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Vol. 14, Issue 8, 3216-3229, August 2003
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* Department of Craniofacial Biology and Cancer Center, University of Colorado
Health Sciences Center, Denver, Colorado 80262;
Division of Immunology, QCB/BioSource International, Hopkinton, Massachusetts
01748; and
Department of Microbiology, University of Virginia Health System,
Charlottesville, Virginia 22908
Submitted November 21, 2002;
Revised March 4, 2003;
Accepted March 14, 2003
Monitoring Editor: David Drubin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Webb et al., 2002).
Initiation of motility requires cell polarization and formation of the leading
edge at the migratory front. The main "organelle" at the leading
edge responsible for motility is the lamellipodium, formed as a result of
nascent polymerization and remodeling of the cortical actin cytoskeleton
(Small et al., 2002).
Lamellipodia formation requires activation of the small GTPase Rac1, which
occurs after engagement and activation of several growth factor and adhesion
receptors (Hall, 1998).
Rac1 signals regulating lamellipodia formation are mediated in part through
multiple pathways that activate members of the WAVE/Scar protein family
(Miki et al., 2000;
Eden et al., 2002).
WAVE/Scar proteins interact with F- and G-actin
(Suetsugu et al.,
2001), and with actin-related protein complex 2/3 (Arp2/3)
complex, the molecular "machine" that nucleates the formation of
branched actin networks (Cooper et
al., 2001; Higgs and
Pollard, 2001). Interaction of WAVE/Scar proteins with Arp2/3
complex leads to enhanced Arp2/3 complex actin nucleation activity, resulting
in increased cortical actin polymerization and lamellipodia protrusion
(Borisy and Svitkina, 2000).
Rac1 targets WAVE/Scar along with several other actin-binding proteins that
regulate actin dynamics in lamellipodia
(Miki et al., 1998,
Mishima and Nishida, 1999;
Kessels et al.,
2000).
Lamellipodia adhere through integrin-based adhesion complexes with the
extracellular matrix. Adhesion complexes within lamellipodia (focal
"contacts") are formed concurrently with actin networks in
response to Rac1 (Nobes and Hall,
1995) and share many of the same proteins that are found in focal
"adhesions," larger, distally occurring cell-substratum contact
sites regulated by RhoA (Ridley and Hall,
1992). Assembly and regulation of focal contacts and adhesions
requires the activity of several tyrosine kinases, including Src
(Craig and Johnson, 1996). Rac1
activation translocates Src into lamellipodia
(Fincham et al.,
1996), where its kinase activity is required for proper
lamellipodia and focal contact formation
(Timpson et al.,
2001; Prasad et al.,
2002), indicating that Src substrates in lamellipodia are
important in regulating lamellipodia dynamics.
Several Src substrates associated with the actin cytoskeleton are not
components of cell-substratum adhesion complexes
(Brown and Cooper, 1996). One
of these is cortactin, a multidomain protein enriched within lamellipodia
(Wu et al., 1991).
Cortactin interacts with Arp2/3 complex
(Weed et al., 2000;
Uruno et al., 2001)
and F-actin (Wu and Parsons,
1993), interactions that are required for Rac1-induced cortactin
translocation to the cortical actin network
(Weed et al., 2000;
Di Ciano et al.,
2002). Cortactin stimulates Arp2/3 actin nucleation activity and
prevents disassembly of Arp2/3-F-actin networks
(Weaver et al., 2001;
Uruno et al., 2001),
suggesting that cortactin plays a direct role in lamellipodia protrusion and
integrity.
Multiple signaling pathways that activate Rac1 also lead to cortactin
tyrosine phosphorylation (Weed and
Parsons, 2001). Src phosphorylates murine cortactin on three
tyrosine residues (421, 466, and 482) within a proline-rich carboxyl-terminal
domain (Huang et al.,
1998), residues that are also phosphorylated by the kinases Fer
(Kapus et al., 2000)
and c-Met (Crostella et al.,
2001). Phosphorylation of cortactin tyrosines 421, 466, and 482 is
required for efficient cell motility in several cell types, indicating that
cortactin tyrosine phosphorylation plays an important role in cell migration
(Huang et al., 1998;
Crostella et al.,
2001). However, the structural and temporal requirements, as well
as the intracellular signaling pathways regulating cortactin tyrosine
phosphorylation are unknown.
We have examined the domain requirements for cortactin tyrosine phosphorylation and determined that phosphorylation of cortactin by Src requires the Arp2/3 and F-actin binding domains. Site-specific antibodies against cortactin phosphotyrosine 421 and 466 indicate that phosphorylation of these residues occurs in a hierarchical manner, is dependent on targeting to the cortical actin network and is regulated by the activation state of Rac1. Tyrosine phosphorylated cortactin is enriched in lamellipodia. We propose that cortactin tyrosine phosphorylation is coordinated with actin remodeling, where it may serve to integrate and transduce signals involved in cell migration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) was used as the template for production of
cortactin constructs containing tyrosine-to-phenylalanine point mutations of
codons 421, 466, and 482. Mutation of individual residues was conducted by
site-directed mutagenesis by using the QuikChange mutagenesis kit (Stratagene,
La Jolla, CA) following the manufacturer's protocol. All primers were designed
as described previously (Huang et
al., 1998), synthesized by Invitrogen (Carlsbad, CA) and
high-performance liquid chromatography purified. Constructs with more than one
mutation were created by successive rounds of mutagenesis. Mutation of each
codon was confirmed individually by DNA sequencing. FLAG-N-term (NT) and
FLAG-C-term (CT) cortactin constructs were as described previously
(Weed et al., 2000).
The FLAG-C-term construct with triple tyrosine to phenylalanine mutations at
codons 421, 466, and 482 (CT-T) was generated as a
KpnI-EcoRI fragment as described previously
(Weed et al., 2000)
by using FLAG-FL with the three phenylalanine mutations (FL-T) as a template,
and subcloned into KpnI-EcoRI digested pcDNA3FLAG2AB
(Devarajan et al.,
1997).
FLAG-tagged coronin1 was generated by polymerase chain reaction (PCR)
amplification of the coronin1 cDNA in pCR2.1
(Okumura et al.,
1998) with the 5' primer containing a KpnI
restriction endonuclease site and the 3' primer containing a stop codon
followed by an EcoRI site. The amplified cDNA was digested with
KpnI and EcoRI and subcloned into
KpnI-EcoRI digested pcDNA3FLAG2AB. For production of the
coronin1-cortactin C-term hybrid construct (FLAG-coronin1-CT), a
KpnI-BamHI PCR fragment spanning full-length coronin1 cDNA
lacking the stop codon was ligated in frame with a
BamHI-EcoRI fragment from pRK5myc-C-term
(Weed et al., 2000)
and KpnI-EcoRI digested pcDNA3FLAG2AB. pRK5myc, pRK5myc
Rac1N17, and pRK5myc Rac1L61 were described previously
(Lamarche et al.,
1996). All PCR-generated constructs were verified by DNA
sequencing.
Antibodies and Western Blotting
Anti-cortactin antibodies 4F11, anti-C-term, and anti-N-term have been
described previously (Wu and Parsons,
1993; Weed et al.,
1998). Anti-FLAG M5 and anti-FLAG M2 affinity resin were purchased
from Sigma-Aldrich (St. Louis, MO). Anti-phosphotyrosine RC-20, anti-activated
epidermal growth factor receptor (EGFR), and pan-reactive EGFR antibodies were
purchased from BD Transduction (San Diego, CA). Anti-phosphotyrosine 4G10
(direct conjugate to horseradish peroxidase) and anti-Rac1 monoclonal
antibodies (mAbs) were purchased from Upstate Biotechnology (Lake Placid, NY).
The anti-cmyc epitope mAb 9E10, anti-SRC2, and anti-Src N16 antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-
-actin
was purchased from Oncogene Sciences (San Diego, CA).
Phosphorylation site-specific antibodies recognizing mouse cortactin phosphorylated on tyrosine 421 (anti-cortactin pY421) or tyrosine 466 (anti-cortactin pY466) were manufactured and are marketed by BioSource International (Camarillo, CA). These rabbit polyclonal antibodies were generated by immunization of 13-mer peptides surrounding the sites of phosphorylation for both pY421 and pY466 and were purified using both negative and positive affinity purification methods to optimize specificity for the targeted phosphoepitope. Specificity was demonstrated using peptide competition assays (our unpublished data) and through the analysis of site-directed mutants for each tyrosine as shown in Figure 3, A and B. Anti-Src pY418 was purchased from Biosource International. Secondary antibodies coupled to horseradish peroxidase were purchased from Amersham Biosciences (Piscataway, NJ) and Pierce Chemical (Rockford, IL). Secondary antibodies coupled to AlexaFlour 488, 594, and 647, and phalloidin conjugated to AlexaFlour 594 were from Molecular Probes (Eugene, OR).
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)
with 5% bovine serum albumin (Fisher Scientific, Pittsburgh, PA) used for
blocking and blotting with phospho-tyrosine–specific antibodies and 5%
nonfat dry milk for all other antibodies. Primary antibodies were used at the
flowing concentrations or dilutions: 4F11 (0.2 µg/ml), anti-N-term or
anti-C-term (1 µg/ml), M5 (5 µg/ml), 4G10 (1:2000), RC-20 (1:1000),
ant-cortactin pY421 (0.05–0.1 µg/ml), anti-cortactin pY466
(0.05–0.5 µg/ml), anti-activated EGFR and pan-reactive EGFR (1:2500),
anti-Src pY418 (0.5 µg/ml), anti-SRC2 and Src N16 (1:100), anti-Rac1
(1:2000), and anti--actin (1:5000). Primary antibodies were detected
with the appropriate horseradish peroxidase-conjugated secondary (1:10,000)
and immunoreactive bands were visualized using a chemiluminescent substrate
(SugperSignal West Pico; Pierce Chemical). Blots were stripped by incubation
in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol at 50°C
for 40 min and washed extensively before blocking and reblotting.
Cell Culture and Transfections
C3H 10T1/2, 5Hd47, and NeoR1 fibroblast cell lines were a gift from Sarah
Parsons (University of Virginia, Charlottesville, VA). COS-1 cells were a gift
from Michael Webber (University of Virginia). NIH 3T3 527F and Rat1
v-src-transformed fibroblasts were a gift from Steve Anderson (University of
Colorado Health Sciences Center, Denver, CO). Cells were cultured with 10%
fetal calf serum (Hyclone Laboratories, Logan, UT) in DMEM supplemented with
50 U/ml penicillin and 50 µg/ml streptomycin (Mediatech, Herndon, VA).
NeoR1 and 5Hd47 cells were maintained in the presence of 400 µg/ml G-418
sulfate (Mediatech). Transfection of FLAG-cortactin and myc-Rac constructs in
C3H 10T1/2, 5Hd47, and COS-1 cells was conducted as described previously
(Weed et al., 2000)
by using the Polyfect reagent (QIAGEN, Valencia, CA) and 4 µg of total DNA.
For immunofluorescence studies, transfected cells were plated onto coverslips
coated with 40 µg/ml fibronectin (Sigma-Aldrich) and processed as described
previously (Weed et al.,
1998). For the production of motile cells, C3H 10T1/2 cells were
plated onto glass coverslips and grown to confluence for 48 h. Monolayers were
scraped with a plastic pipette tip and cells were cultured for 4 h before
fixation and processing for immunofluorescence microscopy. For spreading
cells, C3H 10T1/2 cells were trypsinized, washed, and plated onto
fibronectin-coated coverslips for 30 min as described previously
(Weed et al., 1998)
and Rat1 v-src cells were trypsinized and plated onto glass coverslips
overnight before fixation and immunolabeling. NeoR1 cells were grown to
confluence, starved for 18 h with DMEM/0.5% fetal bovine serum, trypsinized,
and plated onto coverslips in DMEM for 2 h. Cells were treated with 20 ng/ml
epidermal growth factor (EGF) (Upstate Biotechnology) for the times indicated
before fixation and processing for immunofluorescence microscopy.
Immunofluoresence Microscopy
Cells were fixed and immunolabeled as described previously
(Weed et al., 1998)
except that immunofluorescence buffer
(Schafer et al.,
1998) was substituted for phosphate-buffered saline (PBS).
Epitope-tagged constructs were detected with M5 (5 µg/ml) or 9E10 (1:
1000). Endogenous cortactin was detected with either anti-N-term (1 µg/ml)
or 4F11 (0.25 µg/ml). Anti-cortactin pY421 was used at 1–2 µg/ml.
AlexaFlour 594 phalloidin (Molecular Probes) was used at 1:1000. Primary
monoclonal antibodies were detected with anti-mouse AlexaFlour 594 for
double-labeling and anti-mouse AlexaFlour 647 for triple-labeling experiments;
rabbit primary antibodies were detected with anti-rabbit AlexaFlour 488. All
secondary antibodies were used at 1 µg/ml. Labeled coverslips were mounted
with the AntiFade kit (Molecular Probes), and cells were viewed using a Nikon
E600 epifluorescence microscope equipped with a Plan Aprochromat 60x
objective lens. Images were acquired with a Spot RT charge-coupled cooled
device (Diagnostic Instruments, Sterling Heights, MI) driven by native
software and were processed on a Macintosh computer with Adobe Photoshop
software. A minimum of 90 cells were evaluated for each condition, with at
least 85% of the cells demonstrating the represented effects except where
noted.
Cell Lysis and Immunoprecipitation
For evaluation of anti-cortactin pY421 and 466 antibodies, 10T1/2 cells
transfected with pcDNA3FLAG2AB, FLAG-FL, FLAG-421F, FLAG-466F, or FLAG-FL-T
were washed in PBS containing 1 mM NaVO4 and lysed in ice-cold
modified radioimmunoprecipitation assay buffer (RIPA)
(Du et al., 1998)
supplemented with protease inhibitor cocktail (1:200; Sigma-Aldrich). Lysates
were clarified by centrifugation at 14,000g for 10 min, and 50 µg
of total cell protein was resolved by SDS-PAGE and assayed by Western
blotting. EGF-mediated cortactin tyrosine phosphorylation was evaluated by
treatment of serum-starved NeoR1 cells with 5 ng/ml EGF for the indicated time
periods. Cells were lysed in modified RIPA, and 100 µg of protein was
analyzed by Western blotting with anti-pY421, anti-pY466, 4F11, anti-activated
EGFR, and anti-EGFR antibodies; 150 µg of protein was used for Src
analysis
Cells expressing FLAG-cortactin constructs were lysed in modified RIPA, and 1 mg of each clarified lysate was incubated with 25 µl of FLAG M2 affinity resin (Sigma-Aldrich) for 2 h at 4°C. Immune complexes were collected by centrifugation, washed twice with modified RIPA, separated by SDS-PAGE, and Western blotted with antibodies as described. For precipitation of endogenous cortactin, 25 µl of ImmunoPure Plus immobilized protein A agarose beads (50% slurry in PBS; Pierce Chemical) were precharged with 7.5 µg of rabbit anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in 500 µl of PBS for 2 h. Beads were washed twice with PBS, resuspended in 500 µl of PBS and incubated with 5 µg of 4F11 overnight. After washing the beads twice with PBS, cortactin was immunoprecipitated from modified RIPA lysates (500 µg of protein) of C3H 10T1/2, NIH 3T3 527F, and Rat1 v-src cells by incubation with 4F11-charged beads for 2 h. Immune complexes were processed for Western blotting as described above. Protein concentrations were determined using the DC Protein Assay (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.
In Vitro Kinase Assays
For analysis of cortactin phosphorylation by Src, C3H 10T/12 cells
transfected with FLAG-cortactin constructs were lysed in modified RIPA and
immunoprecipitated with FLAG M2 resin as described. Immune complexes were
washed twice with modified RIPA buffer, once with PBS containing 2 M KCl, and
once with kinase buffer (50 mM HEPES, pH 7.3, 5 mM MnCl2).
Complexes were incubated in kinase buffer containing 10 µCi of
[
-32P]ATP (PerkinElmer Life Sciences, Boston, MA) with or
without 1 U of purified Src (Upstate Biotechnology) in a final volume of 50
µl. Reactions were incubated at 30°C for 10 min, stopped with an equal
volume of hot 2x SDS-PAGE sample buffer, resolved by SDS-PAGE, and
transferred to polyvinylidene difluoride membranes (Amersham Biosciences).
After autoradiography, membranes were stripped as described above, exposed to
x-ray film overnight to ensure removal of all radiolabel, and then blotted
with anti-N-term and C-term cortactin antibodies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
To investigate the extent of phosphorylation of each individual tyrosine in
response to c-Src activation in vivo, a panel of all possible combinatorial
mutations of tyrosines 421, 466, and 482 to phenylalanine were produced in a
FLAG-tagged wild-type cortactin expression construct by successive rounds of
site directed mutagenesis. Wild-type FLAG-cortactin (FL) and mutant constructs
were transfected into 5Hd47 fibroblasts, a nontransformed variant of C3H
10T1/2 cells that exhibits 10-fold c-Src overexpression
(Wilson and Parsons, 1990). As
shown in Figure 1B, FL
(wild-type) cortactin gave a robust signal when immunoprecipitated and
analyzed for tyrosine phosphorylation by Western blotting with the
pan-phosphotyrosine antibody 4G10. The single mutation of tyrosine 421 (421F)
to phenylalanine resulted in a significant reduction in cortactin tyrosine
phosphorylation compared with FL cortactin, whereas single mutations of either
tyrosine 466 or tyrosine 482 to phenylalanine (466F and 482F, respectively)
had less of an effect, with phosphotyrosine levels similar to that of FL
cortactin. Cortactin constructs containing the pairwise mutant combinations
421F/466F and 421F/482F exhibited diminished phosphotyrosine levels similar to
that of 421F, whereas the 466F/482F construct was still abundantly
phosphorylated. A cortactin construct containing tyrosine-to-phenylalanine
mutation of all three sites (421F/466F/482F) resulted in further (but not
complete elimination of) detectable phosphotyrosine levels. In agreement with
previous results (Huang et al.,
1998), these data indicate that codons 421, 466, and 482 are the
tyrosine residues on murine cortactin phosphorylated by c-Src, with tyrosine
421 being the primary site of Src-mediated cortactin phosphorylation in
vivo.
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In vitro, Src directly phosphorylates a recombinant cortactin polypeptide
encoding the proline-rich carboxyl-terminal domain
(Huang et al., 1998).
To determine if Src-mediated phosphorylation of cortactin requires the
presence of additional cortactin domains besides the proline-rich region in
vivo, FLAG-tagged cortactin constructs, including constructs encoding either
the amino-(N-term) or carboxyl (C-term)-cortactin domains
(Figure 1A) were transfected
into C3H 10T1/2 and 5Hd47 fibroblasts, and the overexpressed cortactin fusion
proteins were immunoprecipitated with anti-FLAG resin. Tyrosine
phosphorylation of the expressed cortactin proteins was determined by Western
blotting with RC-20 (Figure 1C,
top) and expression of each cortactin peptide was verified by immunoblotting
with anticortactin antibodies (Figure
1C, bottom). While full-length, wild-type cortactin (FL) was
tyrosine phosphorylated when expressed in C3H 10T1/2 cells, its
phosphorylation was significantly enhanced when expressed in 5Hd47 fibroblasts
(Figure 1C; note lower
expression levels of FL in 5Hd47). Expression of FLAG-cortactin 421F/466F/482F
(FL-T) displayed diminished but dectable tyrosine phosphorylation compared
with wild-type cortactin when expressed in either C3H 10T1/2 or 5Hd47 cells.
Interestingly, FLAG-N-term (NT) displayed a low but consistently reproducible
(n = 3) level of tyrosine phosphorylation in C3H 10T1/2 cells that was not
elevated in 5Hd47 cells. Surprisingly, expression of FLAG-C-term (CT), which
contains the tyrosine residues targeted by Src, was not detectably tyrosine
phosphorylated (Figure 1C), as
was a FLAG-C-term construct with the triple tyrosine to phenylalanine
mutations at 421, 466 and 482 (CT-T). The lack of tyrosine phosphorylation in
the CT construct suggested that Src-mediated phosphorylation of the cortactin
carboxyl-terminal domain requires the presence of the cortactin amino
terminus.
To verify that the lack of phosphorylation of the carboxyl-terminal domain
was not due to misfolding and subsequent lack of recognition by Src,
FLAG-cortactin constructs were expressed in C3H 10T1/2 cells,
immunoprecipitated with anti-FLAG resin, and washed with 2 M KCl to remove
associated proteins. Immune complexes were incubated with purified Src and
[-32P]ATP, and resolved by SDS-PAGE. Autoradiography
(Figure 1D, top) demonstrated
efficient phosphorylation of FLAG-FL and the FLAG-CT, with little
phosphorylation of either FLAG-FL-T, FLAG-NT, or FLAG-CT-T. Stripping and
probing of the membrane with anti-cortactin antibodies confirmed precipitation
of the FLAG-cortactin proteins (Figure
1D, bottom). These data support the conclusion that the
amino-terminal cortactin domain is tyrosine phosphorylated independent of Src
activity and that phosphorylation of the C-terminal cortactin domain by Src
requires the presence of the cortactin amino terminus.
Tyrosine Phosphorylation of the Cortactin Carboxyl Terminus Requires
Localization to the Cell Cortex
The amino-terminal cortactin domain is required for Rac1-mediated targeting
of cortactin to the cell periphery, a process that involves direct association
with F-actin and the Arp2/3 complex (Weed
et al., 2000). To determine whether the inability of the
cortactin C-terminal domain to become tyrosine phosphorylated required
Rac1-mediated localization of the cortactin carboxyl-terminal domain to the
cell cortex, a FLAG-tagged construct was created where the C-terminal
cortactin domain was fused in frame to the C termini of the unrelated
actin-binding and lamellipodia protein coronin1
(Figure 2A). Coronin1 also
targets to the cortical actin cytoskeleton in a Rac1-dependent manner and
directly binds Arp2/3 complex but is not tyrosine phosphorylated
(Mishima and Nishida, 1999;
Humphries et al.,
2002). We selected this strategy over examining cortactin tyrosine
phosphorylation after direct perturbation of the actin cytoskeleton with
cytochalasian D or latrunculin B because these drugs lead to robust Src
activation, potentially resulting in aberrant cortactin tyrosine
phosphorylation (Lock et al.,
1998). To determine whether the coronin1-cortactin C-term
(coronin1-CT) construct restored the ability of the cortactin
carboxyl-terminal domain to become tyrosine phosphorylated, COS-1 cells were
transfected with FLAG-coronin1 and FLAG-coronin1-CT, along with FLAG-FL,
FLAG-CT, and FLAG-CT-T as controls. FLAG-tagged proteins were
immunoprecipitated and tyrosine phosphorylation of recombinant proteins was
analyzed by Western blotting with RC-20. Both FLAG-FL and FLAG-coronin1-CT
were tyrosine phosphorylated, whereas FLAG-CT, FLAG-CT-T, and FLAG-coronin1
were not (Figure 2B, top). The
blot was stripped and precipitation of the FLAG-tagged proteins was verified
by immunoblotting with anti-FLAG M5 (Figure
2B, bottom). These data indicate that fusion of the cortactin
carboxy terminus to coronin1 restores the ability of the carboxyl-terminal
cortactin domain to become tyrosine phosphorylated.
To determine whether the FLAG-coronin proteins targeted to the cell
periphery in response to Rac1 activation, C3H 10T1/2 cells were cotransfected
with constitutively active myc-RacL61 and FLAG-FL, FL-T, CT, CT-T, coronin1,
and coronin1-CT. After transfection, cells were trypsinized and plated on
fibronectin-coated coverslips. Under these conditions, C3H 10T1/2 cells
expressing RacL61 exhibit a rounded morphology, and cortactin localization at
the cell periphery is dependent on the presence of the amino-terminal domain
(Weed et al., 2000).
In agreement with these previous results, FLAG-FL and FL-T accumulated at the
cell cortex with endogenous cortactin, whereas FLAG-CT and FLAG-CT-T failed to
localize at the periphery and remained cytoplasmic
(Figure 2C). FLAG-coronin1 and
FLAG-coronin1-CT also localized with cortactin at the cortex, although a large
amount of both of these proteins was found in the cytoplasm
(Figure 2C). In all cases,
endogenous cortactin exhibited efficient cortical localization. Together,
these data suggest that Rac1-induced localization of cortactin to the cell
cortex is required for tyrosine phosphorylation of the carboxyl-terminal
cortactin domain.
Production and Specificity of Phosphorylation-specific Antibodies
against Phosphorylated Tyrosine 421 and Tyrosine 466 of Murine Cortactin
Tyrosine 421 and 466 are the primary residues phosphorylated by c- and
v-Src in murine cortactin (Figure
1B; Huang et al.,
1998). To facilitate the analysis of these sites and to determine
whether these sites are phosphorylated in response to Rac1-mediated cortical
targeting, we developed antibodies specific to phosphotyrosine 421 and 466 of
murine cortactin. To confirm the specificity of each antibody, C3H 10T1/2
cells were transfected with empty FLAG vector, FLAG-FL, FLAG-FL-T, and either
FLAG-FL with an individual tyrosine-to-phenylalanine mutation at codon 421 for
anti-pY421 analysis (421F) or at codon 466 for anti-pY466 analysis (466F)
(Figure 3, A and B,
respectively). Total cell lysates were immunoblotted with either anti-pY421
(Figure 3A) or anti-pY466
cortactin antibodies (Figure
3B). The anti-pY421 cortactin antibody detected endogenous
cortactin as a doublet that migrated between 72 and 78 kDa by SDS-PAGE in
lysates from cells expressing the empty FLAG vector (V), FLAG-421F, and FL-T.
However, an additional band migrating at 
80 kDa was detected in lysates
from cells transfected with FLAG-FL (Figure
3A), indicating that the anti-pY421 cortactin antibody selectively
recognizes cortactin phosphorylated on tyrosine 421. Similar results were
obtained with the anti-pY466 cortactin antibody, although two additional
cross-reactive bands were observed at 125 and 47 kDa
(Figure 3B). Nevertheless, the
anti-pY466 antibody is specific for the cortactin epitope because it also
detected FLAG-FL but failed to recognize either FLAG-466F or FLAG-FL-T
(Figure 3B). The additional
band present in the FL lanes of the anti-421 and 466 blots is FLAG-FL because
this band is due to the slightly slower electrophoretic mobility caused by the
presence of the FLAG-tag in the recombinant protein
(Weed et al., 2000).
Stripping and reprobing of the blots with the anti-cortactin mAb 4F11 resulted
in a tight cortactin doublet in lysates containing FLAG-tagged cortactin
proteins (Figure 3, A and B), with the recombinant FLAG-cortactin proteins constituting the slower migrating
form of the two bands because this band was absent in lysates from cells
transfected with the empty FLAG vector (V). Immunoblotting of lysates with the
anti-M5 mAb (anti-FLAG) directly verified the expression of the FLAG-cortactin
proteins, and equivalent loading of protein amounts was confirmed by stripping
and immunoblotting for -actin (anti--actin)
(Figure 3, A and B). Collectively, these results show that the anti-pY421 and anti-pY466 cortactin
antibodies specifically recognize their respective phosphoepitopes.
To determine that the anti-phosphospecific cortactin antibodies detect
tyrosine phosphorylation of cortactin at codons 421 and 466 as a result of Src
activation, cortactin was immunoprecipitated from murine fibroblasts with
normal levels of c-Src activity (C3H 10T1/2), fibroblasts expressing a
tyrosine-to-phenylalanine–activating mutation in c-Src (3T3 527F) and
Rat1 fibroblasts expressing v-Src (Rat1 v-Src). A substantial increase in
cortactin phosphorylation was observed on tyrosine 421 and tyrosine 466 in
cortactin from 3T3 527F and Rat1 v-Src fibroblasts compared with C3H 10T1/2
cells after immunoblotting with anti-pY421 and anti-pY466 antibodies
(Figure 3C). Stripping and
reprobing of the blots with 4F11 indicated that equivalent amounts of
cortactin were assayed (Figure
3C), with the slower migrating form of cortactin from Rat1 v-Src
cells attributable to the larger size of cortactin in rat versus murine cells
(Ohoka and Takai, 1998). These
results indicate that the anti-pY421 and anti-pY466 cortactin antibodies
detect Src-mediated phosphorylation of murine cortactin at tyrosine 421 and
466 and that these antibodies also detect phosphorylation of rat cortactin on
these residues as expected from sequence analysis of both phosphorylation
sites (Figure 8).
|
Given the specificity of the anti-pY421 and pY-466 cortactin antibodies, we sought to determine whether these sites were in part responsible for the tyrosine phosphorylation within the cortactin carboxy terminus observed in the cortical targeting of the FLAG-coronin1-CT construct (Figure 2B). C3H 10T1/2 cells were transfected with either empty FLAG vector, FLAG-FL, FLAG-FL-T, FLAG-NT, FLAG-CT, FLAG-CT-T, FLAG-coronin1, or FLAG-coronin1-CT. Recombinant proteins were immunoprecipitated with anti-FLAG resin, and tyrosine phosphorylation of FLAG-tagged proteins was analyzed by Western blotting with anti-pY421 and anti-pY466 cortactin antibodies. As shown in Figure 3D, FLAG-FL and coronin1-CT were the only proteins exhibiting phosphorylation of tyrosine 421 and 466. All recombinant proteins were precipitated as determined by anti-FLAG M5 immunoblotting. Phosphorylation of tyrosine 421 and tyrosine 466 in the coronin1-CT fusion protein therefore indicates that these two residues in the cortactin carboxy terminus contribute to the tyrosine phosphorylation observed in this construct in Figure 2B.
Phosphorylation of Tyrosine 421 in Murine Cortactin Is Required for
Tyrosine 466 Phosphorylation
Several Src substrates containing multiple phosphorylation sites can be
differentially regulated with respect to each other
(Guappone et al.,
1998; Schaller and Schaefer,
2001; Ruest et al.,
2001). To determine whether phosphorylation of tyrosine 421 and
466 in cortactin occurs in an independent or interdependent manner in response
to Src activation in vivo, 5Hd47 fibroblasts were transfected with the panel
of FLAG-tagged cortactin constructs containing all possible
tyrosine-to-phenylalanine mutational combinations of codons 421, 466, and 482
(Figure 1B). Expressed proteins
were immunoprecipitated with anti-FLAG resin, and tyrosine 421 and 466
phosphorylation was analyzed by Western blotting with anti-pY421 and
anti-pY466 cortactin antibodies (Figure
4). As expected, the anti-pY421 cortactin antibody detected all
proteins where tyrosine 421 was not mutated to phenylalanine (FL, 466F, 482F,
and 466F/482F), whereas cortactin constructs containing phenylalanine
mutations at codon 421 (421F, 421F/466F, 421F/482F, and 421F/466F/482F) were
not detected by anti-pY421 cortactin
(Figure 4). Interestingly, the
anti-pY466 antibody only detected the FL and 482F proteins, failing to detect
constructs where tyrosine 466 was intact in the context of a phenylalanine
mutation at codon 421 (421F and 421F/482F) as well as constructs with
phenylalanine present at codon 466 (466F, 421F/466F, 466F/482F, and
421F/466F/482F) (Figure 4).
This result indicates that the presence of a phosphorylated tyrosine residue
at position 421 is required for phosphorylation of tyrosine 466 in murine
cortactin in vivo, and that phosphorylation of tyrosine 421 and 466 is
independent of tyrosine 482 phosphorylation.
|
Localization of pY421 Cortactin with Cortical Actin Structures
In nontransformed cells, cortactin localizes with the cortical F-actin
network within lamellipodia and is also concentrated in a cytoplasmic pool
near the perinuclear region (Wu and
Parsons, 1993). In Src-transformed fibroblasts, cortactin
redistributes into cell-substratum contacts known as podosomes
(Wu et al., 1991). To
determine the subcellular localization of tyrosine phosphorylated cortactin in
normal and transformed cell types, C3H 10T1/2 and Rat1 vSrc fibroblasts were
cultured in the presence of serum and immunolabeled with anti-pY421 cortactin
(Figure 5). In both motile
(Figure 5A) and spreading
(Figure 5B) C3H 10T1/2 cells,
anti-pY421 cortactin labeling was concentrated primarily with F-actin and
cortactin within lamellipodia, with labeling of the perinuclear region
observed to a lesser extent relative to the levels of total cortactin in the
cytoplasm. Anti-pY421 cortactin exhibited strong labeling of podosomes in Rat
1 v-Src cells that also overlapped with F-actin and cortactin
(Figure 5C). Overlaying of
individual images for F-actin, pY421 cortactin, and total (pan) cortactin
indicated that pY421 cortactin is enriched with the pool of cortactin that is
associated with cortical actin in lamellipodia and podosomes
(Figure 5, A–C). The
anti-pY466 antibody failed to specifically label cortactin in cells (our
unpublished data), presumably due to the cross-reactivity of this antibody
with other cellular proteins (Figure
3B).
|
EGF Stimulates Phosphorylation of Cortactin on Tyrosine 421 and
Tyrosine 466
EGF is known to stimulate cortactin tyrosine phosphorylation in a
Src-dependent manner (Maa et al.,
1992). To determine whether tyrosine 421 and tyrosine 466 of
cortactin are phosphorylated in response to EGF, serum-starved C3H 10T1/2
cells with 40-fold overexpression of EGFR (NeoR1;
Maa et al., 1995)
were treated with EGF and lysed at various time points over a 1-h period.
Tyrosine phosphorylation of cortactin was subsequently analyzed by Western
blotting of total cell extracts with anti-pY421 and anti-pY466 cortactin
antibodies (Figure 6A).
Serum-starved NeoR1 cells exhibited low levels of pY421 and pY466
phosphorylation that was elevated by EGF treatment by 1 min, increased after 5
min, decreased at 10 min, and increased from 30 min to 1 h
(Figure 6A). The biphasic
response of cortactin tyrosine phosphorylation in NeoR1 cells in response to
EGF is similar to that observed in cells overexpressing c-Src
(Maa et al., 1992)
and in cells treated with fibroblast growth factor 1
(Zhan et al., 1993).
The changes in tyrosine 421 and 466 phosphorylation were not due to
fluctuations in cortactin protein levels, because stripping and reprobing of
the blots with 4F11 did not indicate a change in cortactin amounts over the
course of the experiment (representative cortactin blot shown in
Figure 6A). Immunoblotting of
lysates from stimulated NeoR1 cells at the same time points with antibodies
against activated EGF receptor (phospho-EGFR) and Src (pY418 Src) indicated
that both enzymes attained rapid (within 1 min) and sustained activation over
the course of the experiment (Figure
6A). These data indicate that tyrosine 421 and 466 are
phosphorylated in response to EGF treatment in NeoR1 cells with kinetics
similar to that in other cell systems and that the time course of cortactin
tyrosine 421 and tyrosine 466 phosphorylation parallels the activation of both
EGF receptor and Src.
|
In addition to Src, EGF also activates Rac1, which leads to cortical actin
polymerization that results in the formation of membrane ruffles and
lamellipodia (Ridley et al.,
1992; Bailly et al.,
1999). Because pY421 cortactin localizes to lamellipodia with
cortical F-actin in cells cultured in serum
(Figure 5, A and B) the
subcellular distribution of both cortactin and pY421 cortactin in NeoR1 cells
was examined after EGF treatment (Figure
6B). Serum-starved NeoR1 cells (0 min) were largely devoid of
pY421 cortactin, with only faint labeling present in the cytoplasm where the
majority of the cortactin pool was localized
(Figure 6B). Serum starved
cells treated for 1 min with 20 ng/ml EGF demonstrated nearly exclusive
labeling of pY421 cortactin within broad membrane ruffles, with very little
staining of the perinuclear region in spite of the presence of abundant
cytoplasmic cortactin (Figure
6B). A similar pattern was observed after 5 min of EGF treatment,
although the membrane ruffles containing cortactin and pY421 cortactin were
narrower (Figure 6B),
reminiscent of the phenotype exhibited by cells overexpressing activated Rac1
(Figure 2B). By 30 min, the
majority (60%) of cells were similar to the phenotype observed after 5 min of
EGF stimulation. However, a significant number (34%) of cells exhibited a
polarized phenotype indicative of motility, with a clearly defined leading and
trailing edge. In these cells, pY421 cortactin was enriched almost exclusively
within leading edge lamellipodia as well as in the trailing edge, with little
labeling of the cytoplasm despite the presence of an abundant cortactin pool
in this region (Figure 6B).
These data indicate that pY421 cortactin selectively localizes to membrane
ruffles and lamellipodia in response to EGF receptor activation.
Phosphorylation of Cortactin Tyrosine 421 and Tyrosine 466 Requires
Rac1 Activity
Translocation of cortactin from the cytoplasm into membrane ruffles and
lamellipodia requires Rac1 activity (Weed
et al., 1998). Because pY421 cortactin was selectively
localized to these regions as a result of EGF treatment in NeoR1 cells, we
examined whether phosphorylation of cortactin tyrosine 421 and tyrosine 466
was regulated by the activation state of Rac1. C3H 10T1/2 cells were
cotransfected with FLAG-FL cortactin and with empty myc vector (V), dominant
negative myc-Rac1T17N (N17), or constitutively active myc-Rac1Q61L (L61).
FLAG-FL cortactin was immunoprecipitated with anti-FLAG resin, and tyrosine
phosphorylation of cortactin was assayed by immunoblotting with anti-pY421 and
anti-pY466 antibodies. FLAG-FL cortactin was phosphorylated at basal levels on
tyrosine 421 and tyrosine 466 when coexpressed with the myc vector. However,
phosphorylation of FLAG-FL cortactin tyrosines 421 and 466 was inhibited when
expressed with myc-Rac1N17 (Figure
7A). Conversely, expression of myc-RacL61 enhanced FLAG-FL
cortactin tyrosine 421 and 466 phosphorylation compared with that of cells
transfected with vector alone (Figure
7A). Expression of myc-Rac proteins was verified by immunoblotting
of cell lysates with an anti-Rac1 mAb, which detects both endogenous Rac1 as
well as the slower migrating myc-Rac1 proteins
(Figure 7A). These results
indicate that the activity of Rac1 regulates phosphorylation of cortactin
tyrosine 421 and 466.
|
To evaluate the subcellular distribution and degree of cortactin tyrosine phosphorylation in response Rac1 activity in vivo, C3H 10T1/2 cells transfected with empty myc vector (vector), myc-Rac1N17 and myc-Rac1L61were trypsinized, plated onto fibronectin, and allowed to spread for 2 h. Cells were immunostained with anti-pY421 cortactin and anti-myc 9E10 (Figure 7B) or anti-pY421 cortactin and anti-cortactin 4F11 (Figure 7C). In cells transfected with myc vector, pY421 cortactin localized at the perinuclear region and within lamellipodia with endogenous cortactin (Figure 7, B and C) as observed previously (Figure 5, A and B). Expression of myc-Rac1N17 inhibited the localization of cortactin to the cell periphery and resulted in the loss of pY421 immunolabeling from the cell cortex as well as significantly reducing level of pY421 labeling in the cytoplasm (Figure 7, B and C). In contrast, myc-RacL61 expression resulted in the translocation of cortactin to the cell cortex (Figure 7C) along with increased pY421 cortactin immunolabeling that localized at both the cell periphery and the perinuclear region (Figure 7, B and C). These results suggest that Rac1-induced translocation of cortactin to the cell periphery is required for phosphorylation of cortactin tyrosine 421
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Kapus et al., 2000;
Crostella et al.,
2001). Although phosphorylation of these residues is required for
efficient cell motility (Huang et
al., 1998; Crostella
et al., 2001), the spatial and temporal requirements for
cortactin tyrosine phosphorylation were previously unknown. The results
presented herein demonstrate that phosphorylation of tyrosine residues in the
carboxyl-terminal cortactin domain requires the cortactin amino terminus.
Site-specific antibodies against phosphotyrosine 421 and 466, two sites of
Src-mediated cortactin tyrosine phosphorylation, demonstrate that both of
these tyrosine residues are phosphorylated in response activation of Src,
EGFR, and Rac1. Western blot analysis of cortactin point mutants indicates
that tyrosine 421 phosphorylation is required for phosphorylation of tyrosine
466. Cortactin phosphorylated on tyrosine 421 is enriched in lamellipodia and
podosomes where it colocalizes with cortical F-actin. Phosphorylation of
tyrosine 421 and 466 is dependent on activation of Rac1. Collectively, our
results indicate that tyrosine phosphorylation of cortactin requires
Rac1-mediated targeting to the cortical actin network within lamellipodia,
where it is tyrosine phosphorylated at codons 421 and 466 in a hierarchical
manner. We propose that phosphorylation of cortactin at tyrosine 421 and 466
serves to integrate tyrosine kinase-based signals with signaling pathways that
contribute to actin cytoskeletal assembly and lamellipodia formation.
In agreement with previous results
(Huang et al., 1998),
expression of cortactin tyrosine point mutants in c-Src–overexpressing
cells indicates that tyrosines 421, 466, and 482 are phosphorylated in vivo as
a result of Src activity, with tyrosine 421 as the primary site of
phosphorylation. Although codons 421, 466, and 482 are phosphorylated by Src,
mutation of these three sites to phenylalanine does not completely abolish
cortactin tyrosine phosphorylation. An additional tyrosine at codon 485 has
been reported to be weakly phosphorylated by Src in vitro
(Huang et al., 1998).
Of the 27 tyrosine residues in murine cortactin, 12 are clustered within the
carboxy terminus (Miglarese et
al., 1994) with the remaining residues distributed throughout
the amino terminus. Our analysis indicates that the amino terminal cortactin
domain is tyrosine phosphorylated in a manner independent from Src and is
phosphorylated at a low stoichiometry and/or at a single site. Tyrosine
phosphorylation of the amino-terminal domain is significantly enhanced after
treatment of cells with several growth factors (our unpublished data) pointing
to a potential physiological role. Although the significance of this is
currently unclear, one possibility is that tyrosine phosphorylation of the
cortactin amino terminus may modulate its interaction with Arp2/3 complex
and/or F-actin networks.
The availability of phosphorylation site-specific antibodies against
tyrosine 421 and 466 in murine cortactin allows for the specific analysis of
these phosphorylation sites. Oncogenic transformation by v-Src results in
increased cortactin tyrosine phosphorylation
(Kanner et al., 1990;
Wu et al., 1991), and
our results by using site-specific antibodies indicate that tyrosines 421 and
466 are phosphorylated in v-Src–transformed cells. Analysis of tyrosine
point mutants with site-specific phosphorylation antibodies indicates that
phosphorylation of tyrosine 421 is required for phosphorylation of tyrosine
466 (Figure 4), a conclusion
consistent the results shown in Figure
1B by using a general anti-phosphotyrosine antibody. The amino
acid sequence distal to tyrosine 421 (421YEDA424)
conforms to the consensus binding site for Src homology 2 (SH2) domains
(Songyan et al.,
1993) and is conserved in all vertebrate cortactins identified to
date (Figure 8). Src
coimmunoprecipitates with cortactin in multiple cell systems
(Zhan et al., 1994;
Okamura and Resh, 1995; van Damme et
al., 1997), and the SH2 domain of Src can associate with
tyrosine-phosphorylated cortactin (Okamura and Resh, 1995). Treatment of
Csk–/– fibroblasts with the SH2
inhibitory compound AP22408 inhibits cortactin tyrosine phosphorylation
(Violette et al.,
2001), pointing to a direct requirement for Src SH2 association
with cortactin as a precursor event for cortactin tyrosine phosphorylation.
These results, together with our observations suggests that cortactin is
phosphorylated by Src in a sequential manner involving initial phosphorylation
of tyrosine 421, potentially creating a binding site for the Src SH2 domain
that would allow for Src association and subsequent phosphorylation of
tyrosine 466. The involvement of tyrosine 482 in such a processive mechanism
will require the development of phosphorylation specific antibodies against
this site.
Cortactin tyrosine phosphorylation occurs concurrently with a multitude of
signaling events that induce remodeling of the cortical actin cytoskeleton
(Weed and Parsons, 2001 and
references therein). Our data herein suggest that the ability of cortactin to
regulate the actin cytoskeleton is structurally coupled with phosphorylation
of tyrosine residues in the carboxy terminus. The inability of the carboxyl
terminal cortactin domain to become tyrosine phosphorylated when expressed
alone suggests that localization and association with F-actin and/or Arp2/3
complex at the cell periphery mediated by the amino terminus is required for
phosphorylation of this domain. The ability of coronin1, a cortical actin and
Arp2/3 complex binding protein implicated in lamellipodia formation
(Mishima and Nishida, 1999;
Humphries et al.,
2002) to rescue tyrosine phosphorylation of the cortactin carboxy
terminus supports a model whereby tyrosine phosphorylation of the carboxy
terminus requires targeting of cortactin to and association with Arp2/3
complex and the cortical actin cytoskeleton, although the possibility exists
that an actin-binding amino terminus (from either cortactin or coronin1) is
required for proper conformational folding of the carboxy terminus and
subsequent recognition by Src and other tyrosine kinases in vivo.
The enrichment of cortactin phosphorylated on tyrosine 421 in lamellipodia
and podosomes, dynamic sites of actin remodeling, supports a possible role for
cortactin tyrosine phosphorylation in regulating actin dynamics. Although the
binding of cortactin to F-actin is not influenced by Src phosphorylation
(Wu and Parsons, 1993),
previous work has suggested that phosphorylation of Src by cortactin
down-regulates the ability of cortactin to bundle actin filaments
(Huang et al., 1997),
although other groups have not detected efficient bundling in the presence of
cortactin (Weaver et al.,
2001). Cortactin exists as a monomer
(Weaver et al., 2002)
and possesses a single F-actin binding site
(Weed et al., 2000),
making the mechanism by which cortactin bundles F-actin unclear. Another
possibility is that tyrosine phosphorylation of cortactin may influence the
cortical actin cytoskeleton by creating binding sites for SH 2/3 adaptor
molecules involved in actin remodeling. Tyrosine phosphorylated cortactin
interacts with the SH2 domains of Nck and Crk (Okamura and Resh, 1995), two
adaptor proteins that couple phosphotyrosine signals through interactions with
their SH2 domains to Rac1 and Arp2/3 complex mediated actin reorganization by
SH3 domain-mediated interactions (reviewed in
Li et al., 2001a;
Feller, 2001). Phosphorylation
of cortactin at tyrosine 421 and/or 466 may therefore serve as an indirect
mechanism for regulating the cortical cytoskeleton in addition to the direct
ability of the cortactin amino terminus to regulate Arp2/3 complex actin
nucleation activity.
In agreement with our findings, a recent report has suggested that Rac1
activation enhances cortactin translocation in cells exposed to hyperosmotic
conditions (Di Ciano et al.,
2002). This group reported that Rac1N17 inhibited cortactin
tyrosine phosphorylation in their system, although the level of inhibition was
not to the extent of that observed in our report. This may be due to
differences specific to hyperosmotic shock and the implicated kinase Fer
(Kapus et al., 2000),
which differs from the analysis preformed in our fibroblast system, as well as
the phosphorylation of other tyrosine residues on cortactin that may not be
regulated by the activation state of Rac1. In addition to its association with
the cortical actin cytoskeleton, cortactin phosphorylated on tyrosine 421 was
also present to a lesser extent within the cytoplasmic cortactin pool in
motile and spreading cells (Figure 5, A and
B), and this cytoplasmic distribution was enhanced in cells
expressing Rac1 L61 (Figure 7, B and
C). Di Ciano et al.
(2002) speculate that tyrosine
phosphorylation of cortactin may serve to stimulate the release of cortactin
from Arp2/3 networks as a result of hyperosmolarity, and the presence of
cytoplasmic cortactin phosphorylated on tyrosine 421 in fibroblasts supports
such a hypothesis. However, serum-starved cells treated with EGF, a potent
Rac1 activator, led to the rapid and specific accumulation of cortactin
phosphorylated on tyrosine 421 at the cell cortex, with little phosphorylated
cortactin present in the cytoplasm (Figure
6B). Although our biochemical and localization data strongly
suggest that Rac1 activation is the major determinant in the phosphorylation
of cortactin tyrosine 421 and 466 in fibroblasts, the differential effects
between EGF and serum in regulating the levels of cytoplasmic cortactin
phosphorylated on tyrosine 421 points to the potential participation of
additional signaling pathways contributing to cortactin tyrosine
phosphorylation.
In addition to their localization in lamellipodia, both cortactin and Src
are present at significant levels in perinuclear vesicular compartments within
the cytoplasm, where their distribution demonstrates a degree of overlap
(Maa et al., 1992;
Okamura and Resh, 1995). Previous work demonstrating the requirement for Rac1
activity in the translocation of cortactin and Src to lamellipodia
(Weed et al., 1998;
Timpson et al., 2001)
combined with the data presented in this report suggests that a possible
mechanism for Src-mediated cortactin tyrosine phosphorylation may involve
Rac1-induced targeting of nonphosphorylated cortactin and Src from cytoplasmic
compartments to the cortical actin cytoskeleton. This would allow for the
compartmentalization of cortactin and Src in lamellipodia, where subsequent
juxtaposition of cortactin to activated Src would result in the processive
phosphorylation of cortactin on tyrosines 421, 466, and 482. The requirement
for phosphorylation of cortactin on these tyrosine residues in normal cell
migration (Huang et al.,
1998; Crostella et
al., 2001) and tumor cell metastasis
(Li et al., 2001b)
highlights the importance of cortactin tyrosine phosphorylation in regulating
normal and neoplastic cell movement. By controlling the subcellular
localization of cortactin and Src, Rac1 may serve to integrate actin
cytoskeletal signaling pathways through cortactin by regulating Arp2/3 complex
activity with transmembrane receptor and nonreceptor tyrosine phosphorylation
pathways used during lamellipodia extension and subsequent cell motility.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: Arp2/3, actin related protein 2/3; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; pY, phosphotyrosine; RIPA, radioimmunoprecipitation assay; SH2/3, Src homology 2/3.
Corresponding author. E-mail address:
scott.weed{at}uchsc.edu
