Tyrosine Phosphorylation of Focal Adhesion Kinase Regulation by ...

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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-

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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

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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