tyrosine phosphorylation of p125K. Thrombin reverses these effects of cAMP, and ..... Foster. City, CA) for viewing with a Zeiss Universal. II research microscope.
Tyrosine Phosphorylation of Focal Adhesion Kinase (p1 25FAK): Regulation by cAMP and Thrombin in Mesangial Cells1 Dean
A. Troyer,2
A. Bouton,
R. Bedolla,
and
Robert
Padilla (but
D.A. Troyer. R. Bedolla, thology, University of San Antonio, TX
R. Padilla, Department Texas, Health Science
A. Bouton, Department of Microbiology. Virginia School of Medicine, Charlottesville, (J. Am.
Soc.
Nephrol.
ing
and
of
composed
of actin with
adhesion
converge
of proteins,
a
includ-
to form the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMPelevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine
effects address
Thrombin
of cAMP, and this some of the cellular
regulating
the
This study
loss and
reverses
model can mechanisms
formation
to in
adhesions.
formerly
covered
cells
increases
by the
restores tyrosine
the cells resume
cell.
actin
a condensed traverse the
Addition
filaments
phosphorylation
a flattened
cell area
of thrombin
(stress of
p1
morphology,
to
fibers)
and
25FAK,
and
even
in the
continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole;
Sigma Chemical, the role of these induced D
the
adhesions
Received
24,
February
Science
of
adhesions. thrombin
phosphorylate
an agent
2 correspondence Health
of focal ability
and
nocodazole, 1
St. Louis, MO) were used to examine cytoskeletal elements in thrombin-
restoration
blocked
center,
7703 Floyd
25FAK.
November
Department curl
Drive,
San
of the
American
Society
focal
of
microtubules
14. 1995.
of Pathology, Antonio,
104&6673/0703-041 5$03.00/0 Journal of the American Society of Nephrology copyright © 1996 by the American Society of Nephrology
Journal
restore
The effects
that destabilizes
1995. Accepted
to Dr. D. Troyer,
p1
Cytochalasin to
of Nephrology
University T
78284-7750.
Mesangial
and can be modulated such as thrombin or via
of microtubules. cell,
focal
adhesion
kinase,
cell
shape
-thrombin
are
these
be used involved
of focal
cAMP-treated cells display with slender processes that
these
polymerization
frequently
observed in glomerular in cell-matrix interactions, by integrins, are likely involved
alterations
reports the effects of cAMP and thrombin on cell shape, distribution of actin, formation fibers, and tyrosine phosphorylation of
mesangial of stress
pl25. body
of p125K.
to
coagulation,
cells and
phosphorylation
similar
in this study
(thrombin), in addition to its effects on has hormone-like effects on cells, which include alterations of cell shape and stimulation of cell proliferation ( 1-3). Cellular proliferation (4,5) and accumulation of extracellular matrix (6,7)
kinase (p125F), and integrin areas of close contact between
receptors
are very
discussed
indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the
altered
filaments,
a number
receptor),
The findings
phosphorylation of pl25F by soluble receptor agonists
199#{243}; 7:415-423)
associate
focal
University VA
of thrombin.
Key Words:
ABSTRACT Stress fibers, upon
of PaCenter,
has no known
which
those
of Texas,
mediated
logical
changes have occupancy
integrins
tor
and those patho-
as well (8,9). The cytoplasmic domains of no enzymatic activity, yet integrin-receppromotes the formation of focal adhe-
sions, increases ( 10, 1 1), alkalinizes dylinositol 5-kinase
naling
diseases, including In these
tyrosine phosphorylation of pl25’ pH ( 1 2), and stimulates phosphati(13). Thus, proposed candidate sighave included focal adhesion-associ-
molecules
ated proteins such as p 1 25. However, less is known about the potential for other (nonintegrin) pathways that may regulate the integrated formation offocal adhesions and actin ifiaments. Soluble receptor agonists can modulate the aflIriity of integrin receptors for their ligands
( 1 4), and pathways adhesions tions,
vasopressin, may converge and thereby
including
cell
bombesin, on pl25’ regulate
shape,
growth,
and src-mediated to influence focal diverse cellular funcadhesion,
and
mo-
tiity ( 10, 15, 16). Convergence of these pathways may Involve autophosphorylation of p 1 25FAK and binding to pp60 src or to its normal counterpart, pp59’’#{176}, to direct
these proteins to sites of cellular adhesion (17,18). The studies presented here examine a model in which mesangial cell focal adhesions are rapidly reformed by thrombin after treatment of cells with cAMP-elevating agents. We conclude that thrombmn promotes the integrated formation of actin filaments and focal adhesions and stimulates tyrosmne phosphorylation of p 1 25FAK The effects of nocodazole (Sigma Chemical, St. Louis, MO), a microtubule toxin without
known
to
of thrombin,
those
are intimately fibers and focal
receptor-mediated
involved adhesions.
and
suggest in the
effects, that regulation
are
similar
microtubules of stress
415
Regulation
of
p12SFAK
by cAMP
and
Thrombin
METHODS Cell Culture Human mesangial cells were cultured lated from human kidneys Immediately phrectomy. as described previously (19).
from glomeruli isoafter surgical neCells were grown In Waymouth’s MB752/ 1 medium (Gibco) supplemented with ITS (insulin/transferrin, selenium; Collaborative Research) and 20% fetal calf serum (irvine Scientific). Before use, cells were serum starved for 48 h by incubation in RPM! supplemented with 10 mM N-hydroxyethylplperazIne-N’-2-ethanesulfonic acid (HEPES).
Reagents
and
Experimental
Conditions
The experimental medium was RPMI supplemented with 10 mM HEPES. Methyl-(5-E2-thlenylcarbonyll1H-Benzimldazol-2-yl) carbamate (nocodazole) was prepared as a 10mg/mL stock in dimethyl sulfoxide (DMSO). For experiments, the stock was diluted 1 : 100 into RPM! and used at 10 L/mL for a final concentration of 3.3 M. Thrombin (courtesy of Dr. John Fenton, Wadsworth Center Labs, Albany, NY) was used at a final concentration of 10 nM. SFLL (SFLLRNPNDKYEPF) was prepared as an acid by Peptide Technologies Corporation, Washington, DC, as described ( 19). Cytochalasin D (Sigma) was diluted into DMSO to make a 1 -mg/mL stock. For experiments, cytochalasin D was diluted into RPMI at 1 : 100 (vol/vol) and 10 L/mL used for a final concentration of 0. 1 g/mL. 8-(4-chlorphenylthio)adenosine-3’-5’-cyclic monophosphate (chlor-AMP) and isobutylmethylxanthine were purchased from Sigma. The standard experimental protocol was to treat serum-starved cells for 45 mm with a 1 -mM concentration of the cyclic nucleotide analog, chlor-AMP 18-(4-chlorphenylthlo) adenoslne-3’5’-cyclic monophosphatel and 1 mM MIX (lsobutylmethylxanthine). a potent phosphodlesterase inhibitor (20,2 1). Thrombin (10 nM), nocodazole (3.3 uM), or SFLL (50 M) were added directly to the experimental medium that contained cAMPelevating agents, and cells were incubated for 10 additIonal mm. Cytochalasin D was added directly to the experimental medium 10 min before the addition ofthrombin. Antibodies to pl25 (BC3 for Western blotting and 2A7 for immunoprecipitation and immunofluorescence) were donated courtesy of Dr. Thomas Parsons (University of Virginia).
Immunofluorescence The immunofluorescence-stalning conditions were adapted from a method described for the Immunoelectron microscopy of cultured cells (22). In brief, cells that had been grown on glass coverslips were fixed for 10 mm in 3.7% paraformaldehyde and washed in TBS-0.02% saponin (TBS Is 25 mM Tris, 0.9% NaCl). Cells were then incubated for 1 h in milk-TBS-saponln-sodium azide (TBS with 0.02% saponln, 5% dry milk, and 0.05% sodium azide). For the staining of F-actln, cells were then incubated for 20 mm at room temperature with rhodamine phalloidin and 1 U/200 pL ofphosphate-buffered sailne (PBS; Molecular Probes, Eugene OR). For staining of vinculln, fixed cells were incubated overnight at 4#{176}C with monoclonal antivinculln antibody (Sigma V4505) at a 1 : 100 dilution in milk-TBSsaponin-azide. Cells were washed four times for 15 mm each with TBS-saponin, followed by incubation for 4 h at 4#{176}C with the appropriate biotinylated secondary antibody (Vector Laboratorles, Burlingame, CA). Cells were again washed four times for 15 mm each with TBS-saponln, followed by incubation for 1 h at room temperature in the dark with 25 pg/mL of rhodam(ne-conjugated avidin (Pierce Chemical). Cells were then
416
washed three times with TBS-saponln and one time with TBS for 15 mm each. Coverslips were then rinsed and mounted on glass slides with Crystal Mount (Blomeda, Foster City, CA) for viewing with a Zeiss Universal II research microscope equipped for
epifiuorescence.
A
Zeiss
conical
microscope
(Center
for
Confocal Microscopy, Institute of Biotechnology, University of Texas Health Science Center) was used to localize focal adhesions as detected with the monoclonal antibody (MAb 2A7) to pl25 and secondary biotinylated antibody, followed by avidin-conjugated rhodamine as described above.
Assay
of Actin
Distribution
Cellular actin can be separated Into three pools: (1 ) a Tritoninsoluble, gelsolin-poor pool of filamentous F-actin; (2) a Triton-soluble, gelsolin-rich pool of F-actln, and (3) G-actin (23). The Triton-insoluble F-actln is a minority of F-actin, Is sedimented by 15,000 X g centrifugation, and is associated with a-actinin, but not gelsolin. TritonInsoluble actin likely has a cross-linked supramolecular structure (24). By contrast, gelsolin-assoclated F-actin accounts for the majority of cellular F-actin and requires high g forces (366,000 x g, 5 mm) to sediment. Gelsolin-assoclated Factin is likely present as short actln filaments (23). Finally, G-actin Is monomeric, accounts for the majority of actin in unstimulated cells, and fails to sediment even at high g forces (23). Cells cultured In 100-mm dishes were washed once with PBS at room temperature after experiments. Imidazole buffer (1 .5 mL) was added ( 10 mM imidazole, 10 mM EGTA, 40 mM KC1, 0. 1 mg/mL aprotlnin, 1 mM polymethylsulfonyl fluoride (PMSF), and 1% TrIton X- 100). Aprotinin was added fresh each time buffer was prepared, and PMSF was added from a 100 mM stock In DMSO that was maintained frozen in aliquots. Cells were lysed for exactly 15 min at room temperature. The imidazole buffer that contained Triton-soluble actin was removed, placed In an ultracentrifuge tube, and stored at 4#{176}C. An additional 1 .5-mL aliquot of imidazole buffer was added, which contained 0.5 tL/mL of Benzonase (Sigma), and cells were then lysed for an additional 15 mm. Cells were then scraped into the imidazole buffer and centrifuged at 15,000 x g for 2 mm at room temperature to pellet the Triton-insoluble actin. The supernatant was discarded, and the pellet resuspended in 250 tL of 2 X sample buffer. The Triton-soluble actin (present In the stored buffer that was removed earlier from the monolayer) was sedlmented at 366,000 x g for 7 min at 4#{176}C. The resulting pellet was resuspended in 200 pL of 1 X sample buffer while the supernatant was used to precipitate G-actin by the addition of 15 L of 0. 15% deoxycholate (DOC) to each sample. After being mixed, samples were incubated for 10 min at room temperature, followed by an addition of 15 L of 72% trichloroacetic acid. Samples were then mixed and centrifuged for 10 mm at 15,000 X g. The resulting supernatant was discarded, and the pellet resuspended In 250 L of 1 X sample buffer. Before being loaded onto electrophoresis gels, the samples were boiled for 5 to 10 min in sample buffer that contained 0.05% mercaptoethanol. Equal sample volumes were then loaded onto a 4% stacking sodium dodecyl sulfate-polyacrylamide gel over a 7.5% resolving gel according to manufacturers specifications (BioRad). Initial protein determinations (BCA Protein Assay Kit; Bio-Rad) indicated that total protein content for the Tritoninsoluble fraction was 1 75 to 225 g and 80 to 100 g for the 366,000 x g fraction. We did not attempt to assay the G-actln fraction for protein because of the presence of DOC. Subse-
Volume
7
.
Number
3
‘
1996
Troyer
quently, we targeted loading 0.5 to 1 g of protein for the 15,000 x g Tritoninsoluble fraction and 2.5 to 3 g for the 366,000 x g Triton-Insoluble fraction. Because total proteins varied from sample to sample by less than 10%, we routinely loaded by volume. After electrophoresis for 1 h at 200 V (Blo-Rad MiniProtean II apparatus), samples were blotted onto nitrocellulose overnight at 4#{176}C at 400 milhlamps. Actin was detected using antl-actln antibody, clone C4 (Boehrlnger) at 1 ug/mL. Bound primary antibody was detected using the Vectastain ABC kit (Vector, Burlingame, CA).
Tyrosine
Phosphorylation
Tyrosine phosphorylation of focal adhesion kinase (pl25) was assayed by immunoblotting with or without prior Immunoprecipitation of p125. Cells were lysed with modified RIPA lysis buffer ( 1% Nonidet P40, 20 mM Tris, 150 mM NaCl, 1 mM Na3VPO4, 5 mM EDTA, 0. 1 mg/mL aprotinin, and 1 mM PMSF). Aprotinin was stored as a 10-mM stock in frozen aliquots, and added fresh before each extraction. PMSF was prepared as a 100-mM stock in DMSO, stored as frozen aliquots, and added fresh to a final concentratlon of 1 mM. Cells grown in 100-mm tissue culture dishes were used for experiments. Cells were washed 2 X with cold PBS before the addition of RIPA lysis buffer for 10 min. Cells were scraped and pipetted into pestle eppendorf tubes (VWR Scientific), and subjected to 20 twIsts with the pestle while on ice. After centrifugation of cells for 20 min, supernatants were transferred to fresh eppendorf tubes and protein determined by use of the BCA protein assay kit (Bio-Rad). Twenty i.i.g of this initial lysate was loaded onto gels for electrophoresis and blotting If no immunoprecipitation was done. After immunoprecipitatlon with the antibody 2A7, followed by Western blotting with the BC3 antibody, and In collaboration with Drs. Michael Schaller and Amy Bouton, the amount of p125 that was extracted from control, cAMP, and cAMP+thrombIn-treated cells was the same (not shown). We therefore proceeded with the experiments by assaying tyrosine phosphorylation directly or after immunoprecipitation with the monoclonal antibody 2A7. For use in Immunoprecipitation, protein A Sepharose (Sigma) was prepared as follows. One g of protein A Sepharose was added to 15 mL of PBS + 0.02% sodium azlde and allowed to mix overnight at 4#{176}C. After settling for 30 mm, the PBS was aspirated to obtain a 50% suspension of protein A Sepharose. One hundred L of protein A Sepharose per sample was then aliquoted into an eppendorf tube, centrifuged for 5 mm at 4#{176}C, and the pellet then resuspended in 10% BSA (Sigma) made up in RIPA lysis buffer containing 10% glycerol (BSA buffer). The ratio of sepharose to BSA buffer was 1 :3 (wt/vol). After three washes in the 10% BSA-contalning buffer, the protein A Sepharose was Incubated with rabbit antimouse immunoglobulin G (Sigma) overnight at 4#{176}C. The protein A Sepharose was washed three times with 10% BSA buffer and resuspended in an amount of 10% BSA buffer sufficient to aliquot 100 iL to each immunoprecipitate.
For immunoprecipitation, 500 g of cell lysate was incubated with 4 j.tL ofanti-p 1 25’ antibody, MAb 2A7 (courtesy of Dr. Thomas Parsons) overnight at 4#{176}C. One hundred L of protein A sepharose, prepared as described above, was then added with gentle mixing for 1 #{189} h. Immunoprecipitates were then centrifuged and washed with RIPA/glycerol buffer. The washed pellet was resuspended in 95 pL of 2 X sample buffer. After reduction, samples were loaded onto a 7%
Journal
of the
American
Society
of Nephrology
et al
resolving SDS-PAGE gel with a 4.5% stacking gel. Samples were then electrophoresed at constant current (35 milliamps) for 4 to 5 h at 4#{176}C. The samples were then electroblotted onto nitrocellulose overnight at 4#{176}C at constant current of 400 milliamps. Phosphotyrosine-contalning proteins were detected by immunoblotting. Blots were blocked with blocking buffer (10 mM
Tris
base,
150
mM
NaC1,
and
3%
BSA)
for
2
to
3
h
followed by incubation with antiphosphotyrosine antibody (PY2O: ICN) In 10 mL of wash buffer (10 mM Tris base and 150 mM NaC1) for 2 h at room temperature. After being washed with wash buffer twice, the blot was washed twice with detergent buffer (wash buffer supplemented with 0.5% Tween 20 and 0.5% Nonidet P40). The blot was then incubated with 3 Ci of 1251 protein A (Amersham) in 10 mL of blocking buffer for 1 h, followed by two washes each in wash buffer and detergent buffer. The blot was then developed by autoradiography to detect radiolabelled proteins.
Image
Analysis
Autoradiographs and Western blots were analyzed by using IMAGE, an image-processing and analysis computer program for the Macintosh computer, as previously described (25). Relative band densities are displayed in the accompanying graphs and figures. For the studies of actin distribution that were assayed biochemically, data was accumulated in six to ten separate experiments, with duplicates for each variable for each actin fraction. For statistical analysis, either t test or two-way analysis of variance (ANOVA) (experiment by treatment) was used. All assumptions underlying the ANOVA were well satisfied. Comparison of means was by Duncan’s new multiple range test.
RESULTS Morphological
of cAMP
Changes
and
Induced
by
Elevation
Thrombin
cAMP was elevated by incubating cells for 45 mm with the cyclic nucleotide analog, chlor-AMP (8-14-chlorphenylthlo]adenosine-3’-5’-cyclic monophosphate) ( 1 mM) and the phosphodlesterase inhibitor, isobutylmethyixanthine (also 1 mM). During this period of time, the fiat well-spread cells developed a rounded cell body with thin processes that traverse the area formerly covered by the flatter (non-cAMP-treated) cells. Photomicrographs of untreated control cells stained for actin (Figures 1A and 1B), can be compared with Figures 1C and 1D, which demonstrate the fragmentation of stress fibers in cAMP-treated cells in which actin stains as clumps or short segments. As shown in Figure 2, cAMP also causes loss ofvinculln-containlng focal adhesions. When added to cAMP-treated cells, thrombin restores a flattened cellular morphology and reestablishes stress fibers (Figures 1E and iF). This effect ofthrombin is duplicated by the peptide SFLL (25 SM), which mimics the tethered ligand portion ofthe thrombin receptor ( 19) (not shown).
There
are
qualitative
morphological
differences
between
the control focal adhesions and those formed after thrombin treatment, but areas of concentrated staining for vinculin reemerge, as shown in Figure 2C. This difference between control and cAMP/thrombin focal adhesions may occur because of the fact that focal adhesions were present for a longer period of time in
417
Regulation
of pl25
by cAMP
and
Thrombin
Figure 1 Fluorescence photomicrographs of cells stained with rhodamine phalloidin to detect stress fibers. (A, B) Control cells at low and high magnifications (bar 10 tm). (C, D) Cells treated for 45 mm with cAMP-elevating agents. (E, F) Reversal of the effects of elevated cAMP by treatment of cells with 10 nM thrombin for 10 mm. The general experi.
=
mental follows:
protocol
nuns.
used
for this and
other
experiments
35
0
I add
add
Cyto
thrombin.
assay
or
actin.
SFLL.
agents
I
D
add
cAMP
as
55
45
H elevating
was
P-lye. morphology
for
nocodazole
control cells than for those treated with cAMP-elevating agents followed by treatment with thrombin. In a separate study, we demonstrated that the same protocol as utilized for standard microscopic studies of vinculin localization caused loss and reformation of focal adhesions Images
as
detected by confocaJ microscopy (not shown). taken at 0.25-SM intervals showed loss and re-formation of concentrated areas of staining for p 1 25FAK
that
at the
although
ventral
standard
focal adhesions are morphologicaily
surface
of the
microscopy
cell.
does
formed after treatment smaller than those
This
suggests
indicate
abilized
that
with thrombin of controls, the
concentrated re-form focal as one would
areas of staining (as proteins aggregate to adhesions) are on the ventral cell surface expect. Nocodazole has effects on cAMP-
treated thrombin
that
Actin As
cells
(not
are
essentially
identical
those
shown).
Distribution confirmation
and
extension
of the morphological of cAMP and throm-
studies, we examined the effects bin on the cellular distribution ofactin.
418
to
Three
cellular
Figure
of
2. Fluorescence
photomicrographs
cells stained
for the detection
of fixed
of vinculin.
trol. (B) Cells treated with cAMP-elevating agents 1 . (C) Reversal of shape change after treatment thrombin for 10 mm as in Figure 3. Bar = 10 m.
perme-
(A) Conas in Figure
with 10 nM
pools of actln can be distinguished (23). The first is a cytoskeleton-associated pool present In Triton-X100 lysates centrifuged at 15,000 x g that contains a-actinin but not gelsolin (also referred to as the Triton-insoluble
F-actin). This 15,000 X g ( 15K) fraction is thought to have a cross-linked supramolecular structure (24). The second actin pool contains gelsolin and is pelleted after centrifugation at 366,000 X g (366K), and consists of
Volume
7
-
Number
3
‘
1996
Troyer
short actin Itated
actin ifiaments associated pool contains monomeric by use of trichloroacetic
As shown the relative increasing thrombin
with gelsolin. G-actln, which acid.
et al
The third Is precip-
in Figure 3, elevation of cAMP decreases amount of actin in the 15K fraction while the amount of G-actin. The addition of to cells treated with cAMP-elevating agents
reverses amount G-actin significant alone, treatment. looked
these changes, increasing the relative ofactin in the 15K fraction and decreasing the fraction. For the 366K actin, there was a decline induced by thrombin treatment and a trend toward an increase with cAMP In separate experiments during which we only at the 366K fraction, we demonstrated that elevation of cAMP increased the content of 366K actin relative to controls (control, 61 ± 7; cAMP, 79 ± 5; N 3, P < 0.05) and that addition of thrombin to cells with elevated cAMP decreased the relative content of 366K actin (cAMP, 68 ± 4; cAMP + thrombin, 54 ± 6; N = 3, P < 0.05). These experiments confirmed
the trends for 366K actin, as shown in Figure 3. Thus, the assays of Triton extracts for actin tend to confirm the morphological observations that cAMP leads to fragmentation of actin ifiaments and thrombin reverses this effect.
Tyrosine Phosphorylation Kinase (p125”) signaling
Various pl25
to
of Focal pathways cell shape,
influence
Adhesion
may growth,
converge and
on adhe-
70
4. Fluorescent photomicrograph of fixed permeabilized control cells stained for p125. Focal adhesions stain as short-linear or dagger-shaped areas toward the cell periphery (arrows). Original magnifIcation, x 975. Figure
366K
.-..*---
60 Relative Density
U
50 -0--
15K 0
-
actin
slon. As mesangial
40
to throm
con
cAMP
cAMP
+
throm
and cAMP elevation followed by thrombin (cAMP + throm) on the distribution of actin. Values shown indicate mean ± SE of relative densities derived from Western blots. Closed squares: Decreased 15K actin in cells treated with cAMPelevating agents and Its normalIzatIon by the addition of thrombin (cAMP+throm). indicates that P < 0.05 comparing cAMP treatment with control, thrombin, or cAMP + a-thrombln treatment; N 10. Closed friangles: decreased 366K actln after treatment with thrombin. indicates P < 0.05 comparing thrombin with control, cAMP, or cAMP + thrombin; N = 8. Open squares: increased G-actln after elevation of cAMP, and normalIzation after the addition of thrombin (cAMP + throm). Indicates that for G-actln, cAMP and cAMP + a-thrombin-treated cells were signIficantly different from each other and from control or thrombln alone (P < 0.05); N = 8. Figure
3. Effects
of
thrombin
(throm),
cAMP,
=
*
Journal
of the American
Society
of Nephrology
that
shows
shown in Figure cell focal adhesions of vinculln
the
(compare
analysis
pl2S by the immunopreclpitation.
of
4,
pl25 Is in a distribution with
tyrosine
Immunoblotting A protein
Figure
present in similar 2).
Figure
phosphorylation of cell lysates ofapproximately
5
of without 1 25 kd
demonstrates ment with
decreased phosphorylation after treatcAMP, and increased phosphorylation after treatment with thrombin in the continued presence of cAMP (26). AddItional (separate) studies were carried out after immunoprecipitation with mAb 2A7 as described in “Methods.” Figure 5 shows the appearance of an autoradlograph in which tyrosine phosphorylated proteins are detected either without inununoprecipitation (left three lanes), or after immunopreclpitation (right three lanes). The most abundant tyrosine-phosphorylated protein in the Western blots of lysates Is near kd, and a band at this same location is Immunoprecipitated by the antibody 2A7, shown of Figure 5. Figure 6 shows that lion of p125 immunoprecipitated
125
in the right three lanes tyrosine phosphorylawith 2A7 Is de-
419
Regulation
of p125
