Rapid stimulation of Ser/Thr protein kinases following treatment of Swiss 3T3 cells with bombesin. Involvement of casein kinase-2 in the signaling pathway of bombesin.

Treatment of quiescent Swiss 3T3 mouse fibroblasts with bombesin resulted in a rapid 6-8-fold stimulation of cytosolic Ser/Thr kinase activities toward the S6 peptide (RRLSSLR), myelin basic protein (MBP), and the G peptide (SPQPSRRGSESSEE). Anion exchange Mono Q chromatography resolved multiple S6 peptide- and G peptide kinase activities and two MBP kinase peaks. Both MBP- and several S6 peptide kinase peaks could be inactivated by PCSL (PP2A2) phosphatase action. This indicates that the bombesin-induced activation of these enzymes is mediated by a Ser/Thr phosphorylation event. The S6 peptide kinases as well as the two MBP kinases stimulated in response to bombesin are similar to those activated by epidermal growth factor in Swiss 3T3 fibroblasts which suggests that the early events of the signal transduction pathway mediated by these growth factors in Swiss 3T3 cells may converge in the activation of common Ser/Thr kinases. Bombesin, which acts as a sole mitogen for Swiss 3T3 fibroblasts, also produced a several-fold increase in the kinase activity toward the RRREEESEEE peptide, a specific substrate for CK-2. This kinase activity was heparin-sensitive and also measurable with the G peptide (SPQPSRRGSESSEE) and GS-1 peptide (YRRAAVPPSPSPSLSRHSSPHQSEDEE), which contain consensus sequences for phosphorylation by CK-2. The bombesin-stimulated CK-2 activity could not be measured in whole cytosols but was revealed by the anion exchange chromatography step. The activation of CK-2 was not reversed by PCSL phosphatase action. The implication of CK-2 in the signal transduction pathway of bombesin is discussed.

1 peptide (YRRAAVPPSPSPSLSRHSSPHQSEDEE), which contain consensus sequences for phosphorylation by CK-2. The bombesin-stimulated CK-2 activity could not be measured in whole cytosols but was revealed by the anion exchange chromatography step. The activation of CK-2 was not reversed by PCSL phosphatase action. The implication of CK-2 in the signal transduction pathway of bombesin is discussed.
Cells respond to a variety of extracellular signals through changes in the level of protein phosphorylation. Stimulation of quiescent mammalian cells by mitogens results in a cascade of phosphorylation events involving protein tyrosine kinases as well as protein serine/threonine kinases, leading to DNA synthesis and cell division.
A number of hormones and growth factors such as insulin, insulin-like growth factor 1 (IGF-l),' epidermal growth factor (EGF), platelet-derived growth factor, and colony-stimulating factor 1 (CSF-l), act through receptors with an intracellular tyrosine kinase domain (reviewed in Refs. 1 and 2). Activation of this intrinsic tyrosine kinase upon ligand binding is supposed to be the initial step in the signaling pathway triggered by those growth factors (1, 2). Since most of the subsequent phosphorylations occur on serine and threonine residues, some link should exist between receptor tyrosine kinases and cytosolic serine/threonine kinases. This could point to a direct activation of a serine/threonine kinase by tyrosine phosphorylation although other mechanisms may occur (3-5).
The amphibian tetradecapeptide bombesin, as well as the bombesin-related mammalian peptides including gastrin-releasing peptide and the neuromedins (for a review see Ref. 6), are potent mitogens for Swiss 3T3 cells (6), human small cell lung cancer cells (7), and bronchial epithelial cells (8). The bombesin receptor has been cloned from Swiss 3T3 fibroblasts: it is a member of the family of seven hydrophobicregion, G-protein-linked neuropeptide receptors (9) and its sequence does not reveal a protein kinase domain. Upon binding to its receptor, bombesin evokes a complex cascade of early biochemical events including inositol 1,4,5-triphosphate-induced mobilization of intracellular Ca", Na+, and K+ fluxes, protein kinase C activation, transmodulation of the EGF-receptor, accumulation, and expression of the protooncogenes c-Fos and c-Myc and CAMP production (6). Although this complex signaling pathway is still largely unknown, it promises to have some striking features.
First of all, bombesin by itself, just like platelet-derived growth factor or fibroblast growth factor, can act as a sole mitogen for Swiss 3T3 cells (10). The fact that many hormones and neuropeptides which have similar receptors with seven membrane spanning domains, are by themselves not mitogenic for Swiss 3T3 cells, suggests that bombesin is acting through a rather special signaling pathway.
Second, although its receptor does not contain a cytoplasmic tyrosine kinase domain, bombesin stimulates the tyrosine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts (11). However, as recently reported by Zachary et al. (11) none of these substrates corresponds by molecular weight or by immunological or biochemical criteria t o known tyrosine kinase substrates suggesting that the tyrosine phosphorylation events initiated by this neuropeptide may be different from the classical receptor tyrosine kinase induced phosphorylations.
We were therefore interested to know whether the bombesin-induced mitogenesis in Swiss 3T3 cells encompasses any of the signaling mechanisms common of receptor tyrosine kinases. In particular, we examined the effect of bombesin on the activity of already known growth factor-activated kinases (recently reviewed in Refs. [12][13][14]. The present study shows that, in quiescent Swiss 3T3 fibroblasts, bombesin rapidly stimulates the activity of several s 6 peptide kinases as well as MBP kinases. Furthermore, our results implicate casein kinase 2 (CK-2) in the early signal transduction pathway of this mitogen.

Time-dependent Effect of Bombesin on Soluble Kinase Activities
Cytosolic extracts of either control cells or cells treated for various lengths of time with bombesin (50 nM) were assayed for their ability to phosphorylate a number of peptide-and protein substrates such as the s6 peptide, the G peptide, MBP, and the RRREEESEEE peptide ( Fig. 1, Miniprint). Fig. 1 shows that kinase activities toward the s6 peptide, MBP, and the G peptide peaked after 5-10 min of bombesin treatment. The rate of phosphorylation of the s 6 peptide and the G peptide declined by about 40% at the 40-min time point (Fig. 1, A and B), whereas the 7-fold maximal stimulation of the MBP kinase, seen at 5-min incubation with the growth factor, was more drastically reduced at the later time points (Fig. 1C). Kinase activities toward the RRASVA peptide and the Kemptide followed the pattern of the s6 peptide phosphorylation in the time-course experiment (not shown).
The rate of phosphorylation of the RRREEESEEE peptide (CK-2 activity) did not change very dramatically (Fig. 1D): a 30% increase in CK-2 activity was observed at the very most. Similar results were also obtained using casein as a substrate for CK-2 (not shown).

Dose Dependence of Protein Kinase Activation by Bombesin
In order to characterize the dose dependence of the protein kinase stimulations, quiescent Swiss 3T3 cells were treated for 5 min with various concentrations of bombesin. As shown in Fig. 2, a maximal response was observed with concentrations of bombesin around lo-' to lo-' M, which corresponds to the concentration range which elicits the mitogenic response in Swiss 3T3 fibroblasts (6). The small CK-2 activation measured with the RRREEESEEE peptide as illustrated in Fig. 1 was not always evident. The higher level of activations observed in these experiments as compared to the ones Portions of this paper (including "Experimental Procedures" and Figs. 1, 2, and 6) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. reported in the time courses of Fig. 1 are possibly due to the stabilizing effect of 100 nM microcystin which was included in the extraction buffer of the dose-dependence experiments.

Separation of Bombesin-activated Protein Kinase Activities by Mono Q FPLC Column Chromatography
The kinase activities in extracts from Swiss 3T3 control cells and from cells treated for either 5 or 40 min with bombesin, were resolved by Mono Q FPLC chromatography.
Early Responses (5 Min of Bombesin Treatment)- Fig. 3A shows that the s6 peptide kinase activities in the cells treated for 5 min with bombesin were separated into several peaks: a major part of the activity came in the breakthrough of the column and four overlapping activity peaks eluted early in the gradient: at 0.12,0.14,0.17, and 0.2 M NaCl, respectively.
All the s6 peptide kinase peaks could phosphorylate the Kemptide and, with the exception of the 0.2 M NaCl peak, also the RRASVA peptide (not shown). The breakthrough, the 0.12 M, and the 0.17 M eluate peaks revealed comparable kinase activities toward the G peptide as substrate ( Fig. 3B), suggesting that the activated kinases may phosphorylate both peptides at very similar rates. The stimulated G peptide kinase peak eluting at 0.09 M NaCl was occasionally also seen using the s 6 peptide as substrate. However, the major stimulated G peptide kinase eluted later in the gradient at 0.35 M NaCl where no s6 peptide kinase activity was measurable (Fig. 3A). Usually more G peptide kinase activity (200%) was recovered after Mono Q chromatography than originally applied to the column, suggesting that a large fraction of kinase activity toward this peptide is masked in whole cytosolic extracts.
The MBP kinase activity (

Bombesin-stimulated SerlThr
Protein Kinases recovered in the gradient fractions. Surprisingly, when the RRREEESEEE peptide was used to specifically detect the CK-2 activity in the Mono Q eluted fractions, a many-fold stimulation was observed, coeluting exactly with the G peptide kinase activity at 0.35 M NaCl (Fig. 30). This observation together with the results depicted in Fig. 1C and Fig. 2 0 strongly suggests that the Mono Q chromatography step may separate substances that interfere in the assay: recovery of the bombesin-stimulated CK-2 activity was several-fold greater in the Mono Q-eluted fractions. The degree of CK-2 activation, measured in the gradient fractions, varied between 2-6-fold in a series of four totally independent stimulation experiments. Differences in the level of activation of CK-2 are for instance seen in the experiments illustrated (see Figs. 3 0 and 5). In general it was observed that when cells of later passages were used in similar experiments or when the subculture schedule was not strictly followed, basal levels of CK-2 activity were enhanced severalfold and stimulation by bombesin greatly reduced. This would suggest that partially transformed Swiss 3T3 cells already have the CK-2 enzyme in an activated form.
Late Responses (40 Min of Bombesin Treatment)-As illustrated in Fig. 4, prolonged incubation (40 min) of Swiss 3T3 cells with bombesin resulted in a decrease in the activation level of all kinases. The activities measured in the Mono Q profiles reflected very much the respective kinase levels measured in the cytosolic extracts at the 40-min time points in Fig. 1 (except for the CK-2 activation discrepancy). The most important decrement in s6 peptide kinase activities (Fig. 4A) occurred in the breakthrough fraction (80%) as well as in those peaks eluting very early in the gradient (between 0.12 and 0.17 M NaCl), whereas the last s6 peptide kinase peak was only slightly affected (about 25%). The MBP kinase activations (Fig. 4C) decreased by 80% after 40 min of incubation of cells with bombesin ( Fig. 3C) similarly to what was observed in the extracts (Fig. IC). The coeluting G peptide and RRREEESEEE peptide kinase activities were also reduced after prolonged bombesin treatment to about 45 and 30%. respectively (Fig. 40). were applied to a Mono Q column and processed as described in Fig. 3. Implication of CK-2 in the Signaling Pathway of Bombesin In order to further characterize the RRREEESEEE peptide kinase eluting in the 0.35 M peak on the basis of its site specificity, the GS-1 peptide (34), YRRAAVPPS3bPSLS3'RH-SS4PHQS5EDEE, which contains the specific CK-2 phosphorylation site (Ser') of glycogen synthase, was used to assay kinase activity under similar experimental conditions as in Fig. 3. As shown in Fig. 5C, the GS-1 peptide served as a substrate for three kinase activities corresponding to the stimulated MBP kinases I and I1 (Fig. 5B) and the RRREEE-SEEE peptide kinase (Fig. 5A).
Phosphorylation of the GS-1 peptide by kinase FA/GSK~, which occurs on sites SerSb, -3c, and -4, is absolutely dependent upon prior phosphorylation of site 5 by CK-2 (27,28). Moreover, kinase FA/GSK~ activity measured with the GS-1 peptide previously phosphorylated by CK-2 eluted in the breakthrough fraction of the Mono Q column, and its activity was not changed upon bombesin treatment. The GS-1 peptide does not contain phosphorylation sites for the s 6 peptide kinases, so that its increased phosphorylation at the 0.11 and 0.17 M NaCl elution peaks is either due to the activated MBP kinases themselves or to still unknown coeluting activities. The rate of phosphorylation of the GS-1 peptide by the two MBP kinases was very similar to that observed with the reference substrate (MBP), indicating that both substrates are recognized equally well by these enzymes (Fig. 5, B and  C ) . On the contrary, at the 0.35 M NaCl elution place, the GS-1 peptide was phosphorylated more readily than the RRREEESEEE peptide (Fig. 5, A and C). This observation is consistent with the site specificity studies of CK-2 which demonstrate that this kinase exhibits a very high affinity for the GS-1 peptide (27) and phosphorylates the shorter hexapeptide SEEEEE, reminescent of the glycogen synthase Ser' site, extremely well (33). Kinase activities in the 0.35 M NaCl peak fractions, were completely abolished by the addition of heparin in the phosphorylation assay (not shown).
In conclusion, the overlapping activities toward three different peptide substrates (GS-1-, G-, and RRREEESEEE peptides) containing consensus sequences for CK-2, together with the coinciding elution at 0.35 M NaCl in the Mono Q gradient (19) and the inhibition by heparin, are three strong arguments indicating that these phosphorylations are due to CK-2 itself.

Phosphatase Treatment of the Bombesin-activated Kinases
The addition of microcystin, a powerful inhibitor of ATP, Mg-dependent (type 1)-and PCS (type 2A) phosphatases (34), to the column fractions was absolutely required to maintain the activation of the protein kinases. This strongly suggests that phosphorylations are involved in the mechanism of the kinase activations triggered by bombesin. Therefore, fractions containing activated kinases were subjected to the PC& (type 2A2) phosphatase before addition of microcystin (Fig.  6). After 30 min of phosphatase treatment the MBP I kinase was almost completely inactivated ( Fig. 6 B ) , whereas the MBP I1 kinase activity was reduced by 60% (Fig. 6C). Since microcystin could prevent MBP kinase inactivation (Fig. 6,  B and C) it can be concluded that the activation of both enzymes depends upon a Ser/Thr phosphorylation reaction. On the contrary, the stimulated CK-2 activity toward the RRREEESEEE peptide was totally unaffected by PCSL (PP2A2) phosphatase action (Fig. 6 A ) . Treatment of the activated CK-2 fractions with alkaline phosphatase bound to agarose did not inactivate the enzyme neither (not shown). Pooled CK-2 fractions from control cytosols were also insensitive to PC& (PP2A2) phosphatase action (not shown). This indicates either that the activation of CK-2 is not directly or solely controlled by Ser/Thr phosphorylation or that the phosphorylation sites involved are not accessible to the phosphatases used.
Due to the high instability of the activated s6 peptide kinases in the absence of microcystin, it was not always possible to obtain a reliable measurement of the residual activities after preincubation of the pooled fractions at 30 "C.
Nevertheless, the s6 peptide kinase activities that were separated by the Mono Q gradient between 0.12 and 0.17 M NaCl were completely abolished by type 2A phosphatase action, whereas the more retarded s6 peptide kinase peak (eluting at 0.2 M NaCl) did not seem to be affected (not shown).

DISCUSSION
The present study unambiguously shows that treatment of Swiss 3T3 cells with the neuropeptide bombesin results in a rapid increase in the activity of several cytosolic Ser/Thr kinases toward a number of protein-and peptide substrates. In particular, incubation of cells with bombesin (5 min) produces a several-fold activation of multiple s6 peptide kinases and of two MBP kinases. Interestingly, this mitogen also elevates a kinase activity toward the peptide RRREEESEEE which is only revealed after chromatography of bombesinstimulated cytosols on a Mono Q FPLC column. This activated kinase was furthermore identified as CK-2.
Quiescent Swiss 3T3 fibroblasts can be induced to proliferate by a variety of peptide growth factors and hormones, seemingly involving different early signal transduction events. However, the activation of certain key enzymes appears to be common to several signaling pathways. This is the case for the s 6 kinases and MBP kinases which are established growth-regulated enzymes (12)(13)(14). When comparing our results with published reports it is clear that the signaling pathways of bombesin and EGF involve the activation of similar sets of kinases, both in the early and in the late phases of the cellular response.
A group of five bombesin-stimulated kinases phosphorylate the s6 peptide, and their time course of activation, substrate specificity, chromatographic behavior, and their sensitivity to phosphatase action relates them to the EGF-activated S g peptide kinases described in Ref. (19).
Two distinct peaks of activated MBP kinases are observed in the Mono Q column elution of bombesin-stimulated cytosols. Their chromatographic properties as well as their sensitivity to inactivation by the PCSL (PP2Az) phosphatase, makes them comparable to the EGF-stimulated MBP k' lnases 1 and 2 which also recognize microtubule-associated protein 2 (MAP2) as a substrate (19, 35). The MBP/MAP2 k' lnases have been reported to be growth-stimulated enzymes in various systems (13,14,36), and both kinases activate a s6 peptide kinase from control Swiss 3T3 cells (37). While this work was in progress, Takuwa et al. (38) investigated the role of Ca2+ influx on the mitogenic response induced by bombesin in Swiss 3T3 cells and reported that bombesin stimulated cytosols incorporated more [32P]phosphate into exogenously added MBP and s6 protein than control cytosols. This increase in phosphorylation was found to be independent of the extracellular Ca2+ concentration, but no attempt was made to characterize the enzymes involved.
Probably the most intriguing finding of the present report is the rapid stimulation of CK-2 following treatment of quiescent Swiss 3T3 fibroblasts with bombesin. This ubiquitous kinase is a very well characterized enzyme as far as its subunit structure, substrate specificity, cellular localization, and sensitivity to inhibitors and activators is concerned, but a full comprehension of its physiological regulation is still missing (for a review see Ref. 39). An interesting but puzzling observation in our study is that a chromatography step is necessary to reveal the bombesin-mediated activation of CK-2. The CK-2 activity of control cytosols is quantitatively recovered in the Mono Q column fractions, indicating that the chromatography step by itself does not produce an artificial increment in the CK-2 activity. Therefore, the increased CK-2 activity measured after the ion exchange chromatography of bombesin-treated cells is likely due to the separation of a factor present in stimulated cytosols, which specifically masks the activated CK-2 enzyme. Since a similar level of CK-2 activity is measured in both control and bombesin-stimulated cytosols it is conceivable that the bombesin-generated CK-2 activity is mobilized from a latent enzyme pool. We have currently no evidence for a direct correlation between the bombesin-induced CK-2 activation and a phosphorylation of the enzyme although clearly more information is required to clarify this point.
As mentioned before, Swiss 3T3 cells of later passages tend to undergo spontaneous transformation, and this is reflected in an altered cell morphology and an increase in the basal level of CK-2 activity. It is well documented that the CK-2 activity is increased in transformed cells and proliferating tissues (40)(41)(42)(43)(44) as well as during cell differentiation (45) and in most cases this correlates with an elevated expression of the enzyme. In addition to this, a transient rise in CK-2 activity has been observed upon stimulation of cells by different growth factors: in W138 human lung fibroblasts by serum (46), in 3T3-Ll adipocytes by insulin or EGF (47), in BALB/ c 3T3 fibroblasts by IGF-1 or insulin (48) and in A431 human carcinoma cells by EGF (49). The latter observations have inferred that some short-term modulation of this kinase should also exist, and it has been suggested that a covalent modification of CK-2 underlies its mechanism of regulation (47,49,50). The magnitude of CK-2 activation observed with insulin or EGF in 3T3-Ll mouse adipocytes is rather modest and variable (ranging from 30 to 150%) (47) and not always reproduced in similar cell systems (18,19,48). It is furthermore interesting to note that the highest levels of CK-2 activation are observed when a combination of different growth factors is used to stimulate the growth of BALB/c 3T3 fibrobasts (48) or when quiescent human W138 fibroblasts are induced to proliferate by whole serum (46). In the case of Swiss 3T3 fibroblasts neither insulin nor EGF alone is able to elicit a mitogenic response and CK-2 does not seem to be activated in these signal transduction events (19). On the contrary, bombesin by itself is a potent mitogen for these cells, and our data clearly shows a drastic stimulation of the CK-2 activity upon treatment of Swiss 3T3 cells with bombesin. It is therefore conceivable that the activation of CK-2 is an intrinsic property of the signal transduction pathway of "sole mitogens" like bombesin as it may well be a compulsory event leading to cell division. This opens the intriguing possibility that CK-2 may also integrate multiple signaling pathways initiated by several different growth factors which work synergistically to make cell proliferation possible. The predominant nuclear localization of CK-2 in actively proliferating cells (51) is one more indication that this kinase may play a fundamental role in the control of cell division, and the observation that a number of nuclear proteins, including the oncogene products Myc, Myb, Fos, p53, ElA, the SV40 T antigen (51), and the serum response factor ~6 7 '~~ (52) are substrates for CK-2 tends to confirm this.

The GS-I peptide ( 2 1 ) . YRRAAVPPS PSLS RHSS QS EUEE, Contain$ the specific CK-2 lite (SerS)
of glycogen synthase. P r i m pharpharylatian Of the Scr5 I S 1 prerequisite far the subsequent phosphorylation by klnare F IGSK which then phosphorylates Ser4,-3c. and -3b ( 2 8 ) . Therefore the phospho-form of the G8-1 &?de labeled by CK-2. was used to detect kindle F./GsK: activity. The GS-I peptlde has been reported td 5erve a110 a5 a substrate for a pGline! directed proteln klnare (29). The RRREEESEEE i s I synthetic peptide which 1s specifically On! should note that l n order to prevent mntrlbutiom of PK-A protein knnare C and phorphorylated by CK-2 (30).