5X3, Canada.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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