From the Departments of Molecular Pharmacology and
Anatomy and Structural Biology, Albert Einstein College
of Medicine, Bronx, New York
Received for publication, August 2, 2000, and in revised form, January 10, 2001
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ABSTRACT |
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Phosphoinositide (PI) 3-kinases are
required for the acute regulation of the cytoskeleton by growth
factors. We have shown previously that in the MTLn3 rat adenocarcinoma
cells line, the p85/p110 The regulation of cellular motility is important in a variety of
physiological processes, ranging from wound healing to the metastatic
behavior of transformed cells. We have used a metastatic breast cancer
cell model, the MTLn3 cell, to study the role of phosphoinositide
3'-kinases in EGF1-stimulated
motility (1). The acute regulation of the actin cytoskeleton by EGF
requires the p85/p110 Although the mechanism by which PI 3-kinases affect the cytoskeleton
are not clear, several potential pathways have recently emerged. Rho
family GTPases are central in growth factor-mediated actin
reorganization (5), and p85/p110 has been linked to Rac activation in
platelet-derived growth factor-stimulated cells (6). Furthermore, GTP
exchange factors for Rac and CDC42 contain pleckstrin homology
or (Fab1, YGL023, Vps27, EEAI) domains and are likely targets of
3-phosphoinositides (7-9). Potential effectors for Rac/CDC42-mediated
cytoskeletal signaling include (a) the actin-severing
protein cofilin, whose activity is regulated by phosphorylation in a
Rac- and PI 3-kinase-dependent manner (10-13); (b) the N-Wiskott-Aldrich syndrome protein and Wave
proteins, which mediate the CDC42/Rac-dependent binding and
activation of the Arp2/3 complex of actin-nucleating proteins (14-18);
and (c) the small GTPase Arf6, which is activated by GTP
exchange factors that contain pleckstrin homology domains specific for
phosphatidyl-inositol (3,4,5)P3 (19-21).
PI 3-kinase signaling depends on the production of
phosphatidylinositol (3,4,5)P3 at appropriate
locations within the cell. This localized signaling is likely to be due
in part to the binding of p85 SH2 domains to tyrosine-phosphorylated
kinases or substrates. For example, the binding of p85/p110 to
activated platelet-derived growth factor receptors leads to the
internalization of p85/p110 into endocytic vesicles (22). However, in
addition to the SH2 domains located in the C-terminal half of p85
(residues 333-724), p85 contains additional potential targeting
domains in its N-terminal half (residues 1-333). These include an SH3
domain, two proline-rich domains (PRDs), and a domain homologous to the
breakpoint cluster region (BCR) gene product (23). These domains
may target PI 3-kinase to distinct regions of the cell or couple PI
3-kinase to distinct downstream responses. Consistent with this
possibility, the short isoforms of p85 (p55 To assess the roles of the protein-protein interaction domains within
p85-(1-333) during EGF-stimulated cytoskeletal regulation, we
microinjected recombinant p85 domains into intact cells. We find that a
fragment of p85 (residues 82-333), containing just the PRD and BCR
homology domains, inhibits lamellipod extension but not mitogenic
signaling. These data point to a specific role for residues 82-333 of
p85 in signaling to the actin cytoskeleton.
Recombinant Proteins--
GST-N17Rac was purchased from
Cytoskeleton, Inc. (Denver, CO). Recombinant p85 constructs used in
this study are summarized in Fig. 1.
p85 Cell Culture and Microinjection--
Culture conditions and
microinjection protocols for MTLn3 cells have been described previously
(2, 28). BrdUrd incorporation was measured after 12 h of
EGF stimulation as described (2). EGF-stimulated lamellipod extension
in MTLn3 cells was measured as described previously (2), with the
recovery time after microinjection as indicated. Cells were scored for
lamellipod extension, and the number of cells extending lamellipodia
was expressed as a percentage of the number of cells injected with each
construct. All data are the mean ± S.E. from at least three experiments.
JNK Activation--
Cells were injected as indicated and allowed
to recover for 2 h. The cells were incubated in the absence or
presence of 1 M sorbitol for 30 min, fixed with 10%
paraformaldehyde for 30 min at room temperature, and permeabilized with
methanol on dry ice for 10 min. After blocking in 1% bovine serum
albumin/5% donkey serum, the cells were stained with anti-active JNK
antibodies (Promega) followed by Cy3 anti-donkey antisera, to measure
JNK activation, or FITC anti-mouse antibody, to identify injected cells. The data are representative of 2 separate experiments.
Imaging--
Images were acquired using a Nikon Eclipse 400 fluorescence microscope with Nikon CFI Plan Apo 60 × 1.4 numerical aperture optics and a Cohu charge-coupled device camera
linked to a Scion VG5 frame grabber. Figures were assembled using Adobe PhotoShop.
Statistical Methods--
Statistical analysis was performed
using ANOVA and Tukey HSD tests, using software from
Dr. Richard Lowry, Vassar College.
Inhibition of EGF-stimulated Lamellipod Extension by Recombinant
p85--
Overexpression of p85 has been shown previously to inhibit
membrane ruffling in Ras-transformed fibroblasts (29). Although some of
this inhibition could be due to the titration of intracellular phosphotyrosine residues by the p85 SH2 domains (30), the N-terminal half of p85 also contains numerous protein-protein interaction domains
(SH3, proline-rich, and BCR homology domains). Thus, p85 overexpression
could also inhibit ruffling by saturating the intracellular targets of
these domains.
To study the function of these N-terminal domains of p85, we made
GST fusions of wild-type p85 or p85 containing mutations in the
conserved FLRV motifs in both p85 SH2 domains. We have shown
previously that the R358A/R659A mutations abolish phosphopeptide binding by the p85 SH2 domains (26). We reasoned that even in the
absence of functional SH2 domains, these constructs could interfere
with interactions between p85 and intracellular targets and block PI
3-kinase-dependent signaling. Purified GST-p85 or GST-p85(R358A/R659A) were microinjected into quiescent MTLn3 cells. After a 2-h recovery, the cells were stimulated with EGF for 3 min,
fixed, stained, and scored for lamellipod extension. Both constructs
caused a significant inhibition of lamellipod extension; microinjection
of the mutant GST-p85(R358A/R659A) inhibited lamellipod extension by
~52%, relative to control, whereas GST-p85 inhibited lamellipod
extension by 77% (Fig. 2). These data
suggest that SH2-independent interactions between p85 and intracellular
proteins are required for EGF-stimulated lamellipod extension.
p85-p110 binding is extremely stable (31), and the exchange of p110
between different p85 molecules is presumably slow. Nonetheless, we
wanted to rule out the possibility that endogenous p110 was being
sequestered by mutant p85 during the 2-h period after injection. We
therefore repeated the experiments but stimulated the cells with EGF 10 min after microinjection. Once again, GST-p85(R358A/R659A) inhibited
lamellipod extension nearly as well as wild-type p85 (Fig.
2B).
N-terminal Domains of p85 Are Involved in Cytoskeletal but Not
Mitogenic Signaling--
The data in Fig. 2 show that recombinant p85
inhibited lamellipod extension even in the absence of functional SH2
domains. This suggested that the N-terminal domains of p85 might also
be critical for coupling of p85/p110 to the regulation of the actin cytoskeleton. To test this directly, we constructed a truncated GST-p85-(1-333), which lacks the SH2 and iSH2 domains and cannot bind
p110 (Fig. 1). When microinjected into MTLn3 cells, GST-p85-(1-333) inhibited EGF-stimulated lamellipod extension almost as well as full-length GST-p85. Microinjection of GST had no effect on lamellipod extension (59% of GST-injected cells responded to EGF, as compared with 62% of uninjected cells). However, microinjection of GST-p85 and
GST-p85-(1-333) reduced EGF-stimulated lamellipod extension to 25 and
33%, respectively (Fig.
3A).
We next tested whether the N-terminal fragment of p85 could inhibit
other PI 3-kinase-dependent signaling pathways. We have shown previously that EGF-stimulated BrdUrd incorporation in MTLn3 cells is dependent on class IA PI 3-kinases (2). Basal BrdUrd incorporation in serum-starved MTLn3 cells was ~40% and was
unaffected by microinjection of recombinant proteins (data not shown).
In contrast, EGF-stimulated BrdUrd incorporation is almost completely blocked by microinjection of recombinant p85 (Fig. 3B).
However, microinjection of p85-(1-333) had no significant effect on
EGF-stimulated BrdUrd incorporation.
The BrdUrd incorporation assay requires a longer post-injection
incubation (12 h) than is used in the lamellipod extension assays. To
rule out the possibility that the BrdUrd results reflected the
degradation or inactivation of p85 (1), we conducted lamellipod extension experiments using the same protocol as in the BrdUrd assays.
Quiescent cells were microinjected with GST, GST-p85-(1-333), or
GST-nSH2 domains (a positive control for inhibition of lamellipodia) and then incubated for 12 h prior to stimulation with EGF for 3 min (Fig. 3C). Once again, the p85-(1-333) fragment
markedly inhibited lamellipod extension. Thus, our data show that
GST-p85-(1-333), which lacks SH2 domains but contains SH3, PRD, and
BCR homology domains, can selectively interfere with EGF-stimulated
cytoskeletal signaling but not EGF-stimulated DNA synthesis.
We have shown previously that MTLn3 cells injected with inhibitory
antibodies to p110 Deletional Mapping of N-terminal Domains Involved in
Cytoskeletal Signaling--
To determine which regions of
p85-(1-333) were required for lamellipod extension, we prepared
constructs in which we selectively removed the SH3 domain, the nPRD,
the cPRD, both PRDs, and the BCR homology domain. The proteins were
soluble after expression in BL-21 E. coli, and their
electrophoretic mobility on SDS polyacrylamide gel electrophoresis was
consistent with their predicted size (data not shown). The purified
proteins were injected into MTLn3 cells, and the effects on lamellipod
extension were evaluated. GST-p85-(82-333), which lacks the SH3
domain, could still inhibit lamellipod extension (Fig.
5). In contrast, deletion of either the
nPRD or the cPRD, or both PRDs, eliminated the inhibitory effect
on lamellipod extension (Fig. 5). These relatively small deletions of
11-12 amino acids were unlikely to cause significant structural
perturbation, because their deletion from full-length p85 had no effect
on binding to p110 or phosphopeptide activation of p85/p110 dimers
(data not shown). Preliminary results also show that a construct
lacking the BCR domain was unable to inhibit EGF-stimulated lamellipod extension (data not shown). Although we cannot yet state with certainty
that the BCR and PRD domains are necessary for inhibition of lamellipod
extension, our data show that the nPRD-BCR-cPRD fragment is sufficient
for inhibition of lamellipod extension.
p85 Constructs Do Not Inhibit Rac-dependent Signaling
in MTLn3 Cells--
EGF-stimulated lamellipod extension is blocked by
dominant negative Rac,2
consistent with data from other systems (32). The BCR homology domain
of p85 binds activated Rac and CDC42 (33, 34). Because the
nPRD-BCR-cPRD domain is sufficient to block lamellipod extension, we
considered the possibility that p85-(1-333) was acting as a generalized Rac inhibitor, through the sequestration of activated Rac.
We therefore measured activation of the JNK kinase, which is known to
be Rac-dependent (35, 36). MTLn3 cells were treated with
sorbitol for 30 min and then fixed and stained with a phospho-specific anti-JNK antibody. As reported previously in other systems (37), sorbitol treatment lead to the activation of JNK, which could be
detected in the nucleus of MTLn3 cells (Fig.
6, A and B). The nuclear accumulation of active JNK was unaffected by microinjection of
GST-p85-(82-333) (Fig. 6B, arrows),
whereas it was completely blocked by microinjection of N17-Rac (data
not shown). Thus, the Rac-dependent activation of JNK was
not inhibited by GST-p85-(82-333), suggesting that the affects of this
construct on EGF-stimulated lamellipod extension were not because of a
general sequestration of endogenous activated Rac.
In this paper, we have defined a subset of PI
3-kinase-dependent responses that are inhibited by
microinjection of N-terminal domains of p85. The p85-(1-333) and
p85-(82-333) constructs do not contain the SH2 or iSH2 domains and
therefore should not disrupt endogenous p85/p110 binding or activation
of endogenous p85/p110 by phosphotyrosine-containing proteins. We
therefore presume that these constructs interfere with the targeting of
endogenous p85/p110 molecules to sites involved in cytoskeletal
regulation. Disruption of this targeting apparently does not interfere
with the activation of mitogenic signaling pathways, which may be less
spatially organized. Consistent with this latter idea, we have
demonstrated previously an increase in DNA synthesis by anti-p85
antibodies that activate p85/p110 dimers but that would be unlikely to
cause the targeting of p85/p110 to specific intracellular sites
(28).
Although the nPRD-BCR-cPRD fragment of p85 is sufficient to inhibit
lamellipod extension, the intracellular targets of these domains of p85
are not yet known. Although the BCR homology domain binds to Rac/CDC42
(33, 34), microinjected p85 fragments do not block JNK activation and
are therefore not acting as a global Rac inhibitor. However, the
p85-derived constructs could disrupt the targeting of a subset of
activated Rac to specific regions of the cell.
In addition to Rac and CDC42, a number of cytoskeletal regulatory
proteins interact with p85. Cas and focal adhesion kinase bind
to the SH2 domains of p85 (38, 39) and should not therefore be affected
by p85-(82-333). Similarly, cbl binds to the SH3 domain of p85 and
should be unaffected by p85-(82-333) (40). Tyrosine-phosphorylated ezrin also binds, to the p85 SH2 domains, but additional binding occurs
through the N terminus of ezrin to an unknown region of p85. The GTP
exchange factor Pak interacting exchange factor and the
actin-binding protein profilin also bind to p85 at unknown sites and
could be affected by microinjection of p85-(82-333) (41, 42). Finally,
p85-(82-333) could act by binding to SH3 domains in Src and related
kinases (43, 44), thereby disrupting their interactions with endogenous p85.
In summary, we have demonstrated that different regions of the p85
regulatory subunit are involved in distinct PI
3-kinase-dependent responses. Whereas the SH2 domains of
p85 have pleiotropic effects on multiple pathways, the BCR and
proline-rich domains of p85 are coupled to cytoskeletal signaling but
not DNA synthesis. These data suggest that different isoforms of class
IA regulatory subunit, particularly the full-length (p85)
versus short forms (p55/p50), are involved in different
subsets of PI 3-kinase-dependent signaling events.
PI 3-kinase is required for epidermal
growth factor (EGF)-stimulated lamellipod extension and
formation of new actin barbed ends at the leading edge of the cell. We
have now examined the role of the p85
regulatory subunit in greater
detail. Microinjection of recombinant p85
into MTLn3 cells blocked
both EGF-stimulated mitogenic signaling and lamellipod extension. In
contrast, a truncated p85(1-333), which lacks the SH2 and iSH2
domains and does not bind p110, had no effect on EGF-stimulated
mitogenesis but still blocked EGF-stimulated lamellipod extension.
Additional deletional analysis showed that the SH3 domain was not
required for inhibition of lamellipod extension, as a construct
containing only the proline-rich and breakpoint cluster region (BCR)
homology domains was sufficient for inhibition. Although the BCR domain
of p85 binds Rac, the effects of the p85 constructs were not because of
a general inhibition of Rac signaling, because sorbitol-induced JNK
activation in MTLn3 cells was not inhibited. These data show that the
proline-rich and BCR homology domains of p85 are involved in the
coupling of p85/p110 PI 3-kinases to regulation of the actin
cytoskeleton. These data provide evidence of a distinct cellular
function for the N-terminal domains of p85.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
isoform of PI 3-kinase. MTLn3 breast cancer
cells express similar levels of p85/p110
and p85/p110
. However,
EGF-stimulated lamellipod extension is blocked by microinjection of
inhibitory antibodies to p110
but not p110
(2). Inhibition of
p110
also blocks the production of new barbed ends at the leading
edge of EGF-stimulated cells (2). Isoform-specific regulation of the
cytoskeleton by class IA PI 3-kinases has also been described in
macrophages and fibroblasts (3, 4). These findings suggest that
different PI 3-kinase isoforms signal differently within the same cell.
, p50
, and p55
) do
not contain the N-terminal domains, and appear to have distinct
biological activities in intact cells (24, 25).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
[R358A/R659A] has been described previously (26). p85
BCR
(deletion of residues 146-299) was provided by Dr. Christopher Rudd,
Harvard University. Deletion of the PRDs from full-length p85 was
accomplished by the method of Kunkel et al. (27) using oligonucleotides that deleted residues 84-96 (nPRD), 303-314
(cPRD), or both. These constructs were then amplified by polymerase
chain reaction using forward primers encompassing bases 1-21 of human p85
and reverse primers encompassing bases 1041-1021 and subcloned into pGEX2T (Amersham Pharmacia Biotech). Finally, the 82-333 fragment was amplified by polymerase chain reaction and subcloned into
pGEX2t. Recombinant proteins were produced in BL-21
Escherichia coli and purified by affinity
chromatography on glutathione-Sepharose (Amersham Pharmacia Biotech).
Proteins were extensively dialyzed against phosphate-buffered saline
and concentrated to 3 mg/ml using Centricon concentrators (Millipore).
Protein purity and final concentration was assayed by SDS
polyacrylamide gel electrophoresis and Coomassie Blue staining. The
proteins were mixed with rabbit or mouse IgG (3 mg/ml final
concentration) prior to injection to facilitate identification of
injected cells.
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Fig. 1.
p85 -derived
constructs used in this study.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 2.
Inhibition of lamellipod extension by p85
does not require functional SH2 domains. A, MTLn3 cells
were incubated without serum for 2 h and then injected with rabbit
IgG, GST-p85, or GST-p85(R358A/R659A) as indicated. After an additional
2 h, cells were stimulated with EGF (5 nM) for 3 min,
fixed, and stained with rhodamine-phalloidin and FITC anti-mouse
antibodies (to identify injected cells). The percentage of injected
cells that extended lamellipodia were counted. Ctl, control.
B, as above, except the cells were incubated without serum
for 4 h, injected for 10 min, and stimulated after an additional
10 min. All values are the mean ± S.E. of four determinations.
ANOVA analysis demonstrated significance at p < 0.0001; statistical significance of differences between individual
means and IgG-injected cells (Tukey HSD test) are indicated.
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Fig. 3.
A truncated p85-(1-333) inhibits lamellipod
extension but not BrdUrd incorporation. A, MTLn3 cells
were incubated without serum for 2 h and then injected with GST,
GST-p85, or GST-p85-(1-333) as indicated. After an additional 2 h
the cells were stimulated for 3 min with 10 nM EGF, and
lamellipod extension was determined as described above. Ctl,
control. B, MTLn3 cells were rendered quiescent in medium
containing 1% fetal bovine serum for 24 h and then injected with
GST, GST-p85, or GST-p85-(1-333) as indicated. The cells were then
incubated in the absence or presence of 2 nM EGF for
12 h, followed by BrdUrd for an additional 2h. The cells were
fixed and stained with anti-BrdUrd antibodies or FITC anti-rabbit
antibodies (to identify injected cells), and the percentage of cells
that incorporated BrdUrd was counted. C, MTLn3 cells were
rendered quiescent in medium containing 1% fetal bovine serum for
24 h and then injected with GST, GST-nSH2 domains, or
GST-p85-(1-333) as indicated. The cells were incubated for 12 h
and then stimulated with 10 nM EGF for 3 min. Lamellipod
extension was determined as described above. All values are the
mean ± S.E. of three to five determinations. ANOVA analysis
demonstrated significance at p < 0.01; statistical
significance of differences between individual means and GST-injected
cells (Tukey HSD test) are indicated.
are highly condensed and stain brightly with
rhodamine-phalloidin (2). Cells injected with full-length GST-p85 had a
similar condensed morphology (Fig.
4C). The effect of
microinjected GST-p85-(1-333) was less pronounced. Although GST-p85-(1-333)-injected cells did not extend lamellipodia in response
to EGF, their morphology was somewhat variable. A few cells resembled
GST-p85-injected cells, but many were similar in morphology to
unstimulated control cells (Fig. 4, D-F). The less
pronounced morphological changes in GST-p85-(1-333)-injected cells, as
well as failure of GST-p85-(1-333) to block EGF-stimulated BrdUrd
incorporation, are consistent with the hypothesis that GST-p85-(1-333)
inhibits a subset of p85/p110-dependent signaling processes.
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Fig. 4.
Morphology of cells injected with GST-p85 or
GST-p85-(1-333). Quiescent MTLn3 cells were not injected
(A) or injected with GST (B), GST-p85
(C), or GST-p85-(1-333) (D, E, and
F). Cells were stimulated with EGF for 3 min
(B-F), fixed, and stained with rhodamine-phalloidin and
FITC anti-rabbit antibodies (to identify injected cells).
Arrows indicate injected cells.
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Fig. 5.
Deletional analysis of p85[1-333].
A, quiescent MTLn3 cells were injected with GST,
GST-p85[82-333], GST-p85 nPRD, GST-p85
cPRD, or
GST-p85
nPRD/cPRD, incubated without or with EGF for 3 min, fixed,
and stained as described for Fig. 2. All values are the mean ± S.E. of three determinations. ANOVA analysis demonstrated significance
at p < 0.001; statistical significance of differences
between individual means and GST-injected cells (Tukey HSD test) are
indicated. Ctl, control.
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Fig. 6.
GST-p85-(82-333) does not inhibit
Rac-dependent signaling. Quiescent MTLn3 cells were
not injected (A) or injected (B) with
GST-p85-(82-333). After 2 h the cells were incubated without
(A) or with (B) 1 M sorbitol for 30 min, fixed, and stained with anti-phospho-JNK antibodies followed by
Cy3 anti-rabbit antibodies and FITC anti-mouse antibodies (to identify
injected cells). Arrows indicated injected cells.
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FOOTNOTES |
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* This work was funded in part by a grant from the American Cancer Society and National Institutes of Health Grant RO1 GM556982 (to J. M. B) and by National Institutes of Health Training Grant 5T32 GM07260 (to K. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Established Scientist of the American Heart Association.
¶ Established Scientist of the American Heart Association and recipient of the Hirschl Scholar Award. To whom correspondence should be addressed: Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2153; Fax: 718-430-3749; E-mail: Backer@aecom.yu.edu.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M006985200
2 M. Bailly and J. E. Segall, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; PI, phosphoinositide; PRD(s), proline-rich domain(s); BCR, breakpoint cluster region; GST, glutathione S-transferase; BrdUrd, deoxybromouridine; JNK, c-Jun N-terminal kinase; FITC, fluorescein isothiocyanate; ANOVA, analysis of variance nPRD, N-terminal PRD; cPRD, C-terminal PRD.
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