Stimulation of M3 Muscarinic Receptors Induces
Phosphorylation of the Cdc42 Effector Activated Cdc42Hs-associated
Kinase-1 via a Fyn Tyrosine Kinase Signaling Pathway*
Daniel A.
Linseman
§,
Kim A.
Heidenreich¶, and
Stephen K.
Fisher
From the
Department of Pharmacology and Neuroscience
Laboratory, Mental Health Research Institute, University of Michigan,
Ann Arbor, Michigan 48104-1687 and the ¶ Department of
Pharmacology, University of Colorado Health Sciences Center, and
the Denver Veterans Affairs Medical Center,
Denver, Colorado 80220
Received for publication, July 28, 2000, and in revised form, October 17, 2000
 |
ABSTRACT |
The tyrosine kinase, activated
Cdc42Hs-associated kinase-1 (ACK-1), is a specific effector of the Rho
family GTPase Cdc42. GTP-bound Cdc42 has been shown to facilitate
neurite outgrowth elicited by activation of muscarinic cholinergic
receptors (mAChRs). Because tyrosine kinase activity is a requirement
for neuritogenesis in several cell systems, we investigated whether
endogenous mAChRs (principally of the M3 subtype)
expressed in human SH-SY5Y neuroblastoma cells would signal to ACK-1.
Incubation of cells with the cholinergic agonist oxotremorine-M (Oxo-M)
induced an approximately 6-fold increase in the tyrosine
phosphorylation of ACK-1 which was inhibited by atropine. ACK-1
phosphorylation was blocked by Clostridium difficile toxin
B, an inhibitor of Rho family GTPases. In contrast, disruption of the
actin cytoskeleton with cytochalasin D stimulated ACK-1
phosphorylation, and moreover, addition of Oxo-M to cells preincubated
with this agent elicited a further increase in phosphorylation, indicating that an intact cytoskeleton is not required for mAChR signaling to ACK-1. Although stimulation of M3 mAChRs
induces both an increase in intracellular Ca2+ and
activation of protein kinase C (PKC), neither of these second messenger
pathways was required for receptor-stimulated ACK-1 phosphorylation.
Instead, inhibition of PKC resulted in a 2-fold increase in
Oxo-M-stimulated ACK-1 phosphorylation, whereas acute activation of PKC
with phorbol ester decreased ACK-1 phosphorylation. The agonist-induced
tyrosine phosphorylation of ACK-1 was blocked by inhibitors of Src
family kinases, and ACK-1 was coprecipitated with Fyn (but not Src) in
an agonist-dependent manner. Finally, scrape loading cells
with glutathione S-transferase fusion proteins of either
the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated
ACK-1 tyrosine phosphorylation. The data are the first to show
phosphorylation of ACK-1 after stimulation of a receptor coupled to
neurite outgrowth and indicate that a Rho family GTPase
(i.e. Cdc42) and Fyn are essential upstream elements of
this signaling pathway.
 |
INTRODUCTION |
Synaptic plasticity plays a critical role in the development of
the central nervous system and in mediating the processes of learning
and memory. The phenomena of long term potentiation and long term
depression are considered in vitro models of the latter (1).
Previous studies have demonstrated a requirement for tyrosine kinase
activity in mediating both long term potentiation in the hippocampus
and long term depression in the cerebellum (2, 3). Furthermore,
tyrosine kinase activity has also been shown to play a prominent role
in facilitating neurite outgrowth in several in vitro models
(4, 5). Currently, ~100 protein tyrosine kinases have been identified
(6). These enzymes can be categorized into two distinct classes,
receptors with intrinsic tyrosine kinase activity and nonreceptor
tyrosine kinases. Examples of receptor tyrosine kinases include
classical growth factor receptors such as the receptor for nerve growth
factor (7). The nonreceptor tyrosine kinases consist of approximately
10 families including two related families, the activated
Cdc42Hs-associated kinase (ACK)1family and the focal
adhesion kinase (FAK) family (8). Nonreceptor tyrosine kinases of the
ACK and FAK families are highly expressed in the brain (9-12) and have
been postulated to regulate synaptic changes in the central nervous
system (13, 14).
ACK-1 is one of two known members of the ACK family and was originally
cloned from a human hippocampal cDNA library (9). ACK-1 is a
specific downstream effector of the Rho family small molecular weight
GTPase Cdc42. In contrast to many effectors that also act as
GTPase-activating proteins for their partner G proteins, the
association of ACK-1 with GTP-bound Cdc42 has been shown to inhibit both the intrinsic and GTPase-activating
protein-stimulated GTPase activity of Cdc42, suggesting that
this interaction may sustain Cdc42 in a GTP-bound (active) state (9).
Active Cdc42 promotes the formation of filopodial extensions and actin
microspikes (15, 16). These changes in actin cytoskeletal structure are mediated via activation of other Cdc42 effectors, such as the p21-activated kinases (17, 18) and the Wiskott-Aldrich syndrome proteins (19, 20). In neuronal cells, Rho family GTPases regulate changes in growth cone morphology (21, 22). In particular, Rac and
Cdc42 facilitate neurite outgrowth, whereas Rho induces neurite
retraction and growth cone collapse (23-25). Although ACK family
proteins have been postulated to play a role in growth cone remodeling
(14), the activation of this class of nonreceptor tyrosine kinase has
not yet been investigated in a neuronal cell model.
Cholinergic transmission is a critical element involved in the
maintenance of cognitive functions in the central nervous system such
as learning and memory (26). For example, deficits in muscarinic cholinergic receptor (mAChR) signaling contribute to the
pathophysiology of Alzheimer's disease and other types of age-related
dementia (27-29). The SH-SY5Y cell line is a human neuroblastoma
derived as a subclone from the parental SK-N-SH line (30, 31). SH-SY5Y cells express a high density of mAChRs, principally of the
M3 subtype (32, 33), which are coupled to the phospholipase
C-dependent production of inositol 1,4,5-trisphosphate and
sn-1,2-diacylglycerol (34, 35). These second messenger
molecules lead to an increase in intracellular Ca2+ and
activation of protein kinase C (PKC), respectively. Stimulation of
mAChRs on SH-SY5Y cells has been shown to elicit morphological changes
characteristic of neurite outgrowth (36, 37) and induces a marked
increase in the tyrosine phosphorylation of the nonreceptor tyrosine
kinase FAK (38). Given that Cdc42 has been shown to regulate
mAChR-mediated growth cone remodeling (23), we investigated whether
mAChR stimulation in SH-SY5Y cells couples to activation of the Cdc42
effector ACK-1.
The results demonstrate that agonist occupancy of mAChRs on SH-SY5Y
neuroblastoma cells elicits an enhanced tyrosine phosphorylation of
ACK-1 which is dependent on Cdc42 function, Fyn tyrosine kinase activity, and an interaction between ACK-1 and Fyn. The last may require both the Fyn-SH2 and Fyn-SH3 domains. The results are the first
to show a receptor-mediated activation of ACK-1 in a neuronal cell
system and support a role for this nonreceptor tyrosine kinase in
signaling during growth cone remodeling.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Phorbol 12-myristate 13-acetate (PMA),
bisindolylmaleimide I (GF 109203X), PP1, PP2, PP3, thapsigargin, and
cytochalasin D were obtained from Calbiochem. Atropine and
mecamylamine were from Sigma. Oxotremorine-M (Oxo-M) was purchased from
Research Biochemicals International (Natick, MA). Clostridium
difficile toxin B was generously provided by Dr. Klaus Aktories
and Dr. Fred Hofmann (Albert-Ludwigs-Universität, Freiburg,
Germany). All other chemicals were of reagent grade.
32Pi (10 mCi/ml), reagents for enhanced
chemiluminescence, and peroxidase-conjugated sheep anti-mouse IgG were
purchased from Amersham Pharmacia Biotech. Monoclonal antibodies to
phosphotyrosine Tyr(P) and myristoylated alanine-rich protein
kinase C substrate (MARCKS) were obtained from Upstate Biotechnology
Inc. (Lake Placid, NY). Polyclonal antibodies to FAK and ACK-1,
blocking peptide for ACK-1, monoclonal antibodies to Src, Fyn, and
glutathione S-transferase (GST), agarose-conjugated protein
A/G, and purified GST, GST-Fyn-SH2, and GST-Fyn-SH3 were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Tissue culture supplies were
purchased from Corning Glass Works (Corning, NY) and Sarstedt (Newton,
NC). Dulbecco's modified Eagle's medium was from Life Technologies,
Inc. Fetal calf serum was from Summit Biotechnology (Ft. Collins, CO).
Human SH-SY5Y neuroblastoma cells were obtained from Dr. June Biedler
(Sloan-Kettering Institute, New York).
Cell Culture Conditions--
Human SH-SY5Y neuroblastoma cells
(passages 68-78) were routinely grown in 75-cm2 tissue
culture flasks containing 20 ml of Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum. Cells were grown for 7-14
days at 37 °C in a humidified atmosphere consisting of 90% air and
10% CO2. Cells were subcultured into 35-mm-diameter, six-well culture plates (Becton Dickinson Labware, Lincoln Park, NJ)
for 2-5 days before treatment. All experiments were performed on cells
that had reached confluence.
Incubation Conditions and Immunoprecipitations--
After
removal of the plating medium, confluent cultures of SH-SY5Y cells were
washed once with 2 ml of prewarmed (37 °C) treatment buffer
consisting of 138 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM Na2HPO4, 5 mM
NaHCO3, 5.5 mM glucose, and 20 mM
HEPES (pH 7.4) and allowed to equilibrate at 37 °C in 1 ml of
treatment buffer for 20 min. During the equilibration period cells were preincubated with inhibitors as described in this paper. In some experiments, cells were preincubated for 24 h in culture medium containing toxin B prior to agonist exposure, as described previously (37). Cells were then incubated for the specified duration at 37 °C
with either buffer or 1 mM Oxo-M. After incubation, the buffer was aspirated, and the cells were rinsed once with 2 ml of
ice-cold phosphate-buffered saline (PBS; pH 7.4). Cells were then
incubated on ice and scraped into lysis buffer (200 µl/well) containing 20 mM HEPES (pH 7.4), 1% Triton X-100, 50 mM NaCl, 1 mM EGTA, 5 mM
-glycerophosphate, 30 mM sodium pyrophosphate, 100 µM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin. Cell debris was removed by centrifugation at 6,000 × g for 3 min, and the protein concentration of the
supernatant was determined using a commercially available protein assay
kit (Pierce Chemical Co.). Aliquots (~350 µg) of supernatant
protein were diluted to a final volume of 500 µl with lysis buffer
and transferred to tubes containing either 2 µg of polyclonal
anti-ACK-1 or 2 µg of polyclonal anti-FAK. In some experiments,
varying amounts of the ACK-1 blocking peptide were included during
immunoprecipitation. Samples were then incubated with mixing for 16-20
h at 4 °C. 25 µl of agarose-conjugated protein A/G was added for
an additional 4 h with mixing. Immune complexes were pelleted by
centrifugation and washed three times with ice-cold lysis buffer. The
final agarose pellet was resuspended in an equal volume of 2 × SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, boiled for
5 min, and electrophoresed through 7.5% polyacrylamide gels. Proteins
were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore
Corp., Bedford, MA) and processed for immunoblot analysis.
Coprecipitation of Src Family Kinases and ACK-1--
Cells were
incubated for 5 min with either buffer or Oxo-M and lysed as described
above. Lysates were then immunoprecipitated with 2 µg of either
polyclonal anti-ACK-1 or monoclonal anti-Src or anti-Fyn. Immune
complexes were resolved by SDS-PAGE, transferred to PVDF, and processed
for immunoblot analysis.
Immunoblot Analysis--
Nonspecific binding sites were blocked
in PBS (pH 7.4) containing 0.1% Tween 20 (PBS-T) and 1% bovine serum
albumin for 1 h at room temperature. Primary monoclonal antibodies
were diluted in blocking solution (final concentration of 0.5-1.0
µg/ml) and incubated with the membranes for 1 h. Excess primary
antibody was removed by washing the membranes three times in PBS-T. The blots were then incubated in peroxidase-conjugated, anti-mouse secondary antibody diluted in PBS-T (1:10,000) for 1 h and
subsequently washed three times in PBS-T. Immunoreactive proteins were
detected by enhanced chemiluminescence. In some experiments, membranes were reprobed after stripping in 0.1 M Tris-HCl (pH 8.0),
2% SDS, and 100 mM
-mercaptoethanol for 30 min at
52 °C. The blots were rinsed twice in PBS-T and processed as above
with a different primary antibody. Autoluminograms shown are
representative of two to four independent experiments. Quantitative
analysis of autoluminograms was performed by computer-assisted imaging
densitometry (MCID; Imaging Research, St. Catharines, Ontario, Canada).
32P Labeling of ACK-1 and MARCKS--
Cells were
incubated to isotopic equilibrium at 37 °C in 1 ml of phosphate-free
treatment buffer containing 1 mCi/ml 32Pi for
4 h. The labeling solution was aspirated, and unincorporated 32Pi was removed by washing three times with 2 ml of the same buffer. The cells were then incubated with either
vehicle, Oxo-M or PMA, as described in the legend to Fig. 4, and
either ACK-1 or MARCKS was immunoprecipitated from cell lysates. Immune
complexes were resolved by SDS-PAGE and transferred to PVDF membranes.
32P-Labeled proteins were visualized by PhosphorImager
analysis (Cyclone Storage Phosphor System; Packard Instrument Company, Meriden, CT). Membranes containing ACK-1 immunoprecipitates were subsequently immunoblotted for Tyr(P) as described above.
Scrape Loading with GST Fusion Proteins--
Either GST alone or
GST fusion proteins (Fyn-SH2 or Fyn-SH3) were scrape loaded into cells
at a concentration of 20 µg/ml in scrape-loading buffer containing 10 mM Tris-HCl (pH 7.0), 114 mM KCl, 15 mM NaCl, and 5.5 mM MgCl2 (39).
Scrape-loaded cells were incubated for 30 min in the presence of the
fusion proteins, and then the cells were pelleted, resuspended in
serum-free medium, and seeded into 35-mm culture dishes. After further
incubation at 37 °C in 10% CO2 for 3 h, the cells
had reattached and were incubated with either buffer or Oxo-M for 5 min. ACK-1 immune complexes were isolated and probed for Tyr(P). The
post-ACK-1 immunoprecipitate lysates were also resolved by SDS-PAGE and
were probed for GST to estimate the efficiency of scrape loading.
Data Analysis--
Results shown represent the means ± S.E. for the number (n) of independent experiments
performed. Statistical differences between the means of unpaired sets
of data were evaluated using Student's two-tailed t tests.
 |
RESULTS |
Agonist Occupancy of mAChRs Enhances the Tyrosine Phosphorylation
of ACK-1--
Incubation of SH-SY5Y neuroblastoma cells with the
cholinergic agonist Oxo-M elicited a marked
increase in the tyrosine phosphorylation of ACK-1 (6.2 ± 0.7-fold increase over basal, n = 6, p < 0.001, Fig. 1A, first
lane versus second lane). Immunoprecipitation of ACK-1
was competitively antagonized by coincubation with a blocking peptide
that mimicked the antigenic site on ACK-1 (Fig. 1A).
Agonist-stimulated ACK-1 phosphorylation was blocked by preincubation
with the muscarinic selective antagonist, atropine, but was unaffected
by the nicotinic selective antagonist, mecamylamine (Fig.
1B). The increased tyrosine phosphorylation of ACK-1
elicited by Oxo-M was time- and dose-dependent. The
agonist-stimulated phosphorylation of ACK-1 was rapid and persistent,
increasing within 1 min of agonist exposure, peaking at 5-10 min, and
remaining elevated above the basal level for at least 1 h (Fig.
1C). Furthermore, Oxo-M induced the tyrosine phosphorylation
of ACK-1 with an EC50 of ~100 µM (Fig.
1D, upper panel). The addition of agonist had no
effect on the immunoprecipitation efficiency of ACK-1 (Fig.
1D, lower panel).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 1.
Stimulation of mAChRs on SH-SY5Y
neuroblastoma cells enhances the tyrosine phosphorylation of
ACK-1. A, confluent cultures of SH-SY5Y cells were
incubated for 5 min with either buffer alone or 1 mM Oxo-M.
Triton X-100-soluble extracts were immunoprecipitated (IP)
with 2 µg of polyclonal anti-ACK-1 in either the absence or presence
of varying concentrations of ACK-1-blocking peptide. Immune complexes
were resolved by SDS-PAGE, transferred to PVDF, and immunoblotted
(IB) for Tyr(P) (PTyr), as described under
"Experimental Procedures." B, SH-SY5Y cells were
preincubated for 10 min with either 10 µM atropine
(ATR) or 10 µM mecamylamine (MEC)
prior to incubation with 1 mM Oxo-M for a further 5 min.
ACK-1 immune complexes were probed for Tyr(P). C, SH-SY5Y
cells were incubated with 1 mM Oxo-M for varying times, and
ACK-1 immune complexes were probed for Tyr(P). D, SH-SY5Y
cells were incubated with varying concentrations of Oxo-M for 5 min,
and ACK-1 immune complexes were blotted for Tyr(P) (upper
panel) or ACK-1 (lower panel).
|
|
mAChR-stimulated ACK-1 Tyrosine Phosphorylation Is Dependent on Rho
Family GTPase Activity but Not on an Intact Actin
Cytoskeleton--
ACK-1 has been shown to interact specifically with
the Rho family GTPase Cdc42 (9). To determine whether the mAChR-induced increase in ACK-1 tyrosine phosphorylation was dependent on Cdc42 activity, cells were preincubated with C. difficile toxin B. Toxin B is a monoglucosyltransferase that selectively inhibits the
function of Rho family GTPases (40). After 24 h of incubation with
toxin B, the subsequent C3 exoenzyme-catalyzed ADP-ribosylation of Rho proteins in SH-SY5Y cell lysates is significantly decreased, a result
indicating that toxin B efficiently enters intact SH-SY5Y cells and
inhibits Rho family GTPases in these cells (37). Moreover, SH-SY5Y
cells become rounded, and a marked disruption of the actin cytoskeleton
is observed after incubation with toxin B (37). The latter morphology
is characteristic of cells in which Rho family GTPase function has been
compromised (40). Preincubation with toxin B essentially abolished the
agonist-induced tyrosine phosphorylation of ACK-1 (Fig.
2A), a result consistent with
a requirement for Cdc42 function in signaling from the M3
mAChR to ACK-1.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 2.
Differential effects of C. difficile toxin B and cytochalasin D on the tyrosine
phosphorylation of ACK-1. A, confluent cultures of
SH-SY5Y cells were preincubated for 24 h with medium containing
either vehicle (0.1% bovine serum albumin in PBS) or 200 pg/ml toxin
B. Cells were then incubated with either buffer alone or 1 mM Oxo-M for 5 min. ACK-1 immune complexes were resolved by
SDS-PAGE, transferred to PVDF, and probed for Tyr(P) (PTyr),
as described under "Experimental Procedures." B, SH-SY5Y
cells were preincubated for 30 min with either vehicle (0.2% ethanol)
or 1 µM cytochalasin D (Cyto D). Cells were
then incubated for a further 5 min with either buffer alone or
containing 1 mM Oxo-M. ACK-1 (upper panel) or
FAK (lower panel) immune complexes were immunoblotted for
Tyr(P). C, SH-SY5Y cells were preincubated for 24 h
with medium containing either vehicle or 200 pg/ml toxin B. Cells were
then incubated for a further 30 min with either vehicle or 1 µM Cyto D. ACK-1 immune complexes were isolated and
probed for Tyr(P). IP, immunoprecipitated; IB,
immunoblotted.
|
|
The ACK family of nonreceptor tyrosine kinases (9, 12) shows some
homology to the FAK family, which includes two principal members, FAK
and PYK-2 (11, 41). Stimulation of an enhanced tyrosine
phosphorylation status of either FAK or PYK-2 requires an intact actin
cytoskeleton (39, 42). Therefore, we analyzed the effects of the actin
cytoskeleton-destabilizing agent cytochalasin D on mAChR-stimulated
ACK-1 tyrosine phosphorylation. After a 30-min preincubation with
cytochalasin D, SH-SY5Y cells displayed a significant disruption of
actin stress fibers (results not shown). As documented previously (38),
cytochalasin D pretreatment suppressed the basal tyrosine
phosphorylation of FAK and completely inhibited the mAChR-induced
increase in FAK phosphorylation (Fig. 2B, lower panel). In contrast, the basal tyrosine phosphorylation of ACK-1 was increased dramatically after incubation with cytochalasin D, and
the agonist-induced increase in ACK-1 phosphorylation was sustained
after disruption of the cytoskeleton (Fig. 2B, upper panel).
Interestingly, the ability of cytochalasin D to enhance the tyrosine
phosphorylation of ACK-1 was blunted significantly by preincubation
with toxin B (Fig. 2C). Thus, an intact actin cytoskeleton
is not required for mAChR signaling to ACK-1, and moreover, disruption
of the actin cytoskeleton promotes ACK-1 phosphorylation via a
mechanism involving Rho family GTPase activity.
Identification of a PKC-dependent Negative Feedback
Loop That Limits mAChR Signaling to ACK-1--
Agonist occupancy of
M3 mAChRs leads to the phospholipase
C-dependent generation of inositol 1,4,5-trisphosphate and
sn-1,2-diacylglycerol which, in turn, increase intracellular
Ca2+ and activate PKC, respectively. Given that increases
in intracellular Ca2+ and/or PKC activity can elicit an
increased tyrosine phosphorylation of either PYK-2 or FAK (42, 43), the
potential involvement of these second messengers in mAChR signaling to
ACK-1 was investigated. First, the effects of depletion of
intracellular Ca2+ stores on agonist-induced ACK-1
phosphorylation were assessed. As described previously (44), a 30-min
preincubation of SH-SY5Y cells with 1 µM thapsigargin
completely abolished the inositol 1,4,5-trisphosphate-mediated increase
in intracellular Ca2+ observed after mAChR stimulation.
However, thapsigargin pretreatment did not have a statistically
significant effect on agonist-induced ACK-1 phosphorylation (690 ± 296% increase in control versus 426 ± 194%
increase in thapsigargin-treated, n = 4, p = 0.48, data not shown). This result suggests that
agonist-induced Ca2+ release does not play a significant
role in mAChR signaling to ACK-1.
Next, we evaluated the effects of agents that modulate PKC activity on
mAChR-stimulated ACK-1 tyrosine phosphorylation. Preincubation with the
PKC inhibitor bisindolylmaleimide I, resulted in a statistically significant 2-fold increase in the mAChR-induced tyrosine
phosphorylation of ACK-1 (Fig. 3,
A and B). In
contrast, activation of PKC, via an acute preincubation with phorbol
ester, significantly inhibited agonist-stimulated ACK-1 tyrosine
phosphorylation by ~50% (Fig. 3, A and B).
This latter inhibitory effect of PMA on ACK-1 tyrosine phosphorylation
was prevented by preincubation with the PKC inhibitor bisindolylmaleimide I (Fig. 3, A and B). These
data indicate that, in general, PKC activity is inhibitory for ACK-1
tyrosine phosphorylation, and more specifically, receptor-mediated PKC
activity limits the ability of the mAChR to stimulate ACK-1 tyrosine
phosphorylation. To determine whether the inhibitory effects of PKC on
ACK-1 tyrosine phosphorylation were mediated via direct serine or
threonine phosphorylation of ACK-1 on some inhibitory site(s), the
potential of PMA to increase 32P incorporation into ACK-1
was assessed in 32P-labeled SH-SY5Y cells. Incubation of
32P-prelabeled cells with Oxo-M induced an increase in the
tyrosine phosphorylation of ACK-1 (Fig.
4A) and a corresponding
increase in 32P incorporation into ACK-1 (Fig.
4B). In contrast, the addition of PMA resulted in a decrease
in the basal tyrosine phosphorylation of ACK-1 (Fig. 4A, see
also Fig. 3A, first lane versus
fifth lane) with no detectable increase in
32P incorporation into ACK-1 (Fig. 4B). Under
the latter conditions, incubation with PMA induced phosphorylation of
the PKC substrate MARCKS in SH-SY5Y cells as indicated by the increase
in 32P incorporation into this protein (Fig.
4C). Thus, activation of PKC blunts tyrosine phosphorylation
of ACK-1 without inducing a corresponding increase in either serine or
threonine phosphorylation of ACK-1.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 3.
PKC activity blunts mAChR signaling to
ACK-1. A, confluent cultures of SH-SY5Y cells were
preincubated for 30 min with vehicle (VEH, 0.2% dimethyl
sulfoxide), 5 µM bisindolylmaleimide I (GF),
or 100 nM PMA. Alternatively, some cells were preincubated
for 30 min with bisindolylmaleimide I prior to a further 30-min
incubation with PMA. The cells were then incubated for a further 5 min
with either buffer alone or buffer containing 1 mM Oxo-M.
ACK-1 immune complexes were then immunoblotted for Tyr(P)
(PTyr). B, densitometric analysis of the effects
of bisindolylmaleimide I and PMA on Oxo-M-induced ACK-1 tyrosine
phosphorylation. The results are expressed as a percentage of the net
agonist-induced increase in ACK-1 tyrosine phosphorylation observed in
vehicle-pretreated cells and represent the means ± S.E. of four
independent experiments. Oxo-M increased the tyrosine phosphorylation
of ACK-1 in vehicle-pretreated cells from 0.04 ± 0.02 O.D. units
to 0.22 ± 0.06 O.D. units. The asterisks indicate a
significant difference from the vehicle-pretreated control
(p < 0.05). IP, immunoprecipitated;
IB, immunoblotted.
|
|

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 4.
Activation of PKC does not induce
phosphorylation of ACK-1 in vivo. Confluent
cultures of SH-SY5Y cells were labeled to isotopic equilibrium with
32Pi. Unincorporated label was removed by
washing, and cells were incubated either for 5 min with 1 mM Oxo-M or for 30 min with either vehicle (VEH,
0.2% dimethyl sulfoxide) or 100 nM PMA. Cell lysates were
then immunoprecipitated with either polyclonal anti-ACK-1
(A, B) or monoclonal anti-MARCKS (C),
as described under "Experimental Procedures." Immune complexes were
resolved by SDS-PAGE, transferred to PVDF, and[32P]-ACK-1
(B) or [32P]-MARCKS (C) was
detected by PhosphorImager analysis. Membranes containing ACK-1 were
subsequently immunoblotted for Tyr(P) (PTyr) (A).
The data shown are representative of two separate experiments that
produced similar results. IP, immunoprecipitated;
IB, immunoblotted.
|
|
Requirement for Fyn Tyrosine Kinase in the mAChR-induced
Phosphorylation of ACK-1--
Previous studies have demonstrated that
FAK and PYK-2 each physically interacts with Src family kinases, and
both are prominent substrates for this class of tyrosine kinase
(45-48). Similarly, preincubation of SH-SY5Y cells with the Src family
kinase inhibitor PP1 significantly attenuated the mAChR-stimulated
tyrosine phosphorylation of ACK-1 (Fig.
5A). The inhibitory effects of
PP1 were maximal at 10 µM, a concentration that
essentially abolished Oxo-M-induced ACK-1 phosphorylation. In addition,
a second Src family kinase inhibitor, PP2, also blocked agonist-induced
ACK-1 tyrosine phosphorylation, whereas a negative control compound,
PP3, had no effect (Fig. 5B). Moreover, Fyn tyrosine kinase,
but not Src, was detected in ACK-1 immunoprecipitates obtained from
lysates of agonist-stimulated SH-SY5Y cells (Fig.
6, first versus second
lane of each panel). In contrast,
tyrosine-phosphorylated ACK-1 was not
detectable in Fyn immunoprecipitates isolated from agonist-stimulated
cells (Fig. 6, upper panel, fifth versus sixth
lane).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
Inhibition of Src family kinase activity
blocks mAChR-stimulated tyrosine phosphorylation of ACK-1.
A, confluent cultures of SH-SY5Y cells were preincubated
with varying concentrations of PP1 for 15 min prior to a further 5-min
incubation with either buffer alone or 1 mM Oxo-M. ACK-1
immune complexes were resolved by SDS-PAGE, transferred to PVDF, and
immunoblotted for Tyr(P) (PTyr). B, SH-SY5Y cells
were preincubated for 15 min with either vehicle (VEH, 0.2%
dimethyl sulfoxide) or 10 µM PP1, PP2, or PP3 prior to
the addition of Oxo-M for a further 5 min. ACK-1 immune complexes were
probed for Tyr(P). IP, immunoprecipitation; IB,
immunoblotted.
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 6.
Coprecipitation of ACK-1 and Fyn after mAChR
stimulation. Confluent cultures of SH-SY5Y cells were incubated
for 5 min with either buffer alone or buffer containing 1 mM Oxo-M. Cell lysates were immunoprecipitated
(IP) with either polyclonal anti-ACK-1 or monoclonal
anti-Src or anti-Fyn as shown. Immune complexes were resolved by
SDS-PAGE, transferred to PVDF, and sequentially immunoblotted
(IB) for Tyr(P) (PTyr), Src, and Fyn. The blots
shown are representative of results obtained in three independent
experiments.
|
|
The monoclonal antibody to Fyn used in the previous experiments
recognizes an epitope within the Fyn-SH2 and/or SH3 domains. The latter
result suggested that ACK-1 may interact with Fyn at one or both of
these sites, thus preventing the Fyn antibody from recognizing Fyn that
is associated with ACK-1 in this manner. To test this hypothesis,
SH-SY5Y cells were scrape loaded with GST fusion proteins of either the
Fyn-SH2 or Fyn-SH3 domain, and mAChR-stimulated ACK-1 tyrosine
phosphorylation in these cells was compared with control cells scrape
loaded with GST alone. Agonist-induced ACK-1 phosphorylation was
inhibited significantly in cells scrape loaded with either the Fyn-SH2
domain (approximately a 60% decrease compared with the GST control) or
the Fyn-SH3 domain (approximately a 90% decrease compared with the GST
control) (Fig. 7, A and
C). Immunoblotting of cell lysates for GST showed that scrape loading of the Fyn-SH3 domain (which is smaller than the Fyn-SH2
domain) may have been slightly more efficient than that of the Fyn-SH2
domain (Fig. 7B). Collectively, these data indicate that Fyn
tyrosine kinase is required for mAChR signaling to ACK-1, and moreover,
Fyn may associate with ACK-1 via both SH2 domain- and SH3
domain-mediated interactions.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of scrape-loaded GST-Fyn-SH2 and
GST-Fyn-SH3 on mAChR-mediated ACK-1 tyrosine phosphorylation.
A, confluent cultures of SH-SY5Y cells were scrape loaded
with either GST alone, GST-Fyn-SH2, or GST-Fyn-SH3 (each at a
concentration of 20 µg/ml), as described under "Experimental
Procedures." After resuspension in serum-free medium and replating
for ~3 h, the cells were incubated for 5 min with either serum-free
medium alone or containing 1 mM Oxo-M. Cell lysates were
immunoprecipitated (IP) with polyclonal anti-ACK-1, and the
isolated immune complexes were probed for Tyr(P) (PTyr).
B, the post-ACK-1 immunoprecipitate lysates were resolved by
SDS-PAGE, transferred to PVDF, and immunoblotted (IB) for
GST. 2-µg aliquots of purified GST and fusion proteins were included
as standards. C, densitometric analysis of the effects of
scrape-loaded GST-Fyn-SH2 and GST-Fyn-SH3 on Oxo-M-induced ACK-1
tyrosine phosphorylation. The results are expressed as a percentage of
the net agonist-induced increase in ACK-1 tyrosine phosphorylation
observed in GST control cells and represent the means ± S.E. of
three independent experiments. Oxo-M increased the tyrosine
phosphorylation of ACK-1 in GST control cells from 0.03 ± 0.01 optical density units to 0.30 ± 0.14 optical density
units. Double asterisks indicate a significant difference
from the GST control (p < 0.01).
|
|
 |
DISCUSSION |
A role for the nonreceptor tyrosine kinases, FAK and PYK-2, in the
regulation of synaptic plasticity in the central nervous system has
recently been reviewed (13). Similarly, members of the related ACK
family of nonreceptor tyrosine kinases have also recently been
postulated to regulate neurite outgrowth (14), given their high levels
of expression in brain and their potential capacities to perpetuate the
actin cytoskeletal remodeling activity of the small molecular weight
GTPase Cdc42 (9, 12). In the current study, we demonstrate that
stimulation of neuritogenic M3 mAChRs on SH-SY5Y human
neuroblastoma cells elicits an enhanced tyrosine phosphorylation of
ACK-1 which occurs rapidly (within 1 min) on a time course that
precedes that observed previously for mAChR-mediated neurite outgrowth
(within minutes) in this same cell system (36) (Fig. 1). Given that a
requirement for Cdc42 in the regulation of mAChR-induced neurite
outgrowth has been described previously (23), the results of the
present investigation are consistent with a role for ACK-1 tyrosine
kinase in the process of growth cone remodeling. The mAChR-stimulated
increase in the tyrosine phosphorylation of ACK-1 was dependent on the
activity of a Rho family GTPase, most likely Cdc42, as demonstrated by its inhibition after preincubation with C. difficile toxin B
(Fig. 2A). Recently, we have also described a novel
requirement for Cdc42 function in the regulation of mAChR signaling to
FAK (37). This latter observation, coupled with the recent findings of
Eisenmann et al. (49) that Cdc42 and ACK-1 also
regulate the tyrosine phosphorylation of the FAK substrate,
p130Cas, suggests the potential for cross-talk
between these two families of nonreceptor tyrosine kinases.
In the present study, the actin cytoskeletal disruptive effects of
toxin B were dissociated from its ability to inhibit ACK-1 signaling by
the observation that preincubation with cytochalasin D actually
promoted ACK-1 phosphorylation (Fig. 2B). Moreover, addition
of a mAChR agonist to cells preincubated with cytochalasin D elicited a
further increase in ACK-1 tyrosine phosphorylation (Fig.
2B), suggesting that an intact actin cytoskeleton is not required for mAChR signaling to ACK-1. The above findings are in direct
contrast to the absolute requirement for an intact actin cytoskeleton
in mAChR signaling to FAK (Fig. 2B; Ref. 38). However, they
are in agreement with a previous report that demonstrated that
disruption of the actin cytoskeleton with cytochalasin leads to
disassembly of the extracellular matrix (50). Matrix disassembly has,
in turn, been shown to promote a compensatory activation of Cdc42, an
enhanced formation of filopodia, and an increased tyrosine
phosphorylation of ACK-1 (51). These last findings are further
supported by our observation that ACK-1 phosphorylation induced by
disruption of the cytoskeleton with cytochalasin D was blocked by
inhibition of Rho family GTPase activity with toxin B (Fig.
2C). Furthermore, our results corroborate the observations of Yang et al. (14) who demonstrated that
integrin-mediated ACK-2 tyrosine phosphorylation also does not depend
on the integrity of the actin cytoskeleton, although it is uncertain
why these authors did not observe an increase in ACK-2 phosphorylation
after incubation with cytochalasin D alone.
Although stimulation of M3 mAChRs elicits both an increase
in intracellular Ca2+ and an activation of PKC, neither of
these second messenger pathways was required for mAChR signaling to
ACK-1 in SH-SY5Y cells. Instead, receptor-mediated PKC activation was
found to act as a negative feedback loop to limit signaling from mAChRs
to ACK-1 (Fig. 3). The ability of PKC activity to blunt ACK-1 tyrosine
phosphorylation was apparently not caused by direct phosphorylation of
ACK-1 on inhibitory serine or threonine residues (Fig. 4). This result suggests that the PKC-mediated negative regulation of ACK-1 tyrosine phosphorylation may occur further upstream in the signaling pathway, perhaps at the level of the Cdc42 GTPase. In this context, it is
interesting to note that serine phosphorylation of the small molecular
weight GTPase, Rac1, by Akt kinase has recently been shown to inhibit
the ability of Rac1 to bind GTP (52). This observation suggests the
potential for negative regulation of Rho family GTPase function by
serine-threonine kinases. In addition, activation of PKC in neuronal
cells has been shown to induce a transient shift in actin cytoskeletal
organization from predominantly filopodial projections to lamellipodial
protrusions, indicating that PKC may be a negative regulator of Cdc42
function in vivo (53).
Perhaps the most significant result to come from the current study is
the finding that Fyn, a Src family kinase, is required for receptor
signaling to ACK-1. Inhibition of Src family kinase activity blunted
mAChR-stimulated ACK-1 tyrosine phosphorylation (Fig. 5), and ACK-1
coprecipitated with Fyn tyrosine kinase, but not Src, in an
agonist-dependent manner (Fig. 6). Although we observed a selective interaction of ACK-1 with Fyn rather than Src
after mAChR stimulation in SH-SY5Y cells, this result does not preclude
an interaction between ACK-1 and Src in other cell or receptor systems.
In fact, there is often significant overlap in the interactions of
multiple Src family members with a given substrate. For example, FAK
has been shown to interact in vitro with both Src and Fyn,
although Fyn is the principal regulator of FAK function in
vivo as demonstrated by hypophosphorylation of FAK in neural
tissue isolated from Fyn knockout mice (54). Thus, ACK-1 is similar to
FAK and PYK-2 in that it interacts with Src family kinases and is a
likely substrate for phosphorylation by this class of nonreceptor
tyrosine kinase (45-48). The agonist dependence of the interaction
between ACK-1 and Fyn observed in the present study suggests that
receptor stimulation of Cdc42 activity and the subsequent binding of
Cdc42 to ACK-1 may localize ACK-1 to the plasma membrane in close
proximity to Fyn. This hypothesis is consistent with previous results
of Yang and Cerione (12) who demonstrated that a constitutively
active mutant of Cdc42 failed to stimulate ACK-2 tyrosine
phosphorylation in vitro but that coexpression of this
mutant with ACK-2 resulted in a marked enhancement of ACK-2 tyrosine
phosphorylation and coassociation with Cdc42 in vivo.
Collectively, these results suggest that the activation of ACK proteins
by Cdc42 may be indirect, perhaps via localization of ACK to a site at
which it can interact with other proteins that modulate its tyrosine
phosphorylation and/or activity.
Finally, scrape loading SH-SY5Y cells with GST fusion proteins of
either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR
signaling to ACK-1. The inhibitory effect was most pronounced in cells
scrape loaded with the Fyn-SH3 domain (Fig. 7). These fusion proteins
lack kinase activity and therefore likely inhibit ACK-1 phosphorylation
by competing with endogenous Fyn for binding sites on the ACK-1
protein. Fyn may associate with ACK-1 via an interaction between the
Fyn-SH3 domain and a proline-rich region on ACK-1 which is exposed
after its association with GTP-bound Cdc42. Previous work has suggested
that Cdc42 binding to the Cdc42/Rac-interactive binding domain of ACK-2
may dissociate an intramolecular interaction between the SH3 domain and
a C-terminal proline-rich region of this protein (14). A similar
process may occur after the association of ACK-1 with GTP-bound Cdc42,
thus allowing for ACK-1 to interact with other target proteins via
these structural motifs. Indeed an interaction between ACK-1 and the
SH3 domain(s) of the adaptor protein GRB-2 after epidermal growth
factor receptor stimulation has been described previously (55). Fyn may
also interact with phosphorylated tyrosine residues on ACK-1 via the
Fyn-SH2 domain.
In summary, stimulation of mAChRs on SH-SY5Y human neuroblastoma cells
elicited an enhanced tyrosine phosphorylation of the Cdc42 effector
ACK-1. Agonist-stimulated ACK-1 phosphorylation was dependent on Fyn
tyrosine kinase activity and the association of ACK-1 with Fyn via both
SH2 domain- and SH3 domain-mediated interactions. The results suggest
that the previously documented role of Cdc42 in the regulation of
neurite outgrowth after cholinergic stimulation may also involve
signaling via the nonreceptor tyrosine kinase ACK-1.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Klaus Aktories and Dr. Fred
Hofmann for generously providing the C. difficile toxin B
and Tim Desmond for assistance with image analysis of the immunoblots.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants NS23831 (to S. K. F.) and MH12193 (to D. A. L.), by a
Department of Veterans Affairs merit award (to K. A. H.), and a
research enhancement award (to K. A. H. and D. A. L.).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.
§
Supported by National Institutes of Health Training Grant
GM07767. To whom correspondence should be addressed: Denver VAMC-111H, Rm. 9C120, 1055 Clermont St., Denver, CO 80220. Tel.:
303-399-8020 (ext. 5264); Fax: 303-393-5271; E-mail:
Dan.Linseman@UCHSC.edu.
Published, JBC Papers in Press, November 21, 2001, DOI 10.1074/jbc.M006813300
 |
ABBREVIATIONS |
The abbreviations used are:
ACK, activated
Cdc42Hs-associated kinase;
FAK, focal adhesion kinase;
GST, glutathione
S-transferase;
mAChR, muscarinic cholinergic receptor;
MARCKS, myristoylated alanine-rich protein kinase C substrate;
Oxo-M, 2-butyn-1-ammonium,
N,N,N-trimethyl-4-(2-oxo-1-pyrrolidinyl)iodide;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered
saline;
PKC, protein kinase C;
PMA, phorbol 12-myristate 13-acetate;
PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
PP3, 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine;
PVDF, polyvinylidene
fluoride;
PYK-2, proline-rich tyrosine kinase-2;
Tyr(P), phosphotyrosine..
 |
REFERENCES |
1.
|
Malenka, R. C.
(1994)
Cell
78,
535-538[Medline]
[Order article via Infotrieve]
|
2.
|
O'Dell, T. J.,
Kandel, E. R.,
and Grant, S. G. N.
(1991)
Nature
353,
558-560[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Boxall, A. R.,
Lancaster, B.,
and Garthwaite, J.
(1996)
Neuron
16,
805-813[Medline]
[Order article via Infotrieve]
|
4.
|
Williams, E. J.,
Walsh, F. S.,
and Doherty, P.
(1994)
J. Cell Biol.
124,
1029-1037[Abstract]
|
5.
|
Worley, T. L.,
and Holt, C. E.
(1996)
J. Neurosci.
16,
2294-2306[Abstract]
|
6.
|
Hunter, T.
(1998)
Philos. Trans. R. Soc. Lond-Biol. Sci.
353,
583-605[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Klesse, L. J.,
and Parada, L. F.
(1999)
Microsc. Res. Tech.
45,
210-216[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Neet, K.,
and Hunter, T.
(1996)
Genes Cells
1,
147-169[Abstract/Free Full Text]
|
9.
|
Manser, E.,
Leung, T.,
Salihuddin, H.,
Tan, L.,
and Lim, L.
(1993)
Nature
363,
364-367[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Burgaya, F.,
Menegon, A.,
Menegoz, M.,
Valtorta, F.,
and Girault, J.-A.
(1995)
Eur. J. Neurosci.
7,
1810-1821[Medline]
[Order article via Infotrieve]
|
11.
|
Lev, S.,
Moreno, H.,
Martinez, R.,
Canoll, P.,
Peles, E.,
Musacchio, J. M.,
Plowman, G. D.,
Rudy, B.,
and Schlessinger, J.
(1995)
Nature
376,
737-745[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Yang, W.,
and Cerione, R. A.
(1997)
J. Biol. Chem.
272,
24819-24824[Abstract/Free Full Text]
|
13.
|
Girault, J.-A.,
Costa, A.,
Derkinderen, P.,
Studler, J.-M.,
and Toutant, M.
(1999)
Trends Neurosci.
22,
257-263[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Yang, W.,
Lin, Q.,
Guan, J.-L.,
and Cerione, R. A.
(1999)
J. Biol. Chem.
274,
8524-8530[Abstract/Free Full Text]
|
15.
|
Kozma, R.,
Ahmed, S.,
Best, A.,
and Lim, L.
(1995)
Mol. Cell. Biol.
15,
1942-1952[Abstract]
|
16.
|
Nobes, C. D.,
and Hall, A.
(1995)
Cell
81,
53-62[Medline]
[Order article via Infotrieve]
|
17.
|
Knaus, U. G.,
and Bokoch, G. M.
(1998)
Int. J. Biochem. Cell Biol.
30,
857-862[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Manser, E.,
and Lim, L.
(1999)
Prog. Mol. Subcell. Biol.
22,
115-133[Medline]
[Order article via Infotrieve]
|
19.
|
Bi, E.,
and Zigmond, S. H.
(1999)
Curr. Biol.
9,
R160-R163[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Snapper, S. B.,
and Rosen, F. S.
(1999)
Annu. Rev. Immun.
17,
905-929[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Luo, L.,
Jan, L.,
and Jan, Y. N.
(1996)
Perspect. Dev. Neurobiol.
4,
199-204[Medline]
[Order article via Infotrieve]
|
22.
|
Threadgill, R.,
Bobb, K.,
and Ghosh, A.
(1997)
Neuron
19,
625-634[Medline]
[Order article via Infotrieve]
|
23.
|
Kozma, R.,
Sarner, S.,
Ahmed, S.,
and Lim, L.
(1997)
Mol. Cell. Biol.
17,
1201-1211[Abstract]
|
24.
|
Katoh, H.,
Aoki, J.,
Yamaguchi, Y.,
Kitano, Y.,
Ichikawa, A.,
and Negishi, M.
(1998)
J. Biol. Chem.
273,
28700-28707[Abstract/Free Full Text]
|
25.
|
Kranenburg, O.,
Poland, M.,
van Horck, F. P. G.,
Drechsel, D.,
Hall, A.,
and Moolenaar, W. H.
(1999)
Mol. Biol. Cell
10,
1851-1857[Abstract/Free Full Text]
|
26.
|
Winkler, J.,
Suhr, S. T.,
Gage, F. H.,
Thal, L. J.,
and Fisher, L. J.
(1995)
Nature
375,
484-487[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Levey, A. I.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13541-13546[Abstract/Free Full Text]
|
28.
|
Avery, E. E.,
Baker, L. D.,
and Asthana, S.
(1997)
Drugs Aging
11,
450-459[Medline]
[Order article via Infotrieve]
|
29.
|
Growdon, J. H.
(1997)
Life Sci.
60,
993-998[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Biedler, J. L.,
Helson, L.,
and Spengler, R.
(1973)
Cancer Res.
33,
2643-2652[Medline]
[Order article via Infotrieve]
|
31.
|
Ross, R. A.,
and Biedler, J. L.
(1985)
Cancer Res.
45,
1628-1632[Abstract]
|
32.
|
Lambert, D. G.,
Ghataorre, A. S.,
and Nahorski, S. R.
(1989)
Eur. J. Pharmacol.
165,
71-77[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Wall, S. J.,
Yasuda, R. P.,
Li, M.,
and Wolfe, B. B.
(1991)
Mol. Pharmacol.
40,
783-789[Abstract]
|
34.
|
Lambert, D. G.,
and Nahorski, S. R.
(1990)
Biochem. J.
265,
555-562[Medline]
[Order article via Infotrieve]
|
35.
|
Fisher, S. K.,
Slowiejko, D. M.,
and McEwen, E. L.
(1994)
Neurochem. Res.
19,
549-554[Medline]
[Order article via Infotrieve]
|
36.
|
Rösner, H.,
Vacun, G.,
and Rebhan, M.
(1995)
Eur. J. Cell Biol.
66,
324-334[Medline]
[Order article via Infotrieve]
|
37.
|
Linseman, D. A.,
Hofmann, F.,
and Fisher, S. K.
(2000)
J. Neurochem.
74,
2010-2020[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Linseman, D. A.,
McEwen, E. L.,
Sorensen, S. D.,
and Fisher, S. K.
(1998)
J. Neurochem.
70,
940-950[Medline]
[Order article via Infotrieve]
|
39.
|
Flinn, H. M.,
and Ridley, A. J.
(1996)
J. Cell Sci.
109,
1133-1141[Abstract/Free Full Text]
|
40.
|
Just, I.,
Seizer, J.,
Wilm, M.,
von Eichel-Streiber, C.,
Mann, M.,
and Aktories, K.
(1995)
Nature
375,
500-503[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Schaller, M. D.,
Borgman, C. A.,
Cobb, B. S.,
Vines, R. R.,
Reynolds, A. B.,
and Parsons, J. T.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
5192-5196[Abstract]
|
42.
|
Brinson, A. E.,
Harding, T.,
Diliberto, P. A.,
He, Y.,
Li, X.,
Hunter, D.,
German, B.,
Earp, H. S.,
and Graves, L. M.
(1998)
J. Biol. Chem.
273,
1711-1718[Abstract/Free Full Text]
|
43.
|
Linseman, D. A.,
Sorensen, S. D.,
and Fisher, S. K.
(1999)
J. Neurochem.
73,
1933-1944[Medline]
[Order article via Infotrieve]
|
44.
|
Slowiejko, D. M.,
Levey, A. I.,
and Fisher, S. K.
(1994)
J. Neurochem.
62,
1795-1803[Medline]
[Order article via Infotrieve]
|
45.
|
Calalb, M. B.,
Polte, T. R.,
and Hanks, S. K.
(1995)
Mol. Cell. Biol.
15,
954-963[Abstract]
|
46.
|
Dikic, I.,
Tokiwa, G.,
Lev, S.,
Courtneidge, S. A.,
and Schlessinger, J.
(1996)
Nature
383,
547-550[CrossRef][Medline]
[Order article via Infotrieve]
|
47.
|
Qian, D.,
Lev, S.,
van Oers, N. S.,
Dikic, I.,
Schlessinger, J.,
and Weiss, A.
(1997)
J. Exp. Med.
185,
1253-1259[Abstract/Free Full Text]
|
48.
|
Schaller, M. D.,
Hildebrand, J. D.,
and Parsons, J. T.
(1999)
Mol. Biol. Cell
10,
3489-3505[Abstract/Free Full Text]
|
49.
|
Eisenmann, K. M.,
McCarthy, J. B.,
Simpson, M. A.,
Keely, P. J.,
Guan, J.-L.,
Tachibana, K.,
Lim, L.,
Manser, E.,
Furcht, L. T.,
and Iida, J.
(1999)
Nature Cell Biol.
1,
507-513[CrossRef][Medline]
[Order article via Infotrieve]
|
50.
|
Barry, E. L. R.,
and Mosher, D. F.
(1988)
J. Biol. Chem.
263,
10464-10469[Abstract/Free Full Text]
|
51.
|
Bourdoulous, S.,
Orend, G.,
MacKenna, D. A.,
Pasqualini, R.,
and Ruoslahti, E.
(1998)
J. Cell Biol.
143,
267-276[Abstract/Free Full Text]
|
52.
|
Kwon, T.,
Kwon, D. Y.,
Chun, J.,
Kim, J. H.,
and Kang, S. S.
(2000)
J. Biol. Chem.
275,
423-428[Abstract/Free Full Text]
|
53.
|
Rösner, H.,
and Fischer, H.
(1996)
Neurosci. Lett.
219,
175-178[CrossRef][Medline]
[Order article via Infotrieve]
|
54.
|
Grant, S. G. N.,
Karl, K. A.,
Kiebler, M. A.,
and Kandel, E. R.
(1995)
Genes Dev.
9,
1909-1921[Abstract]
|
55.
|
Satoh, T.,
Kato, J.,
Nishida, K.,
and Kaziro, Y.
(1996)
FEBS Lett.
386,
230-234[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.