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J. Biol. Chem., Vol. 278, Issue 30, 28116-28122, July 25, 2003
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-Cells*
From the Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285
Received for publication, December 18, 2002 , and in revised form, May 7, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-cells
(Konrad, R. J., Dean, R. M., Young, R. A., Bilings, P. C., and Wolf, B. A.
(1996) J. Biol. Chem. 271, 24179–24186). Glucose induced p125
tyrosine phosphorylation in -TC3 insulinoma cells, -HC9 cells, and
in freshly isolated rat islets, whereas increased tyrosine phosphorylation was
not observed with other fuel secretagogues. Initial efforts to identify p125
were unsuccessful, so a new approach was taken. The protein was purified from
TC6,F7 cells via an immunodepletion method. After electrophoresis and
colloidal Coomassie Blue staining, the area of the gel corresponding to p125
was excised and subjected to tryptic digestion. Afterward, mass spectrometry
was performed and the presence of Crk-associated substrate (Cas) was detected.
Commercially available antibodies against Cas were obtained and tested
directly in -cells, confirming glucose-induced tyrosine phosphorylation
of Cas. Further experiments demonstrated that in -cells the
glucose-induced increase in Cas tyrosine phosphorylation occurs immediately
and is not accompanied by increased focal adhesion kinase tyrosine
phosphorylation. Finally, it is also demonstrated via Western blotting that
Cas is present in normal isolated rat islets. Together, these results show
that the identity of the previously described p125 -cell protein is Cas
and that Cas undergoes rapid glucose-induced tyrosine phosphorylation in
-cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-cells (1). Glucose
induced p125 tyrosine phosphorylation in freshly isolated rat islets as well
as in clonal -cells (1).
Interestingly, glucose proved to be the only fuel secretagogue tested that
stimulated p125 tyrosine phosphorylation, both alone and in the presence of
the muscarinic agonist carbachol
(1).
Since that time, we made several attempts to purify p125 and determine its
identity; however, these attempts were not successful. Therefore, a new
approach was taken and LC/MS/MS was used to analyze p125 prepared from
TC6,F7 cells using a method of selective immunodepletion followed by
electrophoretic separation.
This approach identified p125 as Crk-associated substrate (Cas), an adapter
molecule that has been reported to be tyrosine-phosphorylated in numerous
other cell types but whose existence has not previously been described in
-cells (2). To confirm
the MS/MS-assigned identity of p125 as Cas, monoclonal and polyclonal
commercially available anti-Cas antibodies were obtained and tested directly
in -cells, confirming the glucose-induced tyrosine phosphorylation of
Cas. In addition, the presence of Cas in normal rat islets was confirmed via
Western blotting. Together, these results show for the first time that the
previously described p125 -cell protein is in fact Crk-associated
substrate.
In other cell types, Cas functions as an adapter protein and is involved in
such diverse processes as cytoskeletal organization, localization of focal
adhesions, and association with focal adhesion kinase (FAK) and growth
regulation
(3–12).
The mid-portion of the molecule contains at least 15 YXXP repeats
that can become tyrosine-phosphorylated and function to bind Src-homology 2
(SH2) containing proteins while the N terminus contains a SH3 domain. In
addition, the C terminus of Cas binds a variety of proteins including
phosphatidylinositol 3-kinase
(13–16).
In light of the importance of Cas in other cell types, our results suggest
that glucose-induced Cas tyrosine phosphorylation may be important in
-cell function.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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TC6,F7 Cell Culture—Cells were cultured in 10-cm or
15-cm dishes in the presence of Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 2 mM L-glutamine.
Cells were trypsinized and subcloned weekly. Media were changed three times
weekly and on the day prior to an experiment. Cells were maintained at 37
°C under an atmosphere of 95% air, 5% CO2. In each experiment,
preincubation and all subsequent incubations were performed at 37 °C and
under an atmosphere of 95% air, 5% CO2. Islet Isolation—Islets were isolated aseptically from Sprague-Dawley rats as described previously (1). Islets were isolated using Hanks' balanced salt solution (HBSS) and Ficoll containing 5.5 mM glucose, penicillin (25 units/ml), and streptomycin (25 units/ml). During surgery, the pancreas was inflated with 10–20 ml of HBSS. The distended pancreas was excised, and lymph nodes, fat, blood vessels, and bile ducts were removed. Tissue was chopped extensively, rinsed 5–6 times with HBSS, and digested with collagenase P (2 mg/ml tissue) at 37 °C for 3 min. Digested tissue was rinsed four times with HBSS. Islets were purified on a discontinuous Ficoll gradient consisting of 27, 23, 20.5, and 11% Ficoll in 25 mM HEPES-HBSS buffer. Islets were harvested, washed once with HBSS, and washed six times in 3 mM glucose KRB (Krebs-HEPES buffer containing 25 mM HEPES, pH 7.40, 115 mM NaCl, 1 mM NaH2PO4, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2) via hand picking under a stereodissecting microscope prior to use.
Antibodies—PY20 anti-phosphotyrosine antibody, mouse monoclonal anti-Cas antibody, and mouse monoclonal anti-FAK antibody were obtained from Transduction Laboratories (Lexington, KY), whereas rabbit polyclonal anti-Cas (C-20) antibody was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
Incubation of -Cells for Protein Tyrosine
Phosphorylation—Adherent TC6,F7 cells in 10-cm dishes were
washed three times in 0 mM glucose KRB supplemented with 0.1%
bovine serum albumin. Cells were preincubated for 30 min with 0 mM
glucose KRB. Pre-incubation buffer was aspirated, and cells were incubated
with KRB supplemented with the appropriate compound(s). At the end of the
experiment, cells were processed for analysis of tyrosine-phosphorylated
proteins as described below. In certain experiments, supernatants were removed
from the dishes for insulin radioimmunoassay using a rat insulin standard
(Linco Research, Inc, St. Charles, MO). For data analysis, insulin secretion
in the control sample was set at 100% and results were expressed as a percent
of control.
Immunoprecipitation of -Cell Tyrosine-phosphorylated
Proteins and Other Proteins of Interest—At the end of an
experiment, supernatant was aspirated from the dishes. Cells were washed twice
with ice-cold phosphate-buffered saline supplemented with 1 mM
NaVO4. Afterward, phosphate-buffered solution was aspirated and 1
ml of ice-cold lysis buffer (50 mM HEPES, pH 7.40, 150
mM NaCl, 1% Triton X-100, 5 mM EDTA, 5 mM
EGTA, 20 mM NaF, 20 mM
Na4P2O7, 1mM NaVO4, 1
mg/ml bacitracin, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml
leupeptin, 1 µg/ml aprotinin) was added to each dish. Cells were scraped
into 1.5-ml Eppendorf tubes, vortexed, placed on ice for 15 min, and vortexed
again prior to centrifugation at 14,000 x g for 15 min.
Afterward, the supernatant was transferred to a second 1.5-ml conical
Eppendorf tube. Tyrosine-phosphorylated proteins were immunoprecipitated for 2
h on a rocker with 2.5 µg of PY20 antibody. Other proteins of interest were
similarly immunoprecipitated but with their own specific antibodies. After 2
h, 10 µl of protein A-Trisacryl beads (Pierce) were added to the tubes and
the incubation was continued overnight. At the end of the incubation, beads
were washed once with wash buffer 1 (150 mM NaCl, 10 mM
HEPES, pH 7.40, 1% Triton X-100, 0.1% SDS) and once with wash buffer 2 (10
mM HEPES, pH 7.40, 1% Triton X-100, 0.1% SDS). After the final
washing step, 40 µlof2x sample buffer (100 mM Tris, pH
6.80, 4% SDS, 20% glycerol, 20 µg/liter bromphenol blue, 15 mg/ml
dithiothreitol) was added to each tube. Samples were vortexed, boiled for 5
min, vortexed again, and stored at –20 °C prior to subsequent
analysis.
Western Blotting—Samples were loaded onto 7.5% SDS-polyacrylamide gels. Colored molecular weight markers (Amersham Biosciences) were run on each gel. Proteins were separated for 1 h at 175 V at room temperature using a Bio-Rad Mini-PROTEAN II dual slab cell. Proteins were transferred to ECL nitrocellulose paper (Amersham Biosciences) for 1.5 h (100 V, 4 °C). Nitrocellulose blots were blocked for 1 h at room temperature in blocking buffer (5% bovine serum albumin in 10 mM Tris, pH 7.40, 150 mM NaCl, 0.1% sodium azide, 0.05% Tween 20). After blocking, blots were probed with PY20 or other appropriate antibodies (in blocking buffer) for 1 h at room temperature. Blots were washed six times (5 min each) with TBST (10 mM Tris, pH 7.40, 150 mM NaCl, 0.05% Tween 20). After washing, blots were probed with horseradish peroxidase-conjugated sheep anti-mouse antibody (Amersham Biosciences) at a 1:1500 dilution in TBST for 1 h at room temperature. Blots were washed again as above and developed with ECL reagent (Amersham Biosciences). After air-drying, blots were exposed to BioMax x-ray film (Eastman Kodak Co.).
Purification of -Cell p125—TC6,F7 cells
grown in 15-cm dishes were stimulated for 30 min with 15 mM glucose
+ 0.5 mM carbachol (CCH). Afterward, cells were washed twice with
ice-cold phosphate-buffered saline supplemented with 1 mM
NaVO4. 1 ml of ice-cold lysis buffer (50 mM HEPES, pH
7.40, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 5
mM EGTA, 20 mM NaF, 20 mM
Na4P2O7, 1mM NaVO4, 1
mg/ml bacitracin, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml
leupeptin, 1 µg/ml aprotinin) was added next to each dish. Cells were
scraped into 50-ml conical tubes, vortexed, placed on ice for 15 min, and
vortexed again before centrifugation at 14,000 x g for 15 min.
Afterward, the supernatant was transferred to new 50-ml conical tubes, which
were precleared for 2 h with 200 µl of protein A-Trisacryl beads. After
preclearing, p125 was immunoprecipitated on a rocker with 5 µl of PY20
antibody. After 2 h, 10 µl of protein A-Trisacryl beads was added to the
tubes and the incubation was continued overnight. At the end of the
incubation, beads were pooled and transferred to a 1.5-ml Eppendorf tube,
washed once with wash buffer 1 and once with wash buffer 2. After the final
washing step, 25 µlof2x sample buffer (100 mM Tris, pH
6.80, 4% SDS, 20% glycerol, 20 µg/liter bromphenol blue, 15 mg/ml
dithiothreitol) was added to the tube. The sample was vortexed, boiled for 5
min, vortexed again, and loaded onto a single lane of a 7.5%
SDS-polyacrylamide gel. Following one-dimensional electrophoresis as described
above, the gel was stained with colloidal Coomassie Blue (Novex) and the
portion of the gel corresponding to p125 was excised for LC/MS/MS
analysis.
In-gel Digestion of p125—The region of interest from the gel was cut into 2-mm pieces and subjected to reduction, alkylation, and tryptic digestion on a Tecan Genesis 150 robotics platform. One hundred microliters of 50 mM dithiothreitol in 50 mM NH4HCO3, 50% acetonitrile was added to the stained gel pieces and incubated for 1 h at 37 °C. The solution was aspirated, and 100 mM iodoacetamide (in 50 mM NH4HCO3, 50% acetonitrile) was added to the gel pieces and incubated for 15 min at 37 °C. The solution was decanted, and gel pieces were washed twice with 50 mM NH4HCO3 in 50% acetonitrile. One hundred microliters of acetonitrile was added to the gel pieces and incubated for 5 min at room temperature. Acetonitrile was aspirated, and gel pieces were placed in an incubator for 10 min at 37 °C. Ten microliters of 20 µg/ml Promega modified trypsin was added to the dry gel pieces and incubated overnight at 37 °C. The following day, gel pieces were extracted with 70 µl of 0.1% trifluoroacetic acid in water. The sample was desalted on a µC18 Ziptip (Millipore) by passing extract over the Ziptip two times and washing with 10 µl of 0.1% trifluoroacetic acid 10 times. Bound material was eluted into a 96-well 200-µl polypropylene PCR plate with 1 µl of 0.1% trifluoroacetic acid in 50% acetonitrile and diluted with 9 µl of 0.1% trifluoroacetic acid in water. The sample plate was placed in a FAMOS autosampler (LC Packings).
LC/MS/MS Analysis of p125—LC/MS/MS analysis was performed on a LCQ deca-ion trap mass spectrometer (Thermofinnigan) fitted with a custom capillary high pressure liquid chromatography system using a 75-µm PicoFrit capillary high pressure liquid chromatography column containing Aquasil C18 packing (New Objective). Five microliters of the sample was loaded onto the column at 1.5 µl/min, and after 9 min, the flow rate was reduced to 200 nanoliters/min by opening a tee in the flow-path just above the column and running a linear gradient from 5 to 45% acetonitrile over 60 min. The LCQ deca was run in triple play mode with a custom nano electron spray ionization interface. Data from the mass spectrometer were processed for signal quality, and protein identification was performed by Sequest software (Thermofinnigan).
Data Analysis—Films were photographed using a digital camera. Intensities of bands were quantitated with results expressed as the mean ± S.E. using the Windows-compatible version 2.98 of the program FigP (Biosoft, St. Louis, MO). Statistical analysis was performed using the same program. Data were analyzed by one-way analysis of variance followed by comparisons between the means using the least significant difference test. A probability of p < 0.05 was considered to indicate statistical significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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TC6,F7 cells contained p125 that undergoes increased tyrosine
phosphorylation in response to stimulation of the -cells with glucose
and carbachol. The increased tyrosine phosphorylation was apparent either when
crude cell lysates were used for Western blotting or when
tyrosine-phosphorylated proteins were first immunoprecipitated prior to
Western blotting. Fig.
1B demonstrates that glucose and the combination of
glucose plus carbachol both stimulate p125 tyrosine phosphorylation.
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These preliminary data demonstrated that TC6,F7 cells are a suitable
cell source for purification of p125. To purify the protein, cells were grown
in 15-cm dishes and stimulated for 30 min with 15 mM glucose + 0.5
mM CCH. After preclearing, p125 was immunoprecipitated with PY20
antibody and separated electrophoretically, allowing the portion of the
subsequently Coomassie Blue-stained gel to be excised and analyzed via
LC/MS/MS.
LC/MS/MS analysis of the gel slice revealed the presence of peptides
matching those described for a protein known as Cas, an adapter protein that
has been described to be highly tyrosine-phosphorylated in many different cell
types, but had not been so far described to exist in -cells
(2). The sequences of two such
peptides are shown in Table I,
whereas Fig. 2 shows the
LC/MS/MS spectra of the actual peptides.
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In light of the high quality of the MS/MS spectra obtained and the
indication that Cas had been previously described as being highly
tyrosine-phosphorylated in other cells types
(2), commercially available
monoclonal and polyclonal anti-Cas antibodies were obtained to confirm the
identity of the -cell protein as Crk-associated substrate.
Fig. 3 shows that when these
antibodies were used to immunoprecipitate Cas from TC6,F7 cells, glucose
dramatically increased the tyrosine phosphorylation of Cas. This increased
tyrosine phosphorylation was observed using independent monoclonal and
polyclonal anti-Cas antibodies.
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Likewise, when PY20 antiphosphotyrosine antibody was used to
immunoprecipitate tyrosine-phosphorylated proteins from -cells,
increased Cas was detected by Western blotting following glucose stimulation
of the cells (Fig. 3). The
increase in tyrosine-phosphorylated Cas content in PY20 immunoprecipitates was
also observed with Western blotting using independent monoclonal and
polyclonal anti-Cas antibodies. As expected, glucose stimulation did not
affect the total amount of Cas immunoprecipitates from the -cells as
determined by immunoprecipitation of Cas followed by Western blotting with
anti-Cas antibody.
As Fig. 4 (A and B) demonstrates, glucose alone induced a 180.8 ± 11.3% increase in Cas tyrosine phosphorylation (p < 0.05 compared with control) while the muscarinic agonist carbachol alone caused a 123.7 ± 11.6% increase in Cas tyrosine phosphorylation (p = 0.08 compared with control). The combination of glucose and carbachol induced a 224.2 ± 43.5% increase in Cas tyrosine phosphorylation (p < 0.05 compared with control). This increase, while significant compared with control, was not significantly greater than that caused by glucose alone. Fig. 4C shows the insulin secretion data corresponding to Fig. 4 (A and B). Glucose alone induced a 151.8 ± 17.5% increase in insulin secretion (p < 0.05 compared with control), whereas carbachol alone caused a 138.4 ± 3.5% increase in insulin secretion (p < 0.05 compared with control). The combination of glucose and carbachol induced a 365.9 ± 83.2% increase in insulin secretion (p < 0.05 compared with control).
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As Fig. 5 demonstrates, Cas
tyrosine phosphorylation was immediate, occurring within 2 min of stimulation.
Together, the data shown in Figs.
3,
4,
5 confirmed that the protein
that we had previously named p125, which undergoes rapid increased tyrosine
phosphorylation in -cells in response to glucose, is in fact Cas.
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In light of reports in other cell types that increased Cas tyrosine
phosphorylation is also accompanied by increased FAK tyrosine phosphorylation
and that Cas and FAK can co-localize at focal adhesions
(3–7),
we examined FAK tyrosine phosphorylation in -cells. This analysis was
also warranted in light of the fact that the molecular mass of FAK is 125 kDa
(3–7).
To determine whether FAK underwent glucose-induced tyrosine phosphorylation,
TC6,F7 cells were stimulated with glucose prior to immunoprecipitation
with either anti-FAK antibody or PY20 anti-phosphotyrosine antibody.
Subsequent Western blotting demonstrated that FAK immunoprecipitates had no
glucose-induced increase in tyrosine phosphorylation
(Fig. 6). Similarly, PY20
immunoprecipitates did not demonstrate any increase in tyrosine-phosphorylated
FAK content (Fig. 6),
indicating that -cell glucose-induced tyrosine phosphorylation of Cas is
not accompanied by increased FAK tyrosine phosphorylation.
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Although FAK tyrosine phosphorylation did not accompany Cas phosphorylation
in -cells, it was still possible that that an additional substrate or
substrates of similar molecular weight to Cas might be
tyrosine-phosphorylated. To explore this possibility, TC6,F7 cells were
stimulated with glucose and carbachol. Cas immunodepletion was performed next
by incubating the immunoprecipitation lysates with protein A beads
pre-adsorbed with anti-Cas antibody. Following this Cas immunodepletion,
immunoprecipitation and Western blotting were performed with PY20 antibody.
Fig. 7 shows the results from
these experiments. Remarkably, Cas immunodepletion demonstrated that there is
an additional protein of slightly higher molecular weight than Cas that also
undergoes glucose and carbachol-induced tyrosine phosphorylation. We do not
know the identity of this protein and are currently trying to identify it.
Possibilities include a variant of Cas, a post-translationally modified form
of Cas, or an unrelated protein.
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Finally, the expression of Cas in normal isolated rat islets was explored
via Western blotting. As Fig. 8
shows, when rabbit anti-Cas antibody was used to immunoprecipitate Cas from
isolated rat islets and mouse monoclonal anti-Cas antibody was used for
subsequent Western blotting, Cas was clearly observed to be present in
isolated islets. It was noted that in Fig.
8, the absolute intensity of the Cas band was less than that
compared with experiments with TC6,F7 cells. This is to be expected
since each islet tube contained 1000 islets, which are estimated to correlate
to 2 million -cells (assuming 2,000 -cells per islet). In
contrast, experiments with TC6,F7 cells were performed in 10-cm dishes,
which contain 
20–40 million -cells (10–20 times as many
cells).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-cell protein p125, which
undergoes glucose-induced tyrosine phosphorylation, is in fact Cas. Cas has
not previously been described in -cells. Identification of p125 as
Crk-associated substrate by LC/MS/MS analysis following purification of the
protein from TC6,F7 cells was confirmed by immunoprecipitation and
Western blotting with two different commercially available anti-Cas
antibodies. Importantly, the presence of Cas in normal islets is also
demonstrated. Interestingly, in the -cell, glucose-induced Cas tyrosine
phosphorylation was not accompanied by increased tyrosine phosphorylation of
FAK. This is in contrast to some other cell types in which both molecules have
been reported to map to focal adhesions and undergo increased tyrosine
phosphorylation in response to the same stimuli
(3–7).
These results open up a new area of -cell investigation because Cas
has not been previously described in pancreatic -cells. In other cell
types, Cas appears to function as an adapter protein and is implicated in a
number of cellular responses including migration, regulation of growth, focal
adhesion localization, and actin organization
(8–10;
for a detailed review, see Ref.
2). Cas was first described as
forming complexes with v-Src and v-Crk in a tyrosine phosphorylation-dependent
manner and, when subsequently cloned, appeared to be particularly well suited
as an adapter molecule based on its structure
(11,
12). The N-terminal portion of
the protein contains a SH3 domain, whereas the mid-portion of the molecule
contains at least 15 YXXP repeats that can become
tyrosine-phosphorylated and function to bind SH2-containing proteins
(13,
14). Finally, the C terminus
of Cas can also function to bind a variety of proteins, including
phosphatidylinositol 3-kinase and 14-3-3 proteins
(15,
16).
There appear to be several described inducers of Cas tyrosine phosphorylation in other cell types including G-protein receptor agonists such as cholecystokinin, adhesion receptors such as ICAM (intercellular adhesion molecule) and integrins, and ligands of receptor protein tyrosine kinases such as epidermal growth factor (17–24). In addition, non-receptor protein tyrosine kinases such as Src, Pyk2, and FAK have also been reported to associate with and/or tyrosine-phosphorylate Cas (25–28). Once phosphorylated, it appears that Cas is able to interact with a number of SH2-containing proteins including Src and Crk (29–32). In the latter case, Cas appears to allow Crk to interact subsequently with numerous additional proteins involved in intracellular signaling (29–32).
In the -cell, the mechanism by which glucose stimulates increased Cas
tyrosine phosphorylation is unclear. Glucose does not have a receptor and as
such does not fit into any of the categories of stimulators of Cas tyrosine
phosphorylation listed above. We are currently attempting to determine which
-cell kinase or kinases tyrosine-phosphorylate Cas in response to
increased extracellular glucose and how these kinases might be linked to
-cell recognition of glucose. Likewise, it is also unclear which
molecules may lie downstream of tyrosine-phosphorylated Cas in the
-cell. Similarly, we are attempting to identify which -cell
signaling molecules may bind to tyrosine-phosphorylated Cas in the
-cell. Our identification of p125 as Crk-associated substrate opens up a
new and exciting pathway in the -cell.
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FOOTNOTES |
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To whom correspondence should be addressed: Eli Lilly Corporate Center,
Building 88–358C, Indianapolis, IN 46285. Tel.: 317-655-9290; E-mail:
konrad_robert{at}lilly.com.
1 The abbreviations used are: p125, 125-kDa protein; LC, liquid
chromatography; MS, mass spectroscopy; SH, Src homology; KRB, Krebs-HEPES
buffer; CCH, carbachol; HBSS, Hanks' balanced salt solution; Cas,
Crk-associated substrate; FAK, focal adhesion kinase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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