(Received for publication, January 16, 1997, and in revised form, February 28, 1997)
From the Department of Experimental Pathology, The
Holland Laboratory, American Red Cross, Rockville, Maryland 20855, the § Division of Biology, Glaxo and Welcome Research
Institute, Research Triangle Park, North Carolina 27709, and the
Departments of ¶ Pathology and
Anatomy and Cell
Biology, The George Washington University,
Washington, D. C. 20037
Cortactin, a prominent substrate for pp60c-src, is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60c-src phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60c-src moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60c-src can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.
Cortactin (p80/p85) was initially discovered as a major
phosphotyrosine-containing protein in v-Src-transformed chicken embryo fibroblasts (1). The murine homologue was independently isolated as a
signaling molecule involved in the transition from G0 to G1 phase in response to fibroblast growth factor (2, 3), and the human cortactin was found as an oncogene that is frequently amplified in subsets of tumors and tumor cell lines (4, 5). A strong
association of cortactin with F-actin1 has
been described (14). Consistent with its F-actin binding activity,
cortactin primarily localizes within peripheral cell structures such as
lamellipodia, pseudopodia, and membrane ruffles (9, 14), which are
enriched for cytoskeletal proteins. However, unlike many other
F-actin-binding proteins, the protein sequence of cortactin features a
unique structure characterized by six and a half 37-amino acid tandem
repeats and a Src homology 3 (SH3) domain at the carboxyl terminus.
Between the SH3 and the repeat domains are an -helix domain and a
sequence region rich in proline residues.
Recent evidence has indicated that cortactin is a prominent substrate for Src-related protein-tyrosine kinases (1, 6-8). Furthermore, cortactin is implicated in signaling mediated by multiple extracellular stimuli including fibroblast growth factor (3), epidermal growth factor (9), thrombin (10), integrin (11), bacteria-mediated cell invasion (12), and mechanical strain (13). While tyrosine phosphorylation of cortactin is a profound phenomenon in response to many extracellular stimuli, the biological function of cortactin and the physiological role of tyrosine phosphorylation are not clear.
In an attempt to elucidate the function of cortactin, we have examined biochemical properties of cortactin. The study presented here demonstrated that cortactin is a potent F-actin cross-linking protein. Most importantly, the F-actin cross-linking activity is down-regulated upon phosphorylation mediated by pp60c-src. Thus, cortactin may act as an important mediator for intracellular tyrosine kinases in regulating the cytoskeleton reorganization in vivo.
Murine cortactin was expressed in Escherichia coli as a glutathione S-transferase fusion protein in pGEX-2T plasmid and purified by affinity chromatography using glutathione-Sepharose (Pharmacia Biotech Inc.) as described previously (15). The glutathione S-transferase part of the fusion protein was removed by cleavage with bovine thrombin (ICN) in a digestion buffer (50 mM Tris-HCl, pH 8.2, containing 100 mM NaCl and 1 mM CaCl2) for 3~4 h at room temperature. The digested materials were loaded onto a DEAE-Sepharose FF (Pharmacia) column and eluted with 200 ml of elution buffer (20 mM Tris-HCl, pH 7.6, containing 1 mM MgCl2, 1 mM dithiothreitol, and 1 mM EGTA, and KCl with a gradient concentration from 20 to 600 mM). The fractions containing cortactin were pooled, and undigested fusion proteins were removed by additional chromatography using glutathione-Sepharose. The concentration of purified cortactin was determined by the Dc protein assay (Bio-Rad) according to the manufacturer's instructions.
Preparation of ActinActin was purified from an acetone powder of rabbit skeletal muscle according to Pardee and Spudich (16). Pyrene-labeled actin was prepared as described by Kouyama and Mihashi (17). The labeled actin was further purified by chromatography using Sephadex G-150 (Pharmacia). Globular actin was polymerized into filaments by adding KCl, MgCl2, and ATP to the final concentrations of 134, 1, and 1 mM, respectively, and incubated for at least 4 h at room temperature.
Phosphorylation of Cortactin by pp60c-srcRecombinant human
pp60c-src (18) was preactivated and maintained
in a buffer containing 40 µM ATP, 0.8 mM
MgCl2, and 1 mg/ml bovine serum albumin at 0 °C. To
prepare tyrosine-phosphorylated cortactin, purified proteins were
incubated with various amounts of preactivated
pp60c-src in 20 µl of kinase buffer (50 mM Tris-HCl, pH 7.4, containing 5 mM
MgCl2, 5 mM ATP, and 2 µCi of
[-32P]ATP, 6000 ci/mmol) at room temperature for
1 h. For F-actin cross-linking analysis, phosphorylated cortactin
proteins were diluted to a final volume of 50 µl of which the final
concentrations of KCl and MgCl2 were readjusted to 134 and
2 mM, respectively. To confirm and quantitate
phosphorylated cortactin, aliquots of the reactions were combined with
an equal volume of 2 × SDS sample buffer (19) and fractionated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained
with Coomassie Blue, and the phosphorylated proteins were visualized by
autoradiography.
Purified cortactin at the indicated concentrations in 50 µl of TKM buffer (50 mM Tris-HCl, pH 7.4, containing 134 mM KCl and 1 mM MgCl2) was mixed with an equal volume of 8 µM pyrene-labeled F-actin and incubated for 30 min at room temperature. The mixture was then immediately centrifuged at 26,300 × g for 10 min at room temperature in a Beckman TL-100 centrifuge. The supernatant was carefully transferred to a new tube and mixed with 300 µl of TKM buffer containing 2 µM phalloidin (Sigma). The fluorescence in the supernatant was recorded on an LS50B luminescence spectrometer (Perkin-Elmer) at an excitation wavelength of 370 nm with a slit of 2.5 nm and an emission wavelength of 410 nm with a slit 6 nm, respectively. The decrease in the supernatant fluorescence reflects the precipitation of F-actin due to cross-linking. In some experiments, the precipitated pellets were directly analyzed by SDS-PAGE.
Electron Microscopic AnalysisF-actin or the mixture of F-actin and cortactin was mixed with an equal volume of 1% phosphotungstic acid in 0.1 M phosphate buffer, pH 7.4. The stained samples (10 µl) were absorbed to a 0.25% Formvar 15/95E resin- (Sigma) coated film on a gold grid. The grid was then air-dried overnight. Transmission electron micrographs were taken with a Philips M12 microscope.
In an effort to investigate the function of cortactin, the murine
cortactin was expressed in E. coli as a glutathione
S-transferase fusion protein. The glutathione
S-transferase-free cortactin was used for the evaluation of
its F-actin cross-linking activity by virtue of a co-sedimentation
assay (20). To determine optimal conditions for the assay, cortactin
was incubated with F-actin for 30 min and subjected to centrifugation
at different forces from 13,000 to 30,000 × g for 10 min. Under these conditions, neither filaments in the absence of
cortactin (Fig. 1A) nor cortactin alone could
be precipitated (data not shown). However, the presence of both
cortactin and F-actin resulted in a dramatic increase in the amount of
F-actin associated with pellets in a dose-dependent manner
(Fig. 1B). The half-maximum effect requires an approximately 1:100 molecular ratio of cortactin to actin, and the maximum
sedimentation (80% of total F-actin) can be reached in the presence of
a 1:36 ratio of cortactin to actin. The sedimentation of F-actin
induced by cortactin is also correlated with the association of
cortactin with F-actin, as shown by their co-sedimentation at a ratio
of 1 cortactin molecule to approximately 15 actin subunits (Fig. 1C), which is in agreement with the stoichiometry for the
binding of cortactin to actin (14). The two bands of 85 and 90 kDa
shown on the gel most likely reflect different conformations of
cortactin, since only one band of 85 kDa was visualized when SDS-PAGE
was performed in the presence of 5 M urea (data not shown).
A kinetic study further demonstrated that cortactin-mediated F-actin
precipitation is a rapid process (Fig. 1D). Incubation with
cortactin for 2 min resulted in a sedimentation of more than 60% of
F-actin, although the maximum sedimentation (80% of total F-actin)
only occurred at 30 min after interaction. The cross-linked F-actin in
the presence of cortactin was further examined by electron microscopy
after negative staining. Filaments prepared in the absence of cortactin displayed individual single strands (Fig. 2,
A and C). In the presence of cortactin, however,
most F-actin strands became thicker and formed a bundle-like structure
(Fig. 2, B and D).
Since activities of many F-actin cross-linking proteins are dependent
on Ca2+ and pH, we examined the effects of Ca2+
and pH on the activity of cortactin. In the presence of either EGTA or
various concentrations of Ca2+, the F-actin cross-linking
activity of cortactin was not affected (data not shown). However, when
the cross-linking assay was performed at different pH values, the
optimal sedimentation of F-actin was observed in a range from pH 7.3 to
7.5. The F-actin sedimentation induced by cortactin at pH 6.9 and 8.2 is only approximately 30% of that at pH 7.4 (Fig. 3).
The apparent effect of pH on the F-actin cross-linking is not due to
its potential effect on actin polymerization, since pH has little
influence on the stable polymerization of actin (Fig. 3).
The purified cortactin can be efficiently phosphorylated by
pp60c-src exclusively at tyrosine residues in a
manner depending on time and the amount of Src. By incubating cortactin
with 500 nM pp60c-src at room
temperature for 1 h, a maximum phosphorylation of cortactin was
reached (Fig. 4A). When the phosphorylated
cortactin was used in the co-sedimentation assay, a dramatic inhibition
for the F-actin cross-linking was observed (Fig. 4B). The
treatment of cortactin with 62.5 nM
pp60c-src reduced the efficiency of F-actin
sedimentation from near 60% to approximately 38%, and that with 500 nM pp60c-src reduced further the
sedimentation of F-actin to 10%. In a control experiment where
cortactin was treated with the buffer only but in the absence of
pp60c-src, no reduction of the F-actin
cross-linking was detected (Fig. 4B).
The apparent decrease in the F-actin cross-linking was not due to a
possible inhibitory activity of pp60c-src, since
a solution in the presence of pp60c-src itself
did not have any detectable effect on F-actin cross-linking (data not
shown). Furthermore, we examined whether tyrosine phosphorylation is
essential for the inhibition of cortactin's F-actin cross-linking activity. We carried out the phosphorylation of cortactin in a kinase
buffer in the absence of Mg2+, on which the kinase activity
of pp60c-src is dependent (21). As shown in Fig.
5A, the Src-mediated tyrosine phosphorylation
of cortactin was abolished in the absence of Mg2+. When the
Src-treated cortactin in the absence of Mg2+ was mixed with
F-actin and subsequently subjected to the co-sedimentation analysis, a
significant amount of F-actin was detected in the pellet (Fig.
5B, column 4). Although the level of
sedimentation of F-actin induced by cortactin and
pp60c-src in the absence of Mg2+ was
about 33% lower than that by non-Src-treated cortactin (Fig. 5B, compare columns 2 and 4), the
lower efficiency could be the result of a trace amount of
Mg2+ present in the F-actin buffer, which may have
partially restored the kinase activity of
pp60c-src. Indeed, when Src-treated cortactin in
the absence of Mg2+ was further incubated with F-actin,
cortactin was able to be phosphorylated to the extent of approximately
25% of that in the regular kinase buffer (Fig. 5A,
column 4). Taken together, these data demonstrate that the
inhibition of the F-actin cross-linking activity of cortactin by
pp60c-src is dependent on tyrosine
phosphorylation.
Cortactin has been previously described as a potent F-actin-binding
protein (14). Therefore, we also examined the effect of
pp60c-src on its F-actin binding activity by
co-sedimentation at a high centrifugation force of 366,000 × g, at which actin filaments are able to be precipitated. As
shown in Fig. 6, the Src treatment resulted in an
inhibition of the F-actin binding activity of cortactin. However, the
efficiency of the inhibition is apparently less than that for the
F-actin cross-linking. At the concentration of 62.5 nM
pp60c-src, the co-precipitated cortactin with
F-actin was reduced only from 95 to 85%; at 500 nM
pp60c-src, 42% of cortactin was still bound to
F-actin. The moderate inhibition by pp60c-src
may be due to the existence of multiple F-actin binding sites in
cortactin, which are involved in the F-actin cross-linking activity
(30). The lower sensitivity to pp60c-src could
also be the reason for a failure to observe the inhibition of the
F-actin binding activity of cortactin in a system using lysates from
v-Src-transformed cells (14). However, we cannot rule out the
possibility that different phosphorylation sites or additional
kinase(s) may be involved in that system.
The dependence on neutral pH for the optimal cross-linking activity of
cortactin is uncommon. For example, -actinin has the highest
cross-linking activity at pH 6.8 (22). Talin shows a reduced actin
cross-linking activity when pH is increased from 6.5 to 7.3, whereas
its optimal activity is at pH 6.5 (23). EF1
has high
F-actin-cross-linking activity at low pH (6.2-6.5) (24). It has been
well recognized that pH is involved in the regulation of the actin
cytoskeleton (24) and cell motility (25, 26). A recent study also
indicates that the induction of Rho on stress actin filaments is
dependent on Na+/H+ exchange (27). Since both
intracellular pH and tyrosine kinase activities are regulated by a
variety of extracellular signals (28, 29), cortactin could act as an
important mediator for ligands to regulate the cytoskeleton
reorganization.
We thank Nick Greco for critical reading of the manuscript and Diana Norman for expert photographic assistance.