EXPERIMENTAL
CELL RESEARCH
197,245-253
(16%)
Cell Growth Stimulation by EGF: Inhibition through AntisenseOligodeoxynucleotides Demonstrates Important Role of Casein Kinase II R. PEPPERKOK,**-~ P. LOF~ENZ,* R. JAKOBI,* W. ANSORGE,~ AND W. PYERIN**’ *Biochemical Cell Physiology, Institute of Experimental Pathology, German Cancer Research Center; and tBiochemica1 Instrumentation, European Molecular Biology Laboratory, D-6900 Heidelberg, Germany
Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active subunits (a and/or a’) and two presumably regulatory subunits (~9). CKII has numerous cellular functions including a possible role in mitogenic signaling. To address this question, growth-arrested primary human flbroblasts (IMR-90) were exposed prior growth stimulation by epidermal growth factor (EGF) to oligodeoxynucleotides complementary to the translation start region of mRNAs coding for CR11 a and /3subunits. A significant inhibition of growth stimulation (up to 60%) was observed with both antisense-a and antisense8. The inhibition was reversible, became decreased with mutated antisense-oligodeoxynucleotides, and neutralized by simultaneous presence of respective senseoligodeoxynucleotides. The expected down-regulation of CKII protein due to hybrid formation of antisenseoligodeoxynucleotides with target mRNAs was investigated by determination of the .intracellular protein level of CKII B-subunit by immunofluorescence and quantitative image analysis. The protein was revealed to be localized predominantly in the nucleus and to become signitlcantly decreased due to antisensed treatment of cells. The maximum decrease coincided with the early phase (first several hours) of growth stimulation by EGF when antisenseincubation was started 6-2 h before growth stimulation, the period within which application of antisense-ar and antisense-@ caused the maximum of inhibition of growth stimulation. Thus CKII obviously plays, with both subunit a and subunit /3, an important role in the early phase of mitogenic stimulation. 8 1991 Academic Press, Inc.
INTRODUCT1ON
In mammalian cells, external signals are propagated across the plasma membrane and the cytoplasm to the nucleus via complex multistep signaling systems in 1To whom correspondence and reprint requests should be addressed at Biochemical Cell Physiology, Institute of Experimental Pathology, German Cancer Research Center, Im Neuenheimer Feld 260, D-6600 Heidelberg, F.R.G. Telefax: (06221) 401271.
which protein phosphorylation plays a central role [l31. There are indications that casein kinase II (CKII), a highlyconservedubiquitousserine/threonineproteinkinase with numerous cellular functions [4,5], may be one of the key enzymes in the transfer of growth signals. Cytosolic CKII activity is transiently increased following treatment of cells with serum or growth hormones such as epidermal growth factor (EGF), insulin or insulin-like growth factor I [6-lo]. On the other hand, CKII phosphorylates nuclear growth regulatory proteins including oncogene products such as Myc, Myb, Fos, SV40 large T-antigen, adenovirus Ela, human papilloma virus E7, the p53 tumor suppressor protein, the serum response factor, and nucleolin [11-X%]. The human CKII is composed of the two kinase-active subunits (Y and d and the presumably regulatory subunit /3, which form a2&, (YCY’&,or (Y’&~tetramers [4, 5, 191. Unlike regulatory subunits of other protein kinases [l], subunit /3 seems rather to have a stimulatory function; in reconstitution experiments, the @subunit markedly increased activity of a-subunits which were both tissue-derived and recombinant [20-221. Of interest, the B-subunit has been found to become hyperphosphorylated in cells upon growth stimulation by EGF coinciding with CKII activity increase [lo], and subunit @was reported to be a substrate of cdc2 kinase with a positive effect on CK II activity [23]. This supports the idea that CKII acts as a transducer of the EGF signal by a subunit$Lcontrolled change of its cytosolic kinase activity. However, despite these observations, direct evidence is lacking and the involvement of CK II in mitogenie signaling remains a matter of speculation. Molecular cloning and sequencing of cDNAs of the human CKII a! and /3 [24-261 allowed the investigation of the hypothesis that CKII plays a role in mitogenic signaling by antisense-oligodeoxynucleotide methodology [ 27-311. Short oligodeoxynucleotides are intemalized by cells via endocytotic mechanisms [32,33] and by forming hybrids with their target mRNAs, they specifically hinder de nouo protein synthesis and, as a consequence, cause depletion of the respective protein(s) and hence a change in corresponding cellular functions [27, 291.
245 Copyright 0 1991 by Academic Press, Inc. reproduction in any form reserved.
All rights of
246
PEPPERKOK TaroetSeauence CKII u-Subunit
ET AL.
134 144 I I 5 ’ -‘IGI-1Gl~~lux~~l~;-
154 I
I64 I
173 I
I14 124 I I -wxmxmwm~xxx:I~xmx;r5’
134
I 44
I53
3
Anlisense-a Antisense-al
M
Antisense-a3M Sense-a
CKII p-Subunit
Anlisense-p Anlisense-p
1M
I
3’
C~lC~~Iu;I~
3’
C
I
I
3
5’ 5’
.*
Antisense-p3M
3.
C
Sense-p
5’
~~lw;ImAFCI’C
l
5’ 3’
FIG. 1. CKII oligodeoxynucleotides employed and their target sequences. Antisense (3’ to 5’) and sense (5’ to 3’)-oligodeoxynucleotides, 20 bases in length, were prepared symmetrically covering the translation initiation sites of their target sequences, the human CKII subunit a (above), and CKII subunit fi (below). Numbers indicate nucleotide positions in the respective sequence fragments. One or three mutations were introduced into the antisense-oligodeoxynucleotides in a middle position (marked by stars).
We demonstrate here that oligodeoxynucleotides complementary to mRNAs of both CKII subunit Q and CKII subunit /3 significantly inhibit the early phase of mitogenic stimulation of cells by EGF and by serum. The inhibition is shown to be paralleled by a down-regulation of CKIIfl-subunit protein in the early phase of growth stimulation. The data provide evidence for an important role of CKII in the propagation of the mitogenie message in cells. (A preliminary report was presented at the 30th Annual Meeting of the American Society for Cell Biology, 9-13 Dec. 1990, San Diego; J. Cell Bid. 111 342a (19901.) MATERIALS
AND
METHODS
Synthesis and purification of oligodeoxynudeotides. Unmodified oligodeoxynucleotides were synthesized with an Applied Biosystems synthesizer and purified by polyacrylamide gel electrophoresis, gel filtration, and ethanol precipitation. They were lyophilired and redissolved in water. Sequences of the designed oligodeoxynucleotides and their targets are shown in Fig. 1. Cell culture and grourth assay. Human diploid fibroblasts (IMR80) obtained from American Type Culture Collection were grown in Dulbecco’s modified Eagle’e medium (DMEM, GIBCO) supplemented with antibiotics and 10% fetal calf serum at 37’C and 5% CO2. For experiments, 2 x 106 cells were seeded on coverslips (4, 11 mm) in a lo-cm dish and cultured for 3 days. Then medium was changed for DMEM containing 0.5% fetal calf serum. After 3 days less than 1% of the cell population was incorporating 5-bromo-2’deoxyuridine (BrdU, Sigma, final concentration 100 pg/ml) as detected by indirect immunofluorescence (see below). Quiescent cells were growth stimulated by the addition to the culture medium of either fetal calf serum (final concentration 20%) or epidermal growth factor (EGF, Sigma, final concentration 200 ng/
ml). BrdU labeling of cells was between 18 and 24 h after mitogenic stimulation in the absence of oligodeoxynucleotides. Incorporation of BrdU into cells was detected as already described [34]. A monoclonal mouse anti-BrdU antibody (Partec, dilution 19) and a rhodamineconjugated anti-mouse secondary antibody (Sigma, dilution 1:50) were used. Evaluation of the total number of cells was achieved by staining cell nuclei with Hoechst dye 33258 (Sigma, final concentration 1 aglml). Affinity purification of polyckmal antiserum to CKII &wbunit. Anti-CKII&serum 63/7 was employed obtained by injecting rabbits with a fusion protein of CKII&subunit and MS2-polymerase expressed in Escherichia coli (P. Lorenz and W. Pyerin, manuscript in preparation). For immunofluorescence the anti-CKII B-subunit components of the rabbit serum were affinity purified by an immunoblotting procedure [35]. Total cell extract from 107 cells was fractionated on a SDS-polyacrylamide gel and electroblotted onto a nitrocellulose filter (Schleicher and Schtill). Two narrow strips were cut from each side and stained for CKII &subunit using the 6317 antiserum (1:500 dilution) and a peroxidase-conjugated secondary antibody. Then the stained strips were aligned with the unstained part of the filter and the area corresponding to the CKII &subunit band was cut out. After incubation in blocking buffer (3% bovine serum albumin in phosphate-buffered saline plus 0.02% sodium azide) for 24 h at 4°C. the strip was incubated with 63/7 SeNm (1:20 dilution in blocking buffer) for 4 h at room temperature. After washing the filter in PBS, the bound antibody was eluted from the filter with 0.2 M glycine (pH 2.8) and the solution was immediately neutralized with 0.1 M Tris base. Then the antibody solution was concentrated 50X in a Minicon Bl5 concentrator cell. Sodium azide was added to a final concentration of 0.02% and the antibody solution stored at 4°C. The specificity of the antibody was proven by comparative Western blotting (Fig. 2). IMR-90 cell lysates and purified human CKII were electrophorized on a 12% SDS-polyacrylamide gel (Liimmli) and transferred to a PVDF membrane (Immobilon-P, Millipore) by semidry electroblotting. Transferred proteins were stained with 0.3% Ponceau-S in 3% TCA for 5 min. After blocking the membrane with 5% nonfat dry milk in TBS (50 mA4 Tris/HCl, pH 7.4,150 mM NaCl)
CASEIN KINASE
4 67
a+ a'+
247
II: ANTISENSE-OLIGODEOXYNUCLEOTIDES
4 43
gation were growth synchronized (resulting in the same DNA content for every cell), the fluorescent signal for the Hoechst dye should be constant. CKII &subunit content in the cell nuclei was therefore expressed as the ratio of rhodamine fluorescence (proportional to CKII j3-subunit content) to Hoechst dye fluorescence. This has the advantage that any fault measurements due to e.g., sample preparation or camera amplification should be normalized by the Hoechst staining. The same procedure was used to quantify cytoplasmic staining. RESULTS
4 30
P--+ 4 20.1
FIG. 2. Specificity of antiserum and affinity-purified polyclonal antibodies against the CKII@ubunit. One hundred nanograms of purified human CKII (lane 1) and 20 pg protein of total IMR-90 cell lysate (lanes 2-4) were subjected to SDS-PAGE and blotted onto a PVDF membrane. Proteins were visualized by staining with PonceauS (lane 2) and immunoreactivities determined either with antiCKII&serum 63/7 diluted 1:5000 (lanes 1 and 3) or with affinity-purified antibody solution diluted 1:lOO (lane 4) followed by biotin/streptavidin/peroxidase assay.
overnight at 4”C, the blot was incubated with anti-CKIIB serum or the affinity-purified antibodies in washing buffer (TBS/O.l% BSA, 0.05% Tween 20) for 2 h at room temperature. Detection of bound antibody was performed by two consecutive l-h incubations at room temperature with a biotinylated goat-rabbit IgG antibody (Dianova) and streptavidin conjugated with peroxidase (Dianova), followed by reaction with peroxidase substrate I-chloro-1-naphthol [36]. The anti-rabbit IgG antibody was diluted 1:4000, the streptavidin-peroxidase reagent diluted 1:2000 in washing buffer. Zmmurwfhorescence. For immunofluorescence, cells were washed in PBS, fixed for 10 min at room temperature with 3% paraformaldehyde in PBS, permealized for 5 min in PBS containing 0.3% Triton X-100, and quenched for 30 min in 0.1 M glycine-Tris, pH 7.3. Cells were then incubated for 1 h at room temperature with affinity-purified rabbit anti-CKII b-subunit antibody. Then the cells were washed three times with PBS and incubated for 30 min with goat anti-rabbit IgG (1:50 dilution, from Sigma) coupled with rhodamine. The coverslips were washed three times in PBS and finally incubated for 5 min at room temperature with PBS containing Hoechst dye 33258 (Serva, final concentration 1 pglml). After three additional washes in PBS, coverslips were mounted in Mowiol 4.88 (Calbiochem-Behring GmbH). Fluorescence microscopy and photography were performed as described [34]. of immurwstained cells by image analysis. Quantifiation Quantification of CKII ~-subunit expression on immunostained cells was performed on an inverted fluorescence microscope (IM35, Zeiss) equipped with a SIT low light level camera (Hamamatsu) and with an IBAS image analysis system (Zeiss; [37]). From the same field on the coverslip, pictures of cells stained for CKII &subunit or with Hoechst 33258 were loaded separately into the image buffers, background fluorescence next to each cell was subtracted in both cases, and the picture taken for CKII ~-subunit was electronically divided by the picture taken for Hoechst staining. In the resulting image, the mean gray value of the cell nuclei was determined. Since the cells under investi-
CKII Antisense-a and Antisense-@ Oligodeoxynuckotides Specijically Inhibit Growth Stimulation by Epidermal Growth Factor and by Serum Antisense-oligodeoxynucleotides (20 mers) were designed to symmetrically cover the region around the initiation site of the mRNAs coding for the CKII (Ysubunit (antisense-a) and the 0 subunit (antisense-@) (see Fig. 1). When added to the culture medium of quiescent human fibroblasts (IMR-90) 2 h before growth stimulation with EGF, both antisense-a and antisense-p significantly inhibited stimulation of DNA-synthesis as determined by the incorporation of BrdU. On the average, 64% (antisense-@) or 51% (antisense-a) of the cells did not enter S-phase when compared to respective experiments with sense-8 and sense-a at equivalent concentrations (Table 1). Sense-8 and sense-a by themselves had no effect on cell growth and growth stimulation; no difference was observed in the presence and the absence
TABLE
1
Antiproliferative Potential of Oligodeoxynucleotides Complementary to CKII Subunits a and j3 Growth Inhibition (X) Oligodeoxynucleotide
EGF
AntisenseAntisense-61M Antisense-fi3M Antisense-a Antisense-alM Antisense-a3M
64 _t 9 28 + 7 17 + 3 51 f 6 22 k 9 7+8
FCS 44 k 18 + 12 + 31+ 18 + -3 f
3 7 8 6 2 5
Note. IMR-90 cells were rendered quiescent by a low concentration of fetal calf serum (0.5%) for 3 days. Oligodeoxynucleotides were added to the medium (final concentration, 100 pg/ml) 2 h prior to growth stimulation by EGF (final concentration, 200 rig/ml) or fetal calf serum (final concentration 20%). Control experiments were carried out with the respective sense-oligodeoxynucleotides. BrdU labeling of cells was done 18-24 h after growth stimulation in the absence of oligodeoxynucleotides. At least 500 cells were analyzed for DNA synthesis in each experiment. Growth inhibition was calculated from the percentages of BrdU-positive cells in experiments with antisense and the respective sense-oligodeoxynucleotides by the formula: Inhibition (%) = ((%BrdU-positive cells (sense-oligo) - %BrdU-positive cells (antisense-oligo))/%BrdU-positive cells (sense-oligo)) x 100. Shown are the mean + standard deviation.
248
PEPPERKOK 60 2
50
% Z z z
40 30
c $
20
u
10 0
Antisense-a
+
+
+
-
-
-
Sense-a
-
+
-
-
-
+
Antisense-P Sense-p
-
-
+
+ -
+ +
+ -
FIG. 3. Competition between CKII antisense- and sense oligodeoxynucleotides. Different combinations of oligodeoxynucleotides (final concentration 100 ag/ml of each oligodeoxynucleotide) were applied to synchronized cells 2 h before growth stimulation with EGF. Control experiments were carried out with respective sense-oligodeoxynucleotides. Determination of DNA synthesis and calculation of growth inhibition was as described in the legend to Table 1. The mean values + standard deviation of three independent experiments are shown.
of 25,50,100, or 200 pg/ml sense oligodeoxynucleotides. When antisense-oligodeoxynucleotide concentrations were varied, a saturation characteristic of the anti-proliferative effect became evident, plateauing at a concentration of 100 fig/ml (data not shown). This indicates that both the regulatory subunit /3and the kinase-active subunit (Yplay an important role in growth stimulation by EGF. In order to prove the specificity of the observed effect, mutations were introduced into the antisense-oligodeoxynucleotides. A gradual reduction of the growth inhibitory effect was observed paralleling number of mutations (Table 1); antisense-oligodeoxynucleotides with one mutation (antisense-@lM; antisense-alM) were only half as effective as the nonmutated ones, and antisense-oligodeoxynucleotides with three mutations (antisense-P3M; antisense-a3M) showed no significant growth inhibition any more. Further, corresponding antisense- and sense-oligodeoxynucleotides were applied together at equal concentrations which should, if the observed growth inhibition was oligodeoxynucleotidespecific, neutralize the anti-proliferative effect of antisense-oligodeoxynucleotides by competing for hybrid formation with mRNAs coding for the respective CKII subunits. This was, as shown in Fig. 3, exactly what happened, while in controls with noncorresponding oligodeoxynucleotide pairs, inhibition of growth stimulation by antisense-oligodeoxynucleotides was unchanged. Thus, the inhibition of growth stimulation by CKII antisense+ and antisense-cl! is a highly structure-specific event. Similar results as with EGF were obtained, although
ET AL.
less pronounced, when fetal calf serum was used as growth stimulator (Table 1). This would, if the growth stimulation by serum was by its EGF content, indicate an EGF-specific role of CKII. If it was the result (also) of other mitogens contained in the serum, this would indicate that CKII is, in addition to the transduction of the EGF message, important for the transduction of the message of other mitogenic stimuli as well. CKII would then play a rather central role in mitogenic signaling. However, a reliable analysis of mitogenic constituents of the employed serum is lacking and hence a reliable interpretation cannot be provided at the moment. Growth Inhibitory Effect of AntisenseOligodeoxynuckotides Is Transient Are the cells suffering from damage upon incubation with antisense-oligodeoxynucleotides or are they able to re-enter the cell cycle after removal of the oligodeoxynucleotides? To answer this question, antisense- or antisense-a was added to the medium 2 h prior to the growth stimulus and left until 18 h after the stimulation. Then, the oligodeoxynucleotides were removed and the cells were further incubated in the presence of BrdU for various times. As shown in Table 2, the growth inhibition observed 24 h after EGF stimulation was significantly stronger than that 28 h after stimulation, and inhibition became insignificant 34 h after stimulation. Again, similar results were obtained when cells were growth stimulated with fetal calf serum (data not shown). This demonstrates that the inhibition of cell growth stimulation by CKII antisense-oligodeoxynucleotides is reversible and hence not the result of cell damage due to toxic effects of oligodeoxynucleotides. Timing of Antisense-@ Treatment Is Decisive for the Anti-proliferative Effect and Correlates with the Downregulation of CKII &Subunit In the experiments described above, oligodeoxynucleotides were added to the culture medium 2 h before TABLE
2
Persistence of the Growth Inhibitory Effect and Antisense-cY of CKII Antisense+ Growth Time of fixation after growth stimulation (h) 24 28 34
inhibition
(%) by
Antisense-p
Antisense-a
47 + 6 31 + 3 11+ 5
51 t 3 37 + 6 13 f 7
Note. Oligodeoxynucleotides were added to the medium 2 h before growth stimulation of cells with EGF (266 rig/ml). At 18 h after growth stimulation, cells were washed twice with culture medium without oligodeoxynucleotides followed by the addition of BrdU. Cells were fixed and stained at the three given time-points after growth stimulation. Growth inhibition was determined as described in the legend to Table 1. Shown are the means + standard deviation.
CASEIN
g .-L :g a d a r e v
KINASE
60 40 20 0 -20
! -24
-12
-6
-2
0
249
II: ANTISENSE-OLIGODEOXYNUCLEOTIDES
5
Time(h) was FIG. 4. Timing of CKII antisense- incubation. Antisenseadded to the culture medium of synchronized cells (final concentration 100 pg/ml) at different time-points with respect to stimulation with EGF (at 0 h). Control experiments were carried out with sense-b. Determination of DNA synthesis and calculation of growth inhibition was as described in legend to Table 1. Negative numbers in the time scale indicate that oligodeoxynucleotides were added at respective time-points before EGF stimulation. The mean values + standard deviation of three independent experiments are shown.
the growth stimulus. In order to study more precisely the time frame of CKII involvement in the stimulation of cell growth, the time-point of oligodeoxynucleotide addition with respect to EGF stimulation was varied. Since subunit /3 is the presumed controlling element of CKII activity, we concentrated on antisense-& Maximum inhibition of growth stimulation was obtained when incubation of cells with antisense-p had been started between 2 and 6 h prior to EGF stimulation (Fig. 4). Addition to cell medium at 12 or 24 h before EGF had little or no effect on growth stimulation as was the case when antisense- was added simultaneously with EGF or 5 h after EGF. In parallel experiments, the intracellular level of CKII @-subunit protein following antisense-p addition to cell medium was visualized by immunofluorescence using affinity-purified anti-CKII j3-subunit antibody and quantitation by image analysis. The antibody, whose CKII P-specificity had been proven by Western blotting of cell extracts (see Fig. 2), localized the protein in the cytoplasm and in the nucleus with at least a five times higher concentration in the nucleus (Fig. 5A). A similar distribution was found by Western blot analyses of nuclear vs cytoplasmic fractions (data not shown) excluding fixation artifacts. The treatment of cells with antisense-& resulted in a significant decrease of the fluorescence signal (Fig. 5C). In addition, the fluorescent signal could be blocked by preincubation of the anti-CKII P-subunit antibody with purified CKII (Fig. 5E) further strengthening the specificity of the immunofluorescence staining for CKII B-subunit. Staining of cell nuclei with Hoechst dye 33258 ensured same number of cells in the whole experimental series (Figs. 5B, 5D, and 5F).
When antisense- incubation was started at 2 or 6 h before EGF stimulation, a sign&ant down-regulation of CKII &subunit in the nucleus occurred reaching a maximum decrease of approximately 60% and lasting for roughly up to 7 h after the growth stimulation (Fig. 6A). Longer incubation periods showed less of a decrease and CKII B-subunit was back at constitutive level when measured after 24 h in either case. When antisense-8 was applied simultaneously with EGF, the level of nuclear CKII P-subunit showed little change in the early phase of EGF-stimulation; significant decrease was found at 5 and 7 h after EGF addition (Fig. 6A). As expected, neither simultaneous application of antisense-8 and sense-p had any effect on CKII subunit 0 expression nor application of antisense-cu (data not shown). Similar down-regulation kinetics were obtained for the cytoplasmic CKII @-subunit (Fig. 6B). However, the decrease of cytoplasmic CKII P-subunit after incubation with antisense-p was considerably weaker (roughly 30% decrease at maximum) than that obtained for the nuclear p-subunit. On the other hand, the decrease of cytoplasmic CKII p-subunit level appeared to occur faster than that of the nuclear level. Taken together, the time frame of CKII antisense inhibition of growth stimulation matches the down-regulation of CKII protein; time-points for antisense-@ addition leading to maximum inhibition of growth stimulation by EGF show significant reduction of P-subunit protein in the early phase of EGF stimulation whereas time-points leading to insignificant growth inhibition do not. DISCUSSION
The observed inhibition of cell growth stimulation by EGF obtained with the CKII antisense-oligodeoxynucleotides is a highly structure-specific event because (i) inhibition occurs only with antisense-a and antisense-@ but not with the respective sense-oriented oligodeoxynucleotides, (ii) antisense-oligodeoxynucleotides with a single mutation have reduced effects and those with three mutations failed to significantly inhibit cell growth, (iii) inhibition is hindered by the simultaneous presence of sense-oligodeoxynucleotides, (iv) inhibition is concentration-dependent with all characteristics of a saturable effect, and (v) the expected decrease of CKII protein is antisense-specific; subunit /3 becomes downregulated exclusively with antisense-@ but not with antisense-a or any other oligodeoxynucleotide. In addition, toxic effects can be excluded because growth inhibition is reversible; after removal of the oligodeoxynucleotides, the cells re-enter the S-phase of the cell cycle. Thus, our results demonstrate that CKII plays an important role in the mitogenic stimulation of cells by EGF and that both CKII subunit cr and subunit B are critical elements. What kind of role is played by CKII?
250
PEPPERKOK
ET AL.
CASEIN
KINASE
II: ANTISENSE-OLIGODEOXYNUCLEOTIDES
251
n Nwleua(Oh)
bypassed via alternative transduction pathways such as those involving protein kinase A or protein kinase C n [l-3]. The lack of a complete inhibition of growth stimulation by antisense-a and antisense- pretreatments may be due to reasons such as the existence of an d subunit, a potential substitute for subunit LY,and the presence of residual amounts of active subunit proteins synthesized prior to oligodeoxynucleotide treatment. How then does CKII play its role as a signal propagator? The mitogenic message of EGF reaches the cytoplasm by the membrane-anchored EGF receptor. For its propa7 24 2 5 0 1 gation into the nucleus via CKII, two alternative pathTime (h) ways were conceivable. CKII may phosphorylate cytoplasmic protein(s) which then migrate into the nucleus to trigger mitosis, or, as the alternative, CKII may min Wopl~(~) 0 Cytoplasm (-2h) grate itself into the nucleus to phosphorylate there n Cytoplasm k6h) growth regulating protein(s). Both alternatives were compatible with our data and both receive support by the current literature. The functioning as a cytosolic element of the signaling cascade is based on an early and transient change in cytosolic CKII activity (up to fourfold increase) following treatment of cells with growth factors such as EGF [6-lo]. This change is -20 -+ thought to result from an activation of CKII due to a 7 24 1 2 5 0 stimulation of subunit CY through subunit /3.Because the Time (h) antisense-oligodeoxynucleotides cause a decrease of inlevel of CKIIB protein after oligodeoxynuFIG. 6. Intracellular tracellular protein levels of the respective CKII subcleotide incubation. Synchronized cells were incubated with antiunits, they should also reduce the actual CKII activity. sense-p for different periods. At zero time, cells were stimulated with While obvious as a consequence of a decrease of the EGF (200 rig/ml). Nuclear (A) and cytoplasmic (B) level of CKII b-subunit were determined at different time-points after mitogenic kinase-active subunit (Y,antisense+ would be effective stimulation by immunofluorescence and image analysis. Control exonly if subunit /3was indeed a positive regulator of celluperiments were carried out with sense-b. Decrease of CKII b-subunit lar CKII activity. The shown decrease of CKIIB protein by antisense-oligodeoxynucleotides was determined by the formula: level following antisense-@ treatment strengthens this Decrease (%) = ((Protein level (sense-@) - Protein level (antisenseimplication and is in agreement with the recently re&)/Protein level (sense-@)) X 100. Numbers in parentheses indicate the start of oligodeoxynucleotide incubation with respect to EGF stimported reduced CKII activity after transfection of 3T3 ulation (negative numbers indicate time-points prior EGF stimulapreadipocytes with antisense-@ cDNAs [39]. It also tion). Given are mean values + standard deviations of at least three matches the results of reconstitution experiments in independent experiments. which the kinase activity of both tissue-derived and recombinant CKII a-subunit became significantly stimulated when united with subunit fl [20-221. Of note, the The coincidence of maximum protein down-regulainteraction of subunit @with ligands such as polylysine tion and most effective timing of antisense-oligodeoxfurther enhanced CKII activity [22,40,41] making conynucleotide addition indicates that the role played by ceivable tuning of CKII by conformational changes of CKII is limited to the early phase of growth stimulation, subunit @and explaining the stimulation of cytosolic i.e., to the first several hours. Hence functioning as me- CKII activity by the reported hyperphosphorylation [6, diator of the EGF message can be assumed. This CKII8-101. Functioning as a cytosolic element of the mitodependent pathway obviously is the main EGF trans- genie signaling cascade would require the presence of duction system, as otherwise the antisense-oligodeoxappropriate CKII substrate(s) in the cytosol. Such proynucleotide-induced CKII-depletion would have been teins are indeed known. A most prominent example is 0
Nucleus(-2h) Nucbw(-6h)
Synchronized cells were exposed to antisenseor sense-b 2 FIG. 6. Staining of cells for CKII &subunit by indirect immunofluorescence. h before EGF stimulation. Cells were fixed and permeabilized 2 h after EGF addition and CKII @-subunit was visualized with affinity-purified antibody against CKII p-subunit and a rhodamine-conjugated goat anti-rabbit second antibody. Cells incubated (A) with sense-& (C) with antisense-& and (E) with affinity-purified CKII &subunit antibody preincubated with purified CKII (1 pg/pl) for 30 min. (B, D, F) Staining of the corresponding cells with Hoechst dye 33258. A and C as well as B and D were taken at the same exposure times.
252
PEPPERKOK
the oncoprotein Myc, whose presence appears to be essential for cell growth. Myc occurs in the cytosol and, as a phosphoprotein, in the nucleus. Its short-term kinetics of the nuclear cytoplasmic transport appears to depend on phosphorylation by CKII [42]. A number of observations, however, are in favor of a signal transport by CKII itself. As shown here, the level of CKII protein is at least fivefold higher in the nucleus than in the cytoplasm and the accumulation of subunit p in the nucleus is significantly inhibited by antisense-& Moreover, the ratio of nuclear to cytoplasmic CKII subunit p in synchronized cells rapidly increased after EGF or serum stimulation by a factor of two to three (data not shown). This correlates well with the recent finding that CKII is accumulated in the nucleus of growing cells but not in cell cycle-arrested cells [l&38]. Since mainly localized and further accumulated in the nucleus following EGF stimulation of cells, this could mean that CKII migrates into the nucleus to transport the EGF message. In the nucleus, CKII would find a number of known substrates which are of particular importance for cell growth and its stimulation. These include cell regulatory proteins such as the oncogene products Myc, Myb, Fos, SV40 large T-antigen, adenovirus Ela, human papilloma virus E7, the ~53 tumor suppressor protein, or the serum response factor [ll-171. In the connecting postpropagation phase than, CKII would phosphorylate proteins such as nucleolin which had been shown to control rDNA transcription and thus ribosome synthesis, a prerequisite for cell growth [18]. In either case of these alternative signal propagation, CKII would receive the message from the EGF receptor. It remains an open question by which step(s) this would be achieved. It can obviously not be achieved by direct phosphorylation through the receptor-intrinsic kinase activity because this phosphorylates proteins at tyrosine residues [2], while the hyperphosphorylation of CKII subunit fl is a serine/threonine kinase-catalyzed event [lo]. Intermediary serinjthreonine kinase(s) must therefore exist such as cdc2 kinase [23] or those described recently for the EGF propagation cascade in 3T3 cells [43, 441.
ET AL. Lozeman, F. J., Liischer, 4.
2.
3.
Edelman, A. M., Blumenthal, D. K., and Krebs, E. G. (1987) Annu. Rev. Biochem. S&567-613. Hunter, T., Angel, P., Boyle, W. J., Chiu, R., Freed, E., Gould, K. L., Isacke, C. M., Karin, M., Lindberg, R. A., and van der Geer, P. (1988) Cold Spring Harbor Symp. QUXWZ~. Biol. LIII, 131-142. Krebs, E. G., Eisenman, R. N., Kuenzel, E. A., Lichtfield, D. W.,
P. T., and Traugh,
Sommercorn, J., Mulligan, J. A., Lozeman, F. J., and Krebs, E. G. (1987) Proc. Natl. Acad. Sci. USA 84,8834-8838.
7.
Klarlund, J. K., and Czech, M. P. (1988) J. Biol. Chem. 263, 15,872-15,875.
8.
Ackerman, P., and Osheroff, 11,95811,965.
9.
Carroll, D., andMarshak, D. R. (1989) J. Biol. Chem. 264,73457348. Ackerman, P., Glover, C. V. C., and Osheroff, N. (1990) Proc.
10.
N. (1989) J. Biol. Chem. 264,
Natl. Acad. Sci. USA 67.821-825. 11. 12. 13. 14. 15. 16.
Liischer, B., Kuenzel, E. A., Krebs, E. G., and Eisenman, R. N. (1989) EMBO J. 8,1111-1119. Luscher, B., Christenson, E., Litchfield, D. W., Krebs, E. G., and Eisenman, R. N. (1990) Nature 344,517-522. Carroll, D., Santoro, N., and Marshak, D. R. (1988) Cold Spring Harbor Symp. Quant. Biol. LIII, 91-95. Griisser, F. A., Scheidtmann, K. H., Tuazon, P. T.,Traugh, J. A., and Walter, G. (1988) Virology 166, 13-22. Barbosa, M. S., Edmonds, C., Fisher, C., Schiller, J. T., Lowy, D. R., and Vousden, K. H. (1990) EMBO J. 9,153-160. Manak, J. R., de Bisschop, N., Kris, R. M., and Prywes, R. (1990)
Genes Dev. 4.955-967. 17.
Meek, D. W., Simon, S., Kikkawa,
U., and Eckhart,
W. (1990)
EMBO J. 4,3253-3260. 18.
19. 20. 21.
22.
Belenguer, P., Baldin, V., Mathieu, C., Prats, H., Bensaid, M., Bouche, G., and Amalric, F. (1989) Nucleic Acids Res. 1’7,66256636. Pyerin, W., Burow, E., Michaely, K., Kiibler, D., and Kinzel, V. (1987) Biol. Chem. Hoppe Seykr 368,215-227. Cachet, C., and Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406. Pyerin, W., Jakobi, R., Taniguchi, H., Lorenz, P., Hoppe, J., Vol3, H., Kiibler, D., Kinzel, V. (1988) Biol. Chem. Hoppe Seykr 369,896. Grankowski, N., Boldyreff, B., and Issinger, O.-G. (1991) Eur. J.
Biochem. 198,25-30. 23.
Mulner-Lorillon, O., Cormier, P., Labbe, J-C., Do&e, M., Poulhe, R., Osborn, H., and Belle, R. (1990) Eur. J. B&hem.
24.
Meisner, H., Heller-Harrison, R., Buxton, J., and Czech, M. P. (1989) Biochemistry 28,4072-4076. Lozeman, F. J., Litchfield, D. W., Piening, C., Takio, K., Walsh, K. A., and Krehs, E. G. (1990) Biochemistry 29,843~8447. Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem.
193,529-534.
26.
183,227-233.
28. 29.
1.
Tuazon,
6.
27.
REFERENCES
J. P. (1988) Cold
77-84. J. A. (1990) Adv. Sec. Messenger
Phosphoprot. Res. 23, 123-164. 5. Pinna, L. A. (1990) Biochim. Biophys. Actu 1064, 267-284.
25. We thank A. Lampe-Gegenheimer for excellent secretarial assistance and the Deutsche Forschungsgemeinschaft for financial support (grant to W.P.).
B., and Sommerkorn,
Spring Harbor Symp. Quant. Bill. LIII,
Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R., and Neckers, L. M. (1987) Nature 328,445-449. Gewirtz, A. M., and Calahretta, B. (1988) Science 242, 13031306. Anfossi, G., Gewirtz, A. M., and Calabretta, B. (1989) Proc. Natl.
Acad. Sci. USA 86,3379-3383. 30.
31.
Goodcbild, J., Agrawal, S., Civeira, M. P., Sarin, P. S., Sun, D., and Zamecnik, P. C. (1988) Proc. Natl. Acad. Sci. USA 86,55075511. Matsukura, M., Zon, G., Shinozuka, K., Robert-Guroff, M., Shimada, T., Stein, C. A., Mitauya, H., Wong-StaaI, F., Cohen, J. S., and Broder, S. (1989) Proc. Natl. Acad. Sci. USA 86.4244-4248.
CASEIN KINASE
II: ANTISENSE-OLIGGDEOXYNUCLEOTIDES
32. Loke, S. L., Stein, C. A., Zhang, X. H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J. S., and Neckers, L. M. (1989) Proc. Natl. Acad. Sci. USA 88,3414-3478. 33. Yakubov, L. A., Deeva, E. A., Zarytova, V. F., Ivanova, E. M., Ryte, A. S., Yurchenko, L. V., and VIassov, V. V. (1989) Proc. Natl. Acad. Sci. USA 86,6454-6456. 34. Sorrentino, V., Pepperkok, R., Davis, R. L., Ansorge, W., and Philipson, L. (1990) Nature 345,813-815. 35. Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. (1982) EMBO J. 1,1621-1628. 36. Kobayashi, R., and Tashima, Y. (1989) Anal. Biochem. 183, 9-12. 37. Ansorge, W., and Pepperkok, R. (1989) J. Biochem. Biophys. Methods. l&263-292. Received May 16,199l Revised version received August 1, 1991
253
38. FiIhol, O., Cachet, C., and Chambaz, E. M. (1990) Biochemistry 29,9926-9936. 39. Meisner, H., and Czech, M. P. (1991) J. CeU.Biochem. 15B, 261. 40. Traugh, J. A., Lin, W. J., Takada-Axelrod, F., and Tuaxon, P. T. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Ed.), pp. 224-229 Raven Press, New York. 41. Hu, E., and Rubin, C. S. (1990) J. Biol. Chem. 265, 20,60920,615. 42. Rihs, H.-P., Jans, D. A., Fan, H., and Peters, R. (1991) EMBO J. 10,633-639. 43. Ahn, N. G., and Krebs, E. G. (1990) J. Biol. Chem. 265,11,49511,501. 44. Ahn, N., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265,11,487-11,494.
CELL RESEARCH
197,245-253
(16%)
Cell Growth Stimulation by EGF: Inhibition through AntisenseOligodeoxynucleotides Demonstrates Important Role of Casein Kinase II R. PEPPERKOK,**-~ P. LOF~ENZ,* R. JAKOBI,* W. ANSORGE,~ AND W. PYERIN**’ *Biochemical Cell Physiology, Institute of Experimental Pathology, German Cancer Research Center; and tBiochemica1 Instrumentation, European Molecular Biology Laboratory, D-6900 Heidelberg, Germany
Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active subunits (a and/or a’) and two presumably regulatory subunits (~9). CKII has numerous cellular functions including a possible role in mitogenic signaling. To address this question, growth-arrested primary human flbroblasts (IMR-90) were exposed prior growth stimulation by epidermal growth factor (EGF) to oligodeoxynucleotides complementary to the translation start region of mRNAs coding for CR11 a and /3subunits. A significant inhibition of growth stimulation (up to 60%) was observed with both antisense-a and antisense8. The inhibition was reversible, became decreased with mutated antisense-oligodeoxynucleotides, and neutralized by simultaneous presence of respective senseoligodeoxynucleotides. The expected down-regulation of CKII protein due to hybrid formation of antisenseoligodeoxynucleotides with target mRNAs was investigated by determination of the .intracellular protein level of CKII B-subunit by immunofluorescence and quantitative image analysis. The protein was revealed to be localized predominantly in the nucleus and to become signitlcantly decreased due to antisensed treatment of cells. The maximum decrease coincided with the early phase (first several hours) of growth stimulation by EGF when antisenseincubation was started 6-2 h before growth stimulation, the period within which application of antisense-ar and antisense-@ caused the maximum of inhibition of growth stimulation. Thus CKII obviously plays, with both subunit a and subunit /3, an important role in the early phase of mitogenic stimulation. 8 1991 Academic Press, Inc.
INTRODUCT1ON
In mammalian cells, external signals are propagated across the plasma membrane and the cytoplasm to the nucleus via complex multistep signaling systems in 1To whom correspondence and reprint requests should be addressed at Biochemical Cell Physiology, Institute of Experimental Pathology, German Cancer Research Center, Im Neuenheimer Feld 260, D-6600 Heidelberg, F.R.G. Telefax: (06221) 401271.
which protein phosphorylation plays a central role [l31. There are indications that casein kinase II (CKII), a highlyconservedubiquitousserine/threonineproteinkinase with numerous cellular functions [4,5], may be one of the key enzymes in the transfer of growth signals. Cytosolic CKII activity is transiently increased following treatment of cells with serum or growth hormones such as epidermal growth factor (EGF), insulin or insulin-like growth factor I [6-lo]. On the other hand, CKII phosphorylates nuclear growth regulatory proteins including oncogene products such as Myc, Myb, Fos, SV40 large T-antigen, adenovirus Ela, human papilloma virus E7, the p53 tumor suppressor protein, the serum response factor, and nucleolin [11-X%]. The human CKII is composed of the two kinase-active subunits (Y and d and the presumably regulatory subunit /3, which form a2&, (YCY’&,or (Y’&~tetramers [4, 5, 191. Unlike regulatory subunits of other protein kinases [l], subunit /3 seems rather to have a stimulatory function; in reconstitution experiments, the @subunit markedly increased activity of a-subunits which were both tissue-derived and recombinant [20-221. Of interest, the B-subunit has been found to become hyperphosphorylated in cells upon growth stimulation by EGF coinciding with CKII activity increase [lo], and subunit @was reported to be a substrate of cdc2 kinase with a positive effect on CK II activity [23]. This supports the idea that CKII acts as a transducer of the EGF signal by a subunit$Lcontrolled change of its cytosolic kinase activity. However, despite these observations, direct evidence is lacking and the involvement of CK II in mitogenie signaling remains a matter of speculation. Molecular cloning and sequencing of cDNAs of the human CKII a! and /3 [24-261 allowed the investigation of the hypothesis that CKII plays a role in mitogenic signaling by antisense-oligodeoxynucleotide methodology [ 27-311. Short oligodeoxynucleotides are intemalized by cells via endocytotic mechanisms [32,33] and by forming hybrids with their target mRNAs, they specifically hinder de nouo protein synthesis and, as a consequence, cause depletion of the respective protein(s) and hence a change in corresponding cellular functions [27, 291.
245 Copyright 0 1991 by Academic Press, Inc. reproduction in any form reserved.
All rights of
246
PEPPERKOK TaroetSeauence CKII u-Subunit
ET AL.
134 144 I I 5 ’ -‘IGI-1Gl~~lux~~l~;-
154 I
I64 I
173 I
I14 124 I I -wxmxmwm~xxx:I~xmx;r5’
134
I 44
I53
3
Anlisense-a Antisense-al
M
Antisense-a3M Sense-a
CKII p-Subunit
Anlisense-p Anlisense-p
1M
I
3’
C~lC~~Iu;I~
3’
C
I
I
3
5’ 5’
.*
Antisense-p3M
3.
C
Sense-p
5’
~~lw;ImAFCI’C
l
5’ 3’
FIG. 1. CKII oligodeoxynucleotides employed and their target sequences. Antisense (3’ to 5’) and sense (5’ to 3’)-oligodeoxynucleotides, 20 bases in length, were prepared symmetrically covering the translation initiation sites of their target sequences, the human CKII subunit a (above), and CKII subunit fi (below). Numbers indicate nucleotide positions in the respective sequence fragments. One or three mutations were introduced into the antisense-oligodeoxynucleotides in a middle position (marked by stars).
We demonstrate here that oligodeoxynucleotides complementary to mRNAs of both CKII subunit Q and CKII subunit /3 significantly inhibit the early phase of mitogenic stimulation of cells by EGF and by serum. The inhibition is shown to be paralleled by a down-regulation of CKIIfl-subunit protein in the early phase of growth stimulation. The data provide evidence for an important role of CKII in the propagation of the mitogenie message in cells. (A preliminary report was presented at the 30th Annual Meeting of the American Society for Cell Biology, 9-13 Dec. 1990, San Diego; J. Cell Bid. 111 342a (19901.) MATERIALS
AND
METHODS
Synthesis and purification of oligodeoxynudeotides. Unmodified oligodeoxynucleotides were synthesized with an Applied Biosystems synthesizer and purified by polyacrylamide gel electrophoresis, gel filtration, and ethanol precipitation. They were lyophilired and redissolved in water. Sequences of the designed oligodeoxynucleotides and their targets are shown in Fig. 1. Cell culture and grourth assay. Human diploid fibroblasts (IMR80) obtained from American Type Culture Collection were grown in Dulbecco’s modified Eagle’e medium (DMEM, GIBCO) supplemented with antibiotics and 10% fetal calf serum at 37’C and 5% CO2. For experiments, 2 x 106 cells were seeded on coverslips (4, 11 mm) in a lo-cm dish and cultured for 3 days. Then medium was changed for DMEM containing 0.5% fetal calf serum. After 3 days less than 1% of the cell population was incorporating 5-bromo-2’deoxyuridine (BrdU, Sigma, final concentration 100 pg/ml) as detected by indirect immunofluorescence (see below). Quiescent cells were growth stimulated by the addition to the culture medium of either fetal calf serum (final concentration 20%) or epidermal growth factor (EGF, Sigma, final concentration 200 ng/
ml). BrdU labeling of cells was between 18 and 24 h after mitogenic stimulation in the absence of oligodeoxynucleotides. Incorporation of BrdU into cells was detected as already described [34]. A monoclonal mouse anti-BrdU antibody (Partec, dilution 19) and a rhodamineconjugated anti-mouse secondary antibody (Sigma, dilution 1:50) were used. Evaluation of the total number of cells was achieved by staining cell nuclei with Hoechst dye 33258 (Sigma, final concentration 1 aglml). Affinity purification of polyckmal antiserum to CKII &wbunit. Anti-CKII&serum 63/7 was employed obtained by injecting rabbits with a fusion protein of CKII&subunit and MS2-polymerase expressed in Escherichia coli (P. Lorenz and W. Pyerin, manuscript in preparation). For immunofluorescence the anti-CKII B-subunit components of the rabbit serum were affinity purified by an immunoblotting procedure [35]. Total cell extract from 107 cells was fractionated on a SDS-polyacrylamide gel and electroblotted onto a nitrocellulose filter (Schleicher and Schtill). Two narrow strips were cut from each side and stained for CKII &subunit using the 6317 antiserum (1:500 dilution) and a peroxidase-conjugated secondary antibody. Then the stained strips were aligned with the unstained part of the filter and the area corresponding to the CKII &subunit band was cut out. After incubation in blocking buffer (3% bovine serum albumin in phosphate-buffered saline plus 0.02% sodium azide) for 24 h at 4°C. the strip was incubated with 63/7 SeNm (1:20 dilution in blocking buffer) for 4 h at room temperature. After washing the filter in PBS, the bound antibody was eluted from the filter with 0.2 M glycine (pH 2.8) and the solution was immediately neutralized with 0.1 M Tris base. Then the antibody solution was concentrated 50X in a Minicon Bl5 concentrator cell. Sodium azide was added to a final concentration of 0.02% and the antibody solution stored at 4°C. The specificity of the antibody was proven by comparative Western blotting (Fig. 2). IMR-90 cell lysates and purified human CKII were electrophorized on a 12% SDS-polyacrylamide gel (Liimmli) and transferred to a PVDF membrane (Immobilon-P, Millipore) by semidry electroblotting. Transferred proteins were stained with 0.3% Ponceau-S in 3% TCA for 5 min. After blocking the membrane with 5% nonfat dry milk in TBS (50 mA4 Tris/HCl, pH 7.4,150 mM NaCl)
CASEIN KINASE
4 67
a+ a'+
247
II: ANTISENSE-OLIGODEOXYNUCLEOTIDES
4 43
gation were growth synchronized (resulting in the same DNA content for every cell), the fluorescent signal for the Hoechst dye should be constant. CKII &subunit content in the cell nuclei was therefore expressed as the ratio of rhodamine fluorescence (proportional to CKII j3-subunit content) to Hoechst dye fluorescence. This has the advantage that any fault measurements due to e.g., sample preparation or camera amplification should be normalized by the Hoechst staining. The same procedure was used to quantify cytoplasmic staining. RESULTS
4 30
P--+ 4 20.1
FIG. 2. Specificity of antiserum and affinity-purified polyclonal antibodies against the CKII@ubunit. One hundred nanograms of purified human CKII (lane 1) and 20 pg protein of total IMR-90 cell lysate (lanes 2-4) were subjected to SDS-PAGE and blotted onto a PVDF membrane. Proteins were visualized by staining with PonceauS (lane 2) and immunoreactivities determined either with antiCKII&serum 63/7 diluted 1:5000 (lanes 1 and 3) or with affinity-purified antibody solution diluted 1:lOO (lane 4) followed by biotin/streptavidin/peroxidase assay.
overnight at 4”C, the blot was incubated with anti-CKIIB serum or the affinity-purified antibodies in washing buffer (TBS/O.l% BSA, 0.05% Tween 20) for 2 h at room temperature. Detection of bound antibody was performed by two consecutive l-h incubations at room temperature with a biotinylated goat-rabbit IgG antibody (Dianova) and streptavidin conjugated with peroxidase (Dianova), followed by reaction with peroxidase substrate I-chloro-1-naphthol [36]. The anti-rabbit IgG antibody was diluted 1:4000, the streptavidin-peroxidase reagent diluted 1:2000 in washing buffer. Zmmurwfhorescence. For immunofluorescence, cells were washed in PBS, fixed for 10 min at room temperature with 3% paraformaldehyde in PBS, permealized for 5 min in PBS containing 0.3% Triton X-100, and quenched for 30 min in 0.1 M glycine-Tris, pH 7.3. Cells were then incubated for 1 h at room temperature with affinity-purified rabbit anti-CKII b-subunit antibody. Then the cells were washed three times with PBS and incubated for 30 min with goat anti-rabbit IgG (1:50 dilution, from Sigma) coupled with rhodamine. The coverslips were washed three times in PBS and finally incubated for 5 min at room temperature with PBS containing Hoechst dye 33258 (Serva, final concentration 1 pglml). After three additional washes in PBS, coverslips were mounted in Mowiol 4.88 (Calbiochem-Behring GmbH). Fluorescence microscopy and photography were performed as described [34]. of immurwstained cells by image analysis. Quantifiation Quantification of CKII ~-subunit expression on immunostained cells was performed on an inverted fluorescence microscope (IM35, Zeiss) equipped with a SIT low light level camera (Hamamatsu) and with an IBAS image analysis system (Zeiss; [37]). From the same field on the coverslip, pictures of cells stained for CKII &subunit or with Hoechst 33258 were loaded separately into the image buffers, background fluorescence next to each cell was subtracted in both cases, and the picture taken for CKII ~-subunit was electronically divided by the picture taken for Hoechst staining. In the resulting image, the mean gray value of the cell nuclei was determined. Since the cells under investi-
CKII Antisense-a and Antisense-@ Oligodeoxynuckotides Specijically Inhibit Growth Stimulation by Epidermal Growth Factor and by Serum Antisense-oligodeoxynucleotides (20 mers) were designed to symmetrically cover the region around the initiation site of the mRNAs coding for the CKII (Ysubunit (antisense-a) and the 0 subunit (antisense-@) (see Fig. 1). When added to the culture medium of quiescent human fibroblasts (IMR-90) 2 h before growth stimulation with EGF, both antisense-a and antisense-p significantly inhibited stimulation of DNA-synthesis as determined by the incorporation of BrdU. On the average, 64% (antisense-@) or 51% (antisense-a) of the cells did not enter S-phase when compared to respective experiments with sense-8 and sense-a at equivalent concentrations (Table 1). Sense-8 and sense-a by themselves had no effect on cell growth and growth stimulation; no difference was observed in the presence and the absence
TABLE
1
Antiproliferative Potential of Oligodeoxynucleotides Complementary to CKII Subunits a and j3 Growth Inhibition (X) Oligodeoxynucleotide
EGF
AntisenseAntisense-61M Antisense-fi3M Antisense-a Antisense-alM Antisense-a3M
64 _t 9 28 + 7 17 + 3 51 f 6 22 k 9 7+8
FCS 44 k 18 + 12 + 31+ 18 + -3 f
3 7 8 6 2 5
Note. IMR-90 cells were rendered quiescent by a low concentration of fetal calf serum (0.5%) for 3 days. Oligodeoxynucleotides were added to the medium (final concentration, 100 pg/ml) 2 h prior to growth stimulation by EGF (final concentration, 200 rig/ml) or fetal calf serum (final concentration 20%). Control experiments were carried out with the respective sense-oligodeoxynucleotides. BrdU labeling of cells was done 18-24 h after growth stimulation in the absence of oligodeoxynucleotides. At least 500 cells were analyzed for DNA synthesis in each experiment. Growth inhibition was calculated from the percentages of BrdU-positive cells in experiments with antisense and the respective sense-oligodeoxynucleotides by the formula: Inhibition (%) = ((%BrdU-positive cells (sense-oligo) - %BrdU-positive cells (antisense-oligo))/%BrdU-positive cells (sense-oligo)) x 100. Shown are the mean + standard deviation.
248
PEPPERKOK 60 2
50
% Z z z
40 30
c $
20
u
10 0
Antisense-a
+
+
+
-
-
-
Sense-a
-
+
-
-
-
+
Antisense-P Sense-p
-
-
+
+ -
+ +
+ -
FIG. 3. Competition between CKII antisense- and sense oligodeoxynucleotides. Different combinations of oligodeoxynucleotides (final concentration 100 ag/ml of each oligodeoxynucleotide) were applied to synchronized cells 2 h before growth stimulation with EGF. Control experiments were carried out with respective sense-oligodeoxynucleotides. Determination of DNA synthesis and calculation of growth inhibition was as described in the legend to Table 1. The mean values + standard deviation of three independent experiments are shown.
of 25,50,100, or 200 pg/ml sense oligodeoxynucleotides. When antisense-oligodeoxynucleotide concentrations were varied, a saturation characteristic of the anti-proliferative effect became evident, plateauing at a concentration of 100 fig/ml (data not shown). This indicates that both the regulatory subunit /3and the kinase-active subunit (Yplay an important role in growth stimulation by EGF. In order to prove the specificity of the observed effect, mutations were introduced into the antisense-oligodeoxynucleotides. A gradual reduction of the growth inhibitory effect was observed paralleling number of mutations (Table 1); antisense-oligodeoxynucleotides with one mutation (antisense-@lM; antisense-alM) were only half as effective as the nonmutated ones, and antisense-oligodeoxynucleotides with three mutations (antisense-P3M; antisense-a3M) showed no significant growth inhibition any more. Further, corresponding antisense- and sense-oligodeoxynucleotides were applied together at equal concentrations which should, if the observed growth inhibition was oligodeoxynucleotidespecific, neutralize the anti-proliferative effect of antisense-oligodeoxynucleotides by competing for hybrid formation with mRNAs coding for the respective CKII subunits. This was, as shown in Fig. 3, exactly what happened, while in controls with noncorresponding oligodeoxynucleotide pairs, inhibition of growth stimulation by antisense-oligodeoxynucleotides was unchanged. Thus, the inhibition of growth stimulation by CKII antisense+ and antisense-cl! is a highly structure-specific event. Similar results as with EGF were obtained, although
ET AL.
less pronounced, when fetal calf serum was used as growth stimulator (Table 1). This would, if the growth stimulation by serum was by its EGF content, indicate an EGF-specific role of CKII. If it was the result (also) of other mitogens contained in the serum, this would indicate that CKII is, in addition to the transduction of the EGF message, important for the transduction of the message of other mitogenic stimuli as well. CKII would then play a rather central role in mitogenic signaling. However, a reliable analysis of mitogenic constituents of the employed serum is lacking and hence a reliable interpretation cannot be provided at the moment. Growth Inhibitory Effect of AntisenseOligodeoxynuckotides Is Transient Are the cells suffering from damage upon incubation with antisense-oligodeoxynucleotides or are they able to re-enter the cell cycle after removal of the oligodeoxynucleotides? To answer this question, antisense- or antisense-a was added to the medium 2 h prior to the growth stimulus and left until 18 h after the stimulation. Then, the oligodeoxynucleotides were removed and the cells were further incubated in the presence of BrdU for various times. As shown in Table 2, the growth inhibition observed 24 h after EGF stimulation was significantly stronger than that 28 h after stimulation, and inhibition became insignificant 34 h after stimulation. Again, similar results were obtained when cells were growth stimulated with fetal calf serum (data not shown). This demonstrates that the inhibition of cell growth stimulation by CKII antisense-oligodeoxynucleotides is reversible and hence not the result of cell damage due to toxic effects of oligodeoxynucleotides. Timing of Antisense-@ Treatment Is Decisive for the Anti-proliferative Effect and Correlates with the Downregulation of CKII &Subunit In the experiments described above, oligodeoxynucleotides were added to the culture medium 2 h before TABLE
2
Persistence of the Growth Inhibitory Effect and Antisense-cY of CKII Antisense+ Growth Time of fixation after growth stimulation (h) 24 28 34
inhibition
(%) by
Antisense-p
Antisense-a
47 + 6 31 + 3 11+ 5
51 t 3 37 + 6 13 f 7
Note. Oligodeoxynucleotides were added to the medium 2 h before growth stimulation of cells with EGF (266 rig/ml). At 18 h after growth stimulation, cells were washed twice with culture medium without oligodeoxynucleotides followed by the addition of BrdU. Cells were fixed and stained at the three given time-points after growth stimulation. Growth inhibition was determined as described in the legend to Table 1. Shown are the means + standard deviation.
CASEIN
g .-L :g a d a r e v
KINASE
60 40 20 0 -20
! -24
-12
-6
-2
0
249
II: ANTISENSE-OLIGODEOXYNUCLEOTIDES
5
Time(h) was FIG. 4. Timing of CKII antisense- incubation. Antisenseadded to the culture medium of synchronized cells (final concentration 100 pg/ml) at different time-points with respect to stimulation with EGF (at 0 h). Control experiments were carried out with sense-b. Determination of DNA synthesis and calculation of growth inhibition was as described in legend to Table 1. Negative numbers in the time scale indicate that oligodeoxynucleotides were added at respective time-points before EGF stimulation. The mean values + standard deviation of three independent experiments are shown.
the growth stimulus. In order to study more precisely the time frame of CKII involvement in the stimulation of cell growth, the time-point of oligodeoxynucleotide addition with respect to EGF stimulation was varied. Since subunit /3 is the presumed controlling element of CKII activity, we concentrated on antisense-& Maximum inhibition of growth stimulation was obtained when incubation of cells with antisense-p had been started between 2 and 6 h prior to EGF stimulation (Fig. 4). Addition to cell medium at 12 or 24 h before EGF had little or no effect on growth stimulation as was the case when antisense- was added simultaneously with EGF or 5 h after EGF. In parallel experiments, the intracellular level of CKII @-subunit protein following antisense-p addition to cell medium was visualized by immunofluorescence using affinity-purified anti-CKII j3-subunit antibody and quantitation by image analysis. The antibody, whose CKII P-specificity had been proven by Western blotting of cell extracts (see Fig. 2), localized the protein in the cytoplasm and in the nucleus with at least a five times higher concentration in the nucleus (Fig. 5A). A similar distribution was found by Western blot analyses of nuclear vs cytoplasmic fractions (data not shown) excluding fixation artifacts. The treatment of cells with antisense-& resulted in a significant decrease of the fluorescence signal (Fig. 5C). In addition, the fluorescent signal could be blocked by preincubation of the anti-CKII P-subunit antibody with purified CKII (Fig. 5E) further strengthening the specificity of the immunofluorescence staining for CKII B-subunit. Staining of cell nuclei with Hoechst dye 33258 ensured same number of cells in the whole experimental series (Figs. 5B, 5D, and 5F).
When antisense- incubation was started at 2 or 6 h before EGF stimulation, a sign&ant down-regulation of CKII &subunit in the nucleus occurred reaching a maximum decrease of approximately 60% and lasting for roughly up to 7 h after the growth stimulation (Fig. 6A). Longer incubation periods showed less of a decrease and CKII B-subunit was back at constitutive level when measured after 24 h in either case. When antisense-8 was applied simultaneously with EGF, the level of nuclear CKII P-subunit showed little change in the early phase of EGF-stimulation; significant decrease was found at 5 and 7 h after EGF addition (Fig. 6A). As expected, neither simultaneous application of antisense-8 and sense-p had any effect on CKII subunit 0 expression nor application of antisense-cu (data not shown). Similar down-regulation kinetics were obtained for the cytoplasmic CKII @-subunit (Fig. 6B). However, the decrease of cytoplasmic CKII P-subunit after incubation with antisense-p was considerably weaker (roughly 30% decrease at maximum) than that obtained for the nuclear p-subunit. On the other hand, the decrease of cytoplasmic CKII p-subunit level appeared to occur faster than that of the nuclear level. Taken together, the time frame of CKII antisense inhibition of growth stimulation matches the down-regulation of CKII protein; time-points for antisense-@ addition leading to maximum inhibition of growth stimulation by EGF show significant reduction of P-subunit protein in the early phase of EGF stimulation whereas time-points leading to insignificant growth inhibition do not. DISCUSSION
The observed inhibition of cell growth stimulation by EGF obtained with the CKII antisense-oligodeoxynucleotides is a highly structure-specific event because (i) inhibition occurs only with antisense-a and antisense-@ but not with the respective sense-oriented oligodeoxynucleotides, (ii) antisense-oligodeoxynucleotides with a single mutation have reduced effects and those with three mutations failed to significantly inhibit cell growth, (iii) inhibition is hindered by the simultaneous presence of sense-oligodeoxynucleotides, (iv) inhibition is concentration-dependent with all characteristics of a saturable effect, and (v) the expected decrease of CKII protein is antisense-specific; subunit /3 becomes downregulated exclusively with antisense-@ but not with antisense-a or any other oligodeoxynucleotide. In addition, toxic effects can be excluded because growth inhibition is reversible; after removal of the oligodeoxynucleotides, the cells re-enter the S-phase of the cell cycle. Thus, our results demonstrate that CKII plays an important role in the mitogenic stimulation of cells by EGF and that both CKII subunit cr and subunit B are critical elements. What kind of role is played by CKII?
250
PEPPERKOK
ET AL.
CASEIN
KINASE
II: ANTISENSE-OLIGODEOXYNUCLEOTIDES
251
n Nwleua(Oh)
bypassed via alternative transduction pathways such as those involving protein kinase A or protein kinase C n [l-3]. The lack of a complete inhibition of growth stimulation by antisense-a and antisense- pretreatments may be due to reasons such as the existence of an d subunit, a potential substitute for subunit LY,and the presence of residual amounts of active subunit proteins synthesized prior to oligodeoxynucleotide treatment. How then does CKII play its role as a signal propagator? The mitogenic message of EGF reaches the cytoplasm by the membrane-anchored EGF receptor. For its propa7 24 2 5 0 1 gation into the nucleus via CKII, two alternative pathTime (h) ways were conceivable. CKII may phosphorylate cytoplasmic protein(s) which then migrate into the nucleus to trigger mitosis, or, as the alternative, CKII may min Wopl~(~) 0 Cytoplasm (-2h) grate itself into the nucleus to phosphorylate there n Cytoplasm k6h) growth regulating protein(s). Both alternatives were compatible with our data and both receive support by the current literature. The functioning as a cytosolic element of the signaling cascade is based on an early and transient change in cytosolic CKII activity (up to fourfold increase) following treatment of cells with growth factors such as EGF [6-lo]. This change is -20 -+ thought to result from an activation of CKII due to a 7 24 1 2 5 0 stimulation of subunit CY through subunit /3.Because the Time (h) antisense-oligodeoxynucleotides cause a decrease of inlevel of CKIIB protein after oligodeoxynuFIG. 6. Intracellular tracellular protein levels of the respective CKII subcleotide incubation. Synchronized cells were incubated with antiunits, they should also reduce the actual CKII activity. sense-p for different periods. At zero time, cells were stimulated with While obvious as a consequence of a decrease of the EGF (200 rig/ml). Nuclear (A) and cytoplasmic (B) level of CKII b-subunit were determined at different time-points after mitogenic kinase-active subunit (Y,antisense+ would be effective stimulation by immunofluorescence and image analysis. Control exonly if subunit /3was indeed a positive regulator of celluperiments were carried out with sense-b. Decrease of CKII b-subunit lar CKII activity. The shown decrease of CKIIB protein by antisense-oligodeoxynucleotides was determined by the formula: level following antisense-@ treatment strengthens this Decrease (%) = ((Protein level (sense-@) - Protein level (antisenseimplication and is in agreement with the recently re&)/Protein level (sense-@)) X 100. Numbers in parentheses indicate the start of oligodeoxynucleotide incubation with respect to EGF stimported reduced CKII activity after transfection of 3T3 ulation (negative numbers indicate time-points prior EGF stimulapreadipocytes with antisense-@ cDNAs [39]. It also tion). Given are mean values + standard deviations of at least three matches the results of reconstitution experiments in independent experiments. which the kinase activity of both tissue-derived and recombinant CKII a-subunit became significantly stimulated when united with subunit fl [20-221. Of note, the The coincidence of maximum protein down-regulainteraction of subunit @with ligands such as polylysine tion and most effective timing of antisense-oligodeoxfurther enhanced CKII activity [22,40,41] making conynucleotide addition indicates that the role played by ceivable tuning of CKII by conformational changes of CKII is limited to the early phase of growth stimulation, subunit @and explaining the stimulation of cytosolic i.e., to the first several hours. Hence functioning as me- CKII activity by the reported hyperphosphorylation [6, diator of the EGF message can be assumed. This CKII8-101. Functioning as a cytosolic element of the mitodependent pathway obviously is the main EGF trans- genie signaling cascade would require the presence of duction system, as otherwise the antisense-oligodeoxappropriate CKII substrate(s) in the cytosol. Such proynucleotide-induced CKII-depletion would have been teins are indeed known. A most prominent example is 0
Nucleus(-2h) Nucbw(-6h)
Synchronized cells were exposed to antisenseor sense-b 2 FIG. 6. Staining of cells for CKII &subunit by indirect immunofluorescence. h before EGF stimulation. Cells were fixed and permeabilized 2 h after EGF addition and CKII @-subunit was visualized with affinity-purified antibody against CKII p-subunit and a rhodamine-conjugated goat anti-rabbit second antibody. Cells incubated (A) with sense-& (C) with antisense-& and (E) with affinity-purified CKII &subunit antibody preincubated with purified CKII (1 pg/pl) for 30 min. (B, D, F) Staining of the corresponding cells with Hoechst dye 33258. A and C as well as B and D were taken at the same exposure times.
252
PEPPERKOK
the oncoprotein Myc, whose presence appears to be essential for cell growth. Myc occurs in the cytosol and, as a phosphoprotein, in the nucleus. Its short-term kinetics of the nuclear cytoplasmic transport appears to depend on phosphorylation by CKII [42]. A number of observations, however, are in favor of a signal transport by CKII itself. As shown here, the level of CKII protein is at least fivefold higher in the nucleus than in the cytoplasm and the accumulation of subunit p in the nucleus is significantly inhibited by antisense-& Moreover, the ratio of nuclear to cytoplasmic CKII subunit p in synchronized cells rapidly increased after EGF or serum stimulation by a factor of two to three (data not shown). This correlates well with the recent finding that CKII is accumulated in the nucleus of growing cells but not in cell cycle-arrested cells [l&38]. Since mainly localized and further accumulated in the nucleus following EGF stimulation of cells, this could mean that CKII migrates into the nucleus to transport the EGF message. In the nucleus, CKII would find a number of known substrates which are of particular importance for cell growth and its stimulation. These include cell regulatory proteins such as the oncogene products Myc, Myb, Fos, SV40 large T-antigen, adenovirus Ela, human papilloma virus E7, the ~53 tumor suppressor protein, or the serum response factor [ll-171. In the connecting postpropagation phase than, CKII would phosphorylate proteins such as nucleolin which had been shown to control rDNA transcription and thus ribosome synthesis, a prerequisite for cell growth [18]. In either case of these alternative signal propagation, CKII would receive the message from the EGF receptor. It remains an open question by which step(s) this would be achieved. It can obviously not be achieved by direct phosphorylation through the receptor-intrinsic kinase activity because this phosphorylates proteins at tyrosine residues [2], while the hyperphosphorylation of CKII subunit fl is a serine/threonine kinase-catalyzed event [lo]. Intermediary serinjthreonine kinase(s) must therefore exist such as cdc2 kinase [23] or those described recently for the EGF propagation cascade in 3T3 cells [43, 441.
ET AL. Lozeman, F. J., Liischer, 4.
2.
3.
Edelman, A. M., Blumenthal, D. K., and Krebs, E. G. (1987) Annu. Rev. Biochem. S&567-613. Hunter, T., Angel, P., Boyle, W. J., Chiu, R., Freed, E., Gould, K. L., Isacke, C. M., Karin, M., Lindberg, R. A., and van der Geer, P. (1988) Cold Spring Harbor Symp. QUXWZ~. Biol. LIII, 131-142. Krebs, E. G., Eisenman, R. N., Kuenzel, E. A., Lichtfield, D. W.,
P. T., and Traugh,
Sommercorn, J., Mulligan, J. A., Lozeman, F. J., and Krebs, E. G. (1987) Proc. Natl. Acad. Sci. USA 84,8834-8838.
7.
Klarlund, J. K., and Czech, M. P. (1988) J. Biol. Chem. 263, 15,872-15,875.
8.
Ackerman, P., and Osheroff, 11,95811,965.
9.
Carroll, D., andMarshak, D. R. (1989) J. Biol. Chem. 264,73457348. Ackerman, P., Glover, C. V. C., and Osheroff, N. (1990) Proc.
10.
N. (1989) J. Biol. Chem. 264,
Natl. Acad. Sci. USA 67.821-825. 11. 12. 13. 14. 15. 16.
Liischer, B., Kuenzel, E. A., Krebs, E. G., and Eisenman, R. N. (1989) EMBO J. 8,1111-1119. Luscher, B., Christenson, E., Litchfield, D. W., Krebs, E. G., and Eisenman, R. N. (1990) Nature 344,517-522. Carroll, D., Santoro, N., and Marshak, D. R. (1988) Cold Spring Harbor Symp. Quant. Biol. LIII, 91-95. Griisser, F. A., Scheidtmann, K. H., Tuazon, P. T.,Traugh, J. A., and Walter, G. (1988) Virology 166, 13-22. Barbosa, M. S., Edmonds, C., Fisher, C., Schiller, J. T., Lowy, D. R., and Vousden, K. H. (1990) EMBO J. 9,153-160. Manak, J. R., de Bisschop, N., Kris, R. M., and Prywes, R. (1990)
Genes Dev. 4.955-967. 17.
Meek, D. W., Simon, S., Kikkawa,
U., and Eckhart,
W. (1990)
EMBO J. 4,3253-3260. 18.
19. 20. 21.
22.
Belenguer, P., Baldin, V., Mathieu, C., Prats, H., Bensaid, M., Bouche, G., and Amalric, F. (1989) Nucleic Acids Res. 1’7,66256636. Pyerin, W., Burow, E., Michaely, K., Kiibler, D., and Kinzel, V. (1987) Biol. Chem. Hoppe Seykr 368,215-227. Cachet, C., and Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406. Pyerin, W., Jakobi, R., Taniguchi, H., Lorenz, P., Hoppe, J., Vol3, H., Kiibler, D., Kinzel, V. (1988) Biol. Chem. Hoppe Seykr 369,896. Grankowski, N., Boldyreff, B., and Issinger, O.-G. (1991) Eur. J.
Biochem. 198,25-30. 23.
Mulner-Lorillon, O., Cormier, P., Labbe, J-C., Do&e, M., Poulhe, R., Osborn, H., and Belle, R. (1990) Eur. J. B&hem.
24.
Meisner, H., Heller-Harrison, R., Buxton, J., and Czech, M. P. (1989) Biochemistry 28,4072-4076. Lozeman, F. J., Litchfield, D. W., Piening, C., Takio, K., Walsh, K. A., and Krehs, E. G. (1990) Biochemistry 29,843~8447. Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem.
193,529-534.
26.
183,227-233.
28. 29.
1.
Tuazon,
6.
27.
REFERENCES
J. P. (1988) Cold
77-84. J. A. (1990) Adv. Sec. Messenger
Phosphoprot. Res. 23, 123-164. 5. Pinna, L. A. (1990) Biochim. Biophys. Actu 1064, 267-284.
25. We thank A. Lampe-Gegenheimer for excellent secretarial assistance and the Deutsche Forschungsgemeinschaft for financial support (grant to W.P.).
B., and Sommerkorn,
Spring Harbor Symp. Quant. Bill. LIII,
Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R., and Neckers, L. M. (1987) Nature 328,445-449. Gewirtz, A. M., and Calahretta, B. (1988) Science 242, 13031306. Anfossi, G., Gewirtz, A. M., and Calabretta, B. (1989) Proc. Natl.
Acad. Sci. USA 86,3379-3383. 30.
31.
Goodcbild, J., Agrawal, S., Civeira, M. P., Sarin, P. S., Sun, D., and Zamecnik, P. C. (1988) Proc. Natl. Acad. Sci. USA 86,55075511. Matsukura, M., Zon, G., Shinozuka, K., Robert-Guroff, M., Shimada, T., Stein, C. A., Mitauya, H., Wong-StaaI, F., Cohen, J. S., and Broder, S. (1989) Proc. Natl. Acad. Sci. USA 86.4244-4248.
CASEIN KINASE
II: ANTISENSE-OLIGGDEOXYNUCLEOTIDES
32. Loke, S. L., Stein, C. A., Zhang, X. H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J. S., and Neckers, L. M. (1989) Proc. Natl. Acad. Sci. USA 88,3414-3478. 33. Yakubov, L. A., Deeva, E. A., Zarytova, V. F., Ivanova, E. M., Ryte, A. S., Yurchenko, L. V., and VIassov, V. V. (1989) Proc. Natl. Acad. Sci. USA 86,6454-6456. 34. Sorrentino, V., Pepperkok, R., Davis, R. L., Ansorge, W., and Philipson, L. (1990) Nature 345,813-815. 35. Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. (1982) EMBO J. 1,1621-1628. 36. Kobayashi, R., and Tashima, Y. (1989) Anal. Biochem. 183, 9-12. 37. Ansorge, W., and Pepperkok, R. (1989) J. Biochem. Biophys. Methods. l&263-292. Received May 16,199l Revised version received August 1, 1991
253
38. FiIhol, O., Cachet, C., and Chambaz, E. M. (1990) Biochemistry 29,9926-9936. 39. Meisner, H., and Czech, M. P. (1991) J. CeU.Biochem. 15B, 261. 40. Traugh, J. A., Lin, W. J., Takada-Axelrod, F., and Tuaxon, P. T. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Ed.), pp. 224-229 Raven Press, New York. 41. Hu, E., and Rubin, C. S. (1990) J. Biol. Chem. 265, 20,60920,615. 42. Rihs, H.-P., Jans, D. A., Fan, H., and Peters, R. (1991) EMBO J. 10,633-639. 43. Ahn, N. G., and Krebs, E. G. (1990) J. Biol. Chem. 265,11,49511,501. 44. Ahn, N., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265,11,487-11,494.
