NOTES Tyrosyl and Cyclic AMP-Dependent Protein Kinase Activities in BHK Cells That Express Viral pp6Osrc
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MOLECULAR AND CELLULAR BIOLOGY, May 1984, p. 973-977 Vol. 4, No. 5
0270-7306/84/050973-05$02.00/0
Copyright © 1984. American Society for Microbiology
NOTES
Tyrosyl and Cyclic AMP-Dependent Protein Kinase Activities in
BHK Cells That Express Viral pp6Osrc
GAIL M. CLINTON* AND ROBERT ROSKOSKI, JR.
Department of Biochemistry, Louisiana State University Medical Center, New Orleans, Louisiana 70112
Received 2 November 1983/Accepted 19 January 1984
Protein kinase activities were measured in Rous sarcoma virus-infected baby hamster kidney (BHK) cells
that express v-src (BHK [v-src]) and compared with those of revertant and control BHK cells. We observed
about a fivefold-higher tyrosine phosphorylating activity in BHK (v-src) cell extracts, which was due to src
but not other cellular tyrosyl kinase activities since preincubation with anti-src serum reduced the activity to
control cell levels. The cyclic AMP-dependent protein kinase activity was also altered when v-src was
expressed. Resolution of the two cyclic AMP-dependent isozymes from the detergent-soluble fraction of
cells revealed that the type I activity was selectively decreased about fivefold in BHK (v-src) cells.
Phosphorylation of proteins by protein kinases can alter cAMP-independent tyrosine-, threonine-, and serine-specific
the activity of enzymes and the growth properties of cells. as well as cAMP-dependent protein kinase activities in the
The transforming proteins of several retroviruses are associ- different BHK cell lines were compared. The origin and
ated with cyclic AMP (cAMP)-independent, tyrosine-specif- properties of the BHK cells employed, details of their
ic protein kinase activity. The v-src gene product encoded infection with RSV, and measurements of their v-src gene
by Rous sarcoma virus (RSV) is one of the best-described expression have been previously described (4, 10, 17). BHK
tyrosyl kinases (1). Transformation of cells by RSV involves cells that express v-src [BHK (v-src)] also express v-src
amplification of tyrosine phosphorylation in cellular proteins tyrosyl kinase activity, whereas BHK control cell cultures
(20). It is currently unknown whether v-src tyrosyl kinase is which have not been exposed to RSV and the revertant
solely responsible for the amplified phosphotyrosine in cell RSV-infected BIK cell lines do not express v-src. The
proteins or whether other tyrosyl kinases, activated by v- revertant cells contain the RSV proviral genome but do not
src, also phosphorylate proteins in transformed cells. In express v-src mRNA or protein (4, 10). BHK cells were
addition to changes in tyrosine phosphorylation, the degree chosen since the RSV-infected and control BHK cells are
of serine and threonine phosphorylation in some phospho- continuous cell lines with similar growth rates. Differential
proteins is also altered in RSV-transformed cells (6). It is expression of protein kinases, particularly the cAMP-depen-
therefore possible that protein kinase levels (other than dent kinase during the cell cycle (3, 9, 11, 21), should
tyrosyl kinase) might change in cells as a direct or indirect therefore not contribute to differences in enzyme levels in
result of amplified v-src expression. the BHK cells.
The cAMP-dependent protein kinase phosphorylates ser- To determine whether cellular tyrosyl kinases (other than
ine and to a lesser extent threonine in cellular proteins and src) are amplified in response to v-src expression, we first
has been shown to affect cell metabolism by altering activi- measured the total cellular tyrosine-specific kinase activity
ties of several metabolic enzymes (13). The activity of the in the BHK cells. The components of the reaction mix were
cAMP-dependent enzyme is mediated by cAMP levels in adjusted to give maximum levels of 32P-labeled product with
cells. Two types of cAMP-dependent protein kinases exist either RSV-infected or control BHK cell extracts. For these
(8); they contain the same catalytic subunit but have differ- assays, BHK cell extracts were prepared by trypsinizing
ent regulatory subunits which have been postulated to play cultures, washing cells with phosphate-buffered saline, and
separate functional roles as a result of their different charac- disrupting in the components of the final reaction mixture
teristics. The two types are differentially activated during the without ATP or substrate at 107 cells per ml. Equal amounts
changing developmental status of some cells (12, 14, 15, 19) of protein (12 ,ug) from cell extracts were added to give a
and during cell cycle progression (3, 9, 11, 21). final volume of 100 RI containing 10 mM Tris (Sigma
Regulatory interactions between v-src tyrosyl kinase and Chemical Co., St. Louis, Mo.) (pH 8.0), 10 mM MnCl2, 5
the cAMP-dependent protein kinase have not been exten- mM dithiothreitol, 1% Nonidet P-40 (Particle Data Lab, St.
sively investigated. The v-src protein has a serine residue Louis, Mo.), 25 puM sodium vanadate (Fisher Scientific Co.,
that is phosphorylated by the cAMP-dependent protein Pittsburg, Pa.), and 10 puCi of [y-32PIATP (New England
kinase (5), but changes in the cAMP-dependent kinase Nuclear Corp., Boston, Mass.). The substrates used includ-
activity does not apparently affect the v-src tyrosyl kinase ed endogenous cellular proteins or angiotensin II peptide
activity, at least in RSV-transformed vole cells (16). (23). The pH optima of the reactions were found to be from
Studies were initiated to investigate whether v-src expres- 6.5 to 8.8. MnCl2 concentrations of 10 mM were used since
sion affected the activities of cellular protein kinases that are lower levels resulted in decreased tyrosyl kinase activity,
involved in cell metabolism and growth. Overall levels of whereas concentrations higher than 10 mM resulted in
precipitation of protein from the reaction. Substitution of
MgCl, for MnCl2 caused a four- to fivefold decrease in
*
Corresponding author. tyrosine phosphorylation. This was not due to inhibition of
973974 NOTES MOL. CELL. BIOL.
the enzyme, since the addition of MgCl2 to a reaction [32P]angiotensin was not above background. The rate of 32p
containing MnCl2 did not inhibit tyrosyl kinase activity. incorporation into angiotensin was about fivefold higher in
Changes in the concentration of Nonidet P-40 from 0.5 to extracts from BHK (v-src) cells (data not shown). Because
1.5% had little or no effect on the enzyme, nor did alterations angiotensin contains tyrosine but not serine or threonine
in dithiothreitol concentration. The extent but not the initial residues and is a substrate for tyrosine phosphorylation (23),
velocity of tyrosine phosphorylation was increased nearly our results indicated that tyrosyl kinase activity was fivefold
twofold by the addition of 25 ,uM vanadate, a phosphotyro- higher in the BHK (v-src) cells. Thus, the magnitudes of the
syl phosphatase inhibitor, which was included in all reac- difference in tyrosyl kinase activity in the BHK cells were
tions. Most other assays previously conducted for active similar when endogenous cellular proteins and angiotensin
tyrosyl kinase have used an immune complex reaction with were the substrates in the kinase reaction. The results
antibodies directed against single, specific viral enzymes (1). obtained by using soluble substrates to measure tyrosyl
Our assays, on the other hand, contained substrates which kinase activity in cell extracts are consistent with observa-
are phosphorylated by several viral and cellular tyrosyl tions that phosphotyrosine is amplified in vivo in several
kinases in a soluble assay in which the kinetics of the RSV-transformed cells (20) as well as in BHK (v-src) cell
reaction can be investigated. proteins (G. M. Clinton, unpublished data).
The levels of 32p incorporation with endogenous cellular To test directly whether cellular tyrosyl kinases, in addi-
protein substrates in the BHK cell extracts were measured tion to src, may be amplified in BHK (v-src) cells, we
as described above. Maximum 32p incorporation into protein measured the enzyme levels by using angiotensin as a
was reached 5 min after the reaction was initiated, and this substrate in cell extracts which had been reacted with anti-
level was maintained for at least 10 min (data not shown). To src serum that recognizes both viral and cellular src proteins
assess the levels of tyrosine phosphorylating activity, phos- (Clinton, unpublished data). The src-specific serum (but not
phoamino acid analyses of the products at 5 min of incuba- serum from normal rabbits) reduced the tyrosyl kinase
tion were conducted as previously described (4). Quantita- activity in transformed cells about fivefold to the levels
tions of acid-stable, [32P]phosphotyrosine revealed a 5.8- observed in the control BHK cells reacted with either
fold-higher phosphate incorporation with extracts of BHK normal or anti-src serum (Fig. 1). Moreover, when endoge-
(v-src) cells compared with control BHK cells (2.9 versus 0.5 nous proteins were the substrates, tyrosine phosphorylation
fmol/mg of protein per min). The absolute amount of phos- by I3HK (v-src) cell extracts was reduced to the levels
phate transferred to tyrosine, however, is an underestimate observed in the control cells (data not shown). This finding
due to incomplete hydrolysis of phosphopeptides as well as strongly suggests that the activities of cellular tyrosyl protein
hydrolysis of phosphomonoester bonds during acid hydroly- kinases (other than src) are not detectably affected as a result
sis of the 32P-labeled product. When casein was added to the
assay, we observed similar levels of tyrosine phosphoryla-
tion, indicating that endogenous tyrosyl kinase substrates 1200
were in excess of the enzyme (data not shown).
Quantitations of [32P]phosphoserine and [32P]phospho-
threonine in the acid-hydrolyzed endogenous proteins from 1000
the kinase reactions described above revealed that for the
BHK (v-src) cells, 5.0 and 15.2 fmol of phosphate per mg of
protein per min were transferred to threonine and serine a 800
residues in proteins, respectively. For the control BHK
cells, we found that 5.3 and 14.7 fmol of phosphate per mg of 0..
protein per min were transferred to threonine and serine E 600
residues, respectively. Thus, the overall levels of serine and
threonine phosphorylating activities, measured under these E
E
specific reaction conditions, were not greatly altered as a 400
result of v-src expression. When casein was added to the
reaction, we observed similar levels of threonine phosphor-
ylation, but serine phosphorylation was increased by ca. 200
50% in each BHK cell extract (data not shown). Although
these assays may not detect alterations in minor protein
kinases in cells, they suggest that the overall levels of serine
and threonine phosphorylating activities remain undisturbed 0 2.5 5.0 7.5 10.0
by v-src expression. TIME (min)
To determine whethet a substrate other than endogenous FIG. 1. Tyrosyl-protein kinase activity in BHK cells reacted
cellular proteins would reveal additional tyrosyl kinase ac- with anti-src and preimmune rabbit serum. Cell extract protein (12
tivity in BHK (v-src) cells, 100 ,ug of angiotensin II (Asp- ,ug) was reacted at 4°C for 1 h with 5 ,ul of anti-src serum (serum
Arg-Val-Tyr-Ile-His-Pro-Phe; Sigma Chemical Co.) was from RSV-infected, tumor-bearing rabbits that reacts with v-src and
added to the kinase reaction mixture. The reaction was c-src) or 5 p.l of control serum (serum from normal rabbits). Both
incubated at 34°C, and 25-,ul portions were removed, incu- sera had been preincubated at 56°C for 30 min to remove residual
bated in a boiling-water bath for 2 min, and centrifuged in a serum kinase activity. The immunoglobulin G was absorbed by
microfuge for 5 min. Portions (10 ,ul) were spotted on incubation with 25 p.l of a 105% suspension of Staphylococcus aureus
Whatman 3MM filter paper, and the phosphorylated angio- (IgG sorb; The Enzyme Center, Inc., Boston, Mass.). S. aureus
cells were removed by low-speed centrifugation, and the superna-
tensin was resolved from other components by paper elec- tant was assayed in a reaction with angiotensin substrate. Symbols:
trophoresis at pH 3.5 as described previously (23). As a (0) BHK (v-src) cells reacted with control serum, (U) BHK (v-src)
control, a reaction mixture containing either boiled cell cells reacted with anti-src serum, (0) BHK control cells reacted
extract or all components except angiotensin was electro- with control serum, and (-) BHK control cells reacted with anti-src
phoresed, and the 32P-labeled material that comigrated with serum.VOL. 4, 1984 NOTES 975
of v-src expression. Therefore, increased phosphotyrosine TABLE 1. Subcellular distribution of AMP-dependent protein
in several cellular proteins in RSV-transformed cells may be kinase activity in control and BHK (v-src) cellsa
entirely due to the v-src kinase activity or may be due partly Protein kinase activity'
to cellular tyrosyl kinases that are translocated to sites in Cell cAMP cAMP
which substrate proteins are more accessible. The first dependent independent
possibility is supported by observations that the phosphoty- Detergent-soluble
rosine levels in proteins are rapidly reduced to nearly normal BHK 228 ± 8 1,360 ± 112
levels by shifting temperature-sensitive RSV-transformed BHK (v-src) 151 ± 8 800 ± 81'
cells to the nonpermissive temperature (20).
The results obtained with antiserum also illustrate the Detergent-insoluble
validity of the soluble assay for comparing tyrosyl kinase BHK 87 11 425 13
levels in cells. It might have been possible that the differen- BHK (v-src) 87 ± 4 379 ± 24
tial rates of angiotensin or endogenous protein phosphoryla- a Fractions were assayed as described in the text.
tion were due to factors that interfere with measurements of b
Picomoles of phosphate transferred per minute per milligram of
tyrosyl kinase activity such as phosphotyrosyl phosphatase, protein in the unfractionated cells was measured for 5 min at 34°C.
ATPase, protease, or enzyme inhibitors. Because the differ- C
P < 0.01.
ence in tyrosyl kinase activity in the BHK cell lines was
eliminated by immunoprecipitation of a src-related protein
from the cell extract, we concluded that our assays reflect and a wash in 0.42% H3PO4 as previously described (18).
the levels of tyrosyl kinase and are not greatly influenced by The rate of cAMP-dependent and independent serine-pep-
different levels of the above components. tide phosphorylation in both detergent-soluble and insoluble
The active forms of virally coded tyrosyl kinases are fractions was linear for 10 min and reached a plateau by 15
bound to the detergent-insoluble matrix, which contains the min (not shown). In agreement with the levels of serine
cytoskeleton of cells (7), whereas the protein kinase associ- phosphorylation, the cAMP-independent, serine phosphory-
ated with the epidermal growth factor receptor apparently lating activity measured with the serine-peptide substrate
fractionates with the detergent-soluble material, which con- was not altered in activity or association with the cytoskele-
tains soluble cytoplasmic, nuclear, and membrane proteins. ton-containing fraction in the BHK (v-src) cells (Table 1).
BHK (v-src) and control BHK cells were fractionated into Although the cAMP-dependent activity in each cell line was
the detergent-soluble and detergent-insoluble matrix by the the same in the cytoskeleton-containing fraction, there was
method of Brown et al. (2). Monolayer cell cultures (60-mm an apparent twofold decrease of activity in the detergent-
dishes) were washed twice with phosphate-buffered saline, soluble enzyme from the BHK (v-src) cells (Table 1). The
and the detergent-soluble fraction was extracted at 4°C by fraction of cAMP-dependent protein kinase activity in the
the addition of 0.25 ml of extraction buffer containing 100 detergent-soluble fraction was ca. 76% in the control cells
mM KCI, 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) and 67% in the BHK (v-src) cells.
disodium salt (PIPES; Sigma Chemical Co.) (pH 6.8), 0.5%
Nonidet P-40, 300 mM sucrose, 1% trasylol (Aprotonin;
Sigma Chemical Co.), and 3 mM MgClI. After 3 min at 4°C,
the soluble fraction was removed, and the residue was
washed with 0.5 ml of extraction buffer which was then
discarded. The detergent-insoluble material was scraped C
from the plate in 0.25 ml of extraction buffer, and both
detergent-soluble and insoluble fractions were assayed for C
protein kinase activity. Equal amounts of protein from the r-
soluble fraction and an equivalent portion from the insoluble .E
fraction were assayed. By using endogenous cellular pro- E
teins or endogenous cellular proteins with added casein as
the substrate, we found that ca. 80% of the tyrosine phos-
phorylating activity was associated with the cytoskeleton- LYJ
containing, insoluble fraction in BHK (v-src) cells. This is in
agreement with previously published results that show the
majority of v-src protein to be associated with the detergent- 5 10 15 20
insoluble fraction of cells. As was found by using whole-cell FRA CT ION
extracts, the serine and threonine phosphorylating activities FIG. 2. cAMP-dependent protein kinase profiles of extracts from
in fractionated cells were not detectably affected by v-src control and BHK (v-src) cells. The cells were dispersed in 10 mM
expression (data not shown). potassium phosphate buffer (pH 7) with three 10-s bursts on a
To test whether v-src kinase activity may affect the Tekman Tissuemizer (70 setting). The extract was centrifuged at
activity and localization of the cAMP-dependent protein 20,000 x g for 10 min, and a portion of the supernatant was applied
kinase, the enzyme was measured in BHK (v-src) and to a column of DE-52 (0.5 by 1 cm) equilibrated with the same
control BHK cells. Enzyme activity was measured in a total buffer. After the column was washed with 5 ml of buffer, a linear
volume of 50 ,ul containing the following: 50 mM morpholin- gradient of 16 ml (total ) of 0 to 0.4 M KCI (in 10 mM phosphate; pH
opropane sulfonic acid (pH 7.0), 10 mM MgCl2, 100 mM 7) was used to elute the enzyme in 1.0-ml fractions. Enzyme activity
KCI, 0.25 mM ethylene glycol-bis(,-aminoethylether)-N,N- (25 ,ul) was measured + cAMP by using serine-peptide as the
tetraacetic acid 1 mg of bovine serum albumin per ml, 100 substrate. Incubations were for 10 or 15 min at 30°C. The type I
p.M y-32P]ATP (250 cpm/pmol), and 100 p.M Leu-Arg-Ala- enzyme, by convention, is the enzyme which elutes at the lower KCl
concentration. The protein applied to the column was determined by
Ser-Leu-Gly in the presence of 20 p.M cAMP (when speci- using the Bio-Rad protein assay kit (Bio-Rad Laboratories. Rock-
fied). The phosphorylated peptide was resolved from ATP ville Centre, N.Y.). Symbols: (0) BHK cell extracts and (0) BHK
and its metabolites by application to phosphocellulose strips (v-src) cell extracts.976 NOTES MOL. CELL. BIOL.
Because different cAMP-dependent enzyme activities and support the concept that amplified v-src expression may
were found in the soluble fraction of the two cell lines, we directly or indirectly affect cAMP-mediated phosphorylation
determined the proportion of the type I and II isozymes by processes.
DEAE-cellulose chromatography. In the control BHK cells, This work was supported by Public Health Service grants CA-
there were approximately equal amounts of the type I and II 34517, NS-15997, and HL 24791 from the National Institutes of
isoenzyme forms (Fig. 2; Table 2). Coinciding with v-src Health and by a grant from the E. G. Schlieder Foundation.
expression, however, there was a four- to fivefold decrease We thank Patricia Ritchie, Tina Tan, David J. Tate, Jr., and Joelle
in the activity of the type I enzyme (Table 2). A modest and Whelen for valuable technical assistance. We also thank Herman
statistically significant 38% decrease in the activity of the Oppermann for the generous gift of the pp6Osrc antiserum. We
type II enzyme was also observed. To test further whether gratefully acknowledge the secretarial assistance of Wanda Santa
the decrease in the cAMP-dependent enzyme was due to Marina and Mildred Williams.
elevated v-src expression, the isozymes were measured in LITERATURE CITED
revertant RSV-infected BHK cells which contain the RSV
genome but do not express v-src. The amounts of type I and 1. Bishop, J. M., and H. Varmus. 1982. Functions and origins of
type II enzymes in the revertant BHK cells were indistin- retroviral transforming genes, p. 999-1108. In R. Weiss, N.
guishable from the control, uninfected BHK cells (data not Teich, H. Varmus, and J. Coffin (ed.), Molecular biology of
tumor viruses: RNA tumor viruses, 2nd ed. Cold Spring Harbor
shown). Laboratory, Cold Spring Harbor, N.Y.
To determine whether these differences in activity could 2. Brown, S., W. Levinson, and J. A. Spudich. 1976. Cytoskeletal
be related to the presence of a soluble activator in the control elements of chick embryo fibroblasts revealed by detergent
BHK cells or to an inhibitor in the BHK (v-src) cells, extraction. J. Supramol. Struct. 5:119-130.
extracts from the two cell lines were mixed and assayed for 3. Byus, C. V., G. R. Klimpel, D. 0. Lucas, and D. H. Russell.
enzyme. In samples from the DEAE-cellulose chromatogra- 1977. Type I and Type II cyclic AMP-dependent protein kinases
phy or from detergent-soluble extracts, the activities were as opposite effectors of lymphocyte mitogenesis. Nature (Lon-
additive. This makes the presence of a soluble activator or don) 268:63-64.
an inhibitor as a mechanism for the decrease observed in the 4. Clinton, G. M., N. G. Guerina, H.-Y. Guo, and A. S. Huang.
BHK cells with elevated v-src protein unlikely. 1982. Host-dependent phosphorylation and kinase activity asso-
ciated with vesicular stomatitis virus. J. Biol. Chem. 257:3313-
The mechanism whereby the reduction in type I cAMP- 3319.
dependent kinase activity occurred is unknown. A direct 5. Collett, M. S., E. Erikson, and R. L. Erikson. 1979. Structural
effect of src on cAMP kinases has not apparently been analysis of the avian sarcoma virus transforming protein: sites
investigated, since the presence or absence of phosphotyro- of phosphorylation. J. Virol. 29:770-781.
sine in the regulatory or catalytic subunits in RSV-trans- 6. Cooper, J. A., and T. Hunter. 1981. Four different classes of
formed cells has not been reported. It is possible that the retroviruses induce phosphorylation of tyrosines present in
alteration in the proportion of the isozymes that we observed similar cellular proteins. Mol. Cell. Biol. 1:394-407.
7. Cooper, J. A., and T. Hunter. 1982. Discrete primary locations
may not be caused by tyrosine phosphorylation of the of a tyrosine protein kinase and of three proteins that contain
cAMP-dependent enzymes but may well be a secondary phosphotyrosine in virally transformed chick fibroblasts. J. Cell
event following amplified expression of v-src. Biol. 94:287-296.
Several observations indicate that an alteration in the 8. Corbin, J. D., S. L. Keely, and C. R. Parks. 1975. The distribu-
proportion of type I kinase may alter cell differentiation and tion and dissociation of cyclic adenosine 3':5'-monophosphate-
growth. For example, alterations in the ratios of type I and dependent protein kinases in adipose, cardiac, and other tissue.
type II cAMP-dependent kinases have been observed in J. Biol. Chem. 250:218-225.
differentiating cells (12, 14, 15, 19), in cells progressing 9. Costa, M., E. W. Gerner, and D. H. Russell. 1976. Cell cycle-
through the cell cycle (3, 9, 11, 21), in the growth arrest and specific activity of type I and type II cyclic adenosine 3':5'-
reverse transformation of some cells, caused by elevated monophosphate-dependent protein kinases in Chinese hamster
ovary cells. J. Biol. Chem. 251:3313-3319.
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However, a functional role for either enzyme has not been Characteristics of virus specific RNA in avian sarcoma virus-
clearly defined. An alteration in the proportion of the type I transformed BHK-21 cells and revertants. Virology 76:313-330.
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phorylation. tial expression of Type I and Type ll cyclic AMP-dependent
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of v-src in BHK cells coincides with alterations in the growth arrest. Proc. Natl. Acad. Sci. U.S.A. 77:3445-3449.
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Independent expression of cardiac type I and II cyclic AMP-
dependent protein kinase during murine embryogenesis and
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b Picomoles of phosphate transferred per minute per milligram of 412.
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''P < 0.01. infected with avian sarcoma virus. J. Mol. Biol. 143:363-393.VOL. 4, 1984 NOTES 977
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Enzymol. 99:1-6. cyclic AMP required for onset of lymphocyte DNA synthesis.
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