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Home , Kinase, Phosphoprotein, Protein kinase, Protein Kinase C, Ribosomal s6 kinase

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12110)-12114, December 1995 Cell

Activation of a / signaling pathway by transforming type ( AZEDDINE ATFI*, KATIA LEPAGE*, PIERRE ALLARDt, ALCIDE CHAPDELAINEt, AND SIMONE CHEVALIER*tt *McGill University. Department of Surgery, Urology Division and The Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G 1A4; and tUniversity of Montreal, Departments of Medicine and and Research Center, Maisonneuve-Rosemont Hospital, 5415 I'Assomption, Montreal, Quebec, Canada HIT 2M4 Communicated by Seymour Lieberman, St. Luke's-Roosevelt Institute for Health Sciences, New York, NY, August 17, 1995

ABSTRACT Transforming growth factor type 13 (TGF-f3) ylated by receptor 11 (8, 9). of receptor I on is a multifunctional factor that regulates proliferation and both serine and threonine residues correlates with activation of differentiation of many cell types. TGF-fJ mediates its effects its kinase activity (9). Receptor complexes in which receptor I by binding to and activating cell surface receptors that possess is unphosphorylated, because of either a mutation in the type serine/threonine kinase activity. However, the intracellular II receptor kinase domain or mutations in the GS domain of signaling pathways through which TGF-,B receptors act re- the type I receptor (i.e., the site of phosphorylation by type II main largely unknown. Here we show that TGF-f3 activates a receptor), are unable to transmit signals (9). 78-kDa (p78) serine/threonine kinase as evidenced by A fundamental aspect of the antiproliferative action of an in-gel kinase assay. -induced activation of the kinase TGF-,3 is its ability to promote arrest in G, (13). was near-maximal 5 min after TGF-,B addition to the cells and Recently, progress has been made in deciphering the identity occurred exclusively on serine and threonine residues. This and function of putative and that play a role in kinase is distinct from TGF-f3 receptor type II, as well as GI progression (14). In certain cell types, TGF-,B has been several cytoplasmic serine/threonine kinases of similar size, shown to regulate the expression and/or the activity of - including C, Raf, mitogen-activated protein dependent kinases, including cdks and cdc2 (13, 15). While kinase kinase kinase, and . Indeed, these physiological regulation of cdks and cdc2 is likely to be kinases can be separated almost completely from p78 kinase important for TGF-,B actions, the pathways that bridge the gap by immunoprecipitation with specific antibodies. Further- between the TGF-,B receptors and these kinases remain un- more, using different cell lines, we demonstrate that p78 known. A common feature of mitogenic signals relayed by kinase is activated only in cells for which TGF-.8 can act as a growth factor receptors is that they are initiated by ligand- growth inhibitory factor. These data raise the interesting induced activation of the intrinsic kinase activity of the recep- possibility that protein serine/threonine kinases contribute tor (16-18). In the case of receptor kinases, ligand to the intracellular relay of biological signals originating from activation of protein kinase triggers the phosphorylation of receptor serine/threonine kinases such as the TGF-,B recep- multiple cellular on tyrosine, including the receptor tors. itself (18, 19). These initial tyrosine phosphorylation events are followed by activation of several protein serine/threonine The transforming growth factor types ,3 (including TGF-f31, kinases that act in sequence, with one phosphorylating and TGF-f32, and TGF-,B3) are a family of multifunctional cyto- activating the next (16, 17). kines that control an array of functions in animal cells from As these findings suggest that plays virtually every lineage (1, 2). TGF-f was originally described a pivotal role in growth regulation, the isolation of additional as a factor that stimulates the growth of normal rat protein kinases should help to define the mechanisms through fibroblasts in soft agar (3). However, it was later shown to be which TGF-,B receptors regulate normal and a potent growth inhibitor for a wide variety of cell types induce differentiation. In searching for such proteins, we have including most normal and transformed cells (4, 5). TGF-,B identified a 78-kDa protein (p78) serine/threonine kinase also exerts many other biological effects including regulation distinct from TGF-,B type I and type II receptors, and several of cell adhesion and migration, stimulation of extracellular cytoplasmic serine/threonine kinases, including protein kinase matrix production, and modulation of important immune and C (PKC), Raf, mitogen-activated protein kinase kinase kinase endocrine functions (1, 2, 5). Alterations in the activity of this (MEKK), and ribosomal S6 kinase (RSK). We find that factor have been implicated in fibrosis, immunosuppression, treatment of cells with TGF-,B rapidly induces increases in the , and other human disorders (2, 6). kinase activity, indicating that this may be involved in TGF-f3 signals through a heteromeric complex between the TGF-,B pathways. type I and type II receptors (7-9). Cell mutants that fail to bind TGF-f3 via these receptors lack not only the growth inhibitory response but also the responses typically induced by MATERIALS AND METHODS TGF-f3 (1, 9-12). Molecular cloning of the type I and type II Materials. TGF-,31 and staurosporine were obtained from receptors and characterization of their in vitro kinase activities Sigma. Polyclonal antibodies to TGF-f3 receptor II, Raf-1, and has shown that both receptor types are transmembrane serine/ RSK, and TGF-f3 type II receptor competing peptide were threonine kinases (1). The type II receptor, which is a consti- from Upstate Biotechnology. Monoclonal anti-PKC (a, 13, and tutively active kinase, can directly bind ligand, but it is inca- 6) and anti-MEKK antibodies were purchased from Transduc- pable of mediating TGF-,B responses in the absence of a type tion Laboratories, Lexington, KY. Anti-TGF-,B antibody (a I receptor (8, 9). Bound TGF-,B is then recognized by receptor rabbit polyclonal antibody raised against porcine TGF-,1 and I, which is recruited into the complex and becomes phosphor- Abbreviations: MEKK, mitogen-activated protein kinase kinase ki- The publication costs of this article were defrayed in part by page charge nase; PKC, ; RSK, ribosomal S6 kinase; TGF-3, payment. This article must therefore be hereby marked "advertiselment" in transforming growth factor type (B. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 12110 Downloaded by guest on September 24, 2021 Cell Biology: Atfi et al. Proc. Natl. Acad. Sci. USA 92 (1995) 12111 TGF-132) was from R & D Systems. [y-32P]ATP (7000 Ci/ RESULTS mmol; 1 Ci = 37 GBq) was from ICN. Cell and Culture. PC3, DU145, NIH 3T3, CHO, COS, TGF-j3 Stimulates Serine/Threonine Protein Phosphory- HeLa, HepG2, and LNCaP cells were cultured in Dulbecco's lation. We first tested whether incubation of PC3 prostatic modified Eagle's medium (DMEM) supplemented with 5 mM carcinoma cells with TGF-f3 induces any change in protein glutamine and 10% dialyzed fetal calf serum (dFCS) according kinase activity that can be detected by an in-gel kinase assay. to routine procedures. For experiments, cells were grown to Whole cell extracts were prepared from untreated and TGF- 50-70% confluency, washed with DMEM containing 10% ,3-treated cells and subjected to SDS/PAGE. After electro- dFCS, and then treated with TGF-f3 in the same medium. phoresis, the proteins in the gel were allowed to renature and Metabolic Labeling of Proteins, Immunoprecipitation, and the gel was incubated with [-y-32P]ATP to reveal kinase activ- Western Blot Analysis. Cell lysates were prepared as described ities that presumably represent . As (20). Samples of either [35S]methionine-labeled or unlabeled shown in Fig. 1, a protein kinase with an apparent molecular proteins were immunoprecipitated with the indicated antibody mass of 78 kDa (p78) was detected in PC3 cell extracts, and its and immune complexes were resolved by SDS/PAGE. For activity was greatly increased in cells treated with TGF-,B. A 35S-labeled samples, the gels were treated with EN3HANCE variety of other bands were also detected, but there was no (NEN) and exposed to x-ray film at -80°C. For Western blot measurable effect of TGF-,B on the activity of these species. analysis, proteins were electrophoretically transferred to Im- The TGF-,3-induced activation of p78 occurred rapidly after mobilon-P membranes (Millipore) and probed with antibod- addition of TGF-,B, with maximal stimulation at 5 min followed ies; bands were visualized by an enhanced chemiluminescent by a progressive decline at later times (Fig. 1A). Similar results detection system according to the manufacturer's instructions were obtained with CHO and DU145 cell lines, suggesting (ECL; Amersham). similarities in stimulus-response coupling mechanisms of In-Gel Kinase Assay and Phosphoamino Acid Analysis. TGF-,B receptors (A.A. and S.C., unpublished data). Exami- Immunoprecipitates and cell lysates were fractionated by nation of the TGF-,B dose-response revealed detectable p78 electrophoresis on SDS/9% polyacrylamide gel. For denatur- activation at 20 pM and maximal activation at =40 pM (Fig. ation and renaturation of the separated proteins, the gels were 1B). Significantly, the dose-response of p78 activation by treated according to the procedures developed by Durocher et TGF-,B was similar to that for TGF-fB-induced growth inhibi- al. (21). For identification of the autophosphorylated kinases, tion of PC3 cells (A.A. and S.C., unpublished data; see also ref. the gels were immersed for 2 h at 22°C in buffer containing 10 22). It is important to also note that the induction of p78 mM Hepes (pH 7.4), 10 mM 2-mercaptoethanol, 20 mM phosphorylating activity by TGF-,3 was inhibited by addition of MgCl2, 5 mM MnCl2, 100 ,uM Na3VO4, and 100 ,Ci of polyclonal anti-TGF-(3 antibody, which competes with TGF-,B ['y-32P]ATP. After incubation, the gels were washed three receptor for TGF-f binding (20, 22), indicating that ligand times with' 200 ml of 5% trichloroacetic acid containing 1% binding was required for stimulation of p78 activity (A.A. and sodium pyrophosphate and 1% sodium . The S.C., unpublished data). washed gels were dried and exposed to Kodak x-ray films for autoradiography. To establish the identity of the amino acids A B phosphorylated in p78, segments (corresponding to autora- diographically localized autophosphorylated kinase) of the 94 - 94 - polyacrylamide gels were excised and the phosphoamino acid -40- content was determined by published procedures (21). 67 - 67 - TGF-f8 Type II Receptor Kinase Activity. Kinase reactions were carried out by adding immunoprecipitates into buffer that 43 - 43 - contained 50 mM Tris HCl (pH 7.4), 10 mM MgCl2, 1 mM 30 - 30 - CaCl2, and 20 ,uCi of ['y-32P]ATP in a vol of 30 ,ul. Reaction 0 200 mixtures were incubated at 37°C for 30 min and the samples 0 2 5 10 30 5 10 20 40 were analyzed on SDS/7.5% polyacrylamide gel (9). time (min) TGFi(pM) Cell Fractionation. Cells were washed with phosphate- buffered saline (PBS), scraped from the plates, and pelleted at Control TGFP 200 x g. The cell pellet was resuspended in a buffer containing 25 mM Hepes (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, P-.Ser 4.i 1 mM EDTA, 10 mM benzamidine, 10 jig of aprotinin per ml, P.T...I 2 ,g of pepstatin per ml, and 0.08% 2-mercaptoethanol. The w cells were homogenized by 20 strokes in a tight-fitting Dounce P-h I w,, homogenizer, and nuclei were removed by a 30-min centrifu- gation at 3000 x g (4°C). The supernatant was resedimented at 105,000 x g, and the resulting supernatant was saved as the P-Tvr cytosolic fraction. The pellet was then resuspended in the same buffer 0.5% Triton X-100 and 0.25 M and containing KCI, FIG. 1. TGF-j3 activates a 78-kDa protein serine/threonine kinase. rocked gently for 1 h at 4°C to extract membrane proteins. The (A) PC3 cells were treated with 40 pM TGF-1 for the indicated times extracted material was then sedimented at 105,000 x g for 1 h and cell lysates were fractionated on SDS gel and assayed for kinase and the supernatant was saved as the particulate fraction. activity in situ as described. 32P-labeled proteins were detected by [3H] Incorporation. Cells were seeded in six-well autoradiography. (B) PC3 cells were treated with increasing concen- plates in DMEM supplemented with 10% dFCS, grown over- trations of TGF-,B (as indicated) for 10 min and cell lysates were night, and then incubated for 3 days with 40 pM TGF-f3. Cells fractionated on SDS gel and assayed for kinase activity in situ. were labeled with 1 ,uCi of [3H]thymidine per ml for the last Molecular mass markers are indicated on the left; arrow indicates the 78-kDa protein kinase whose activity was increased by TGF-f. (C) The 4 h of incubation. Cells were washed three times with cold PBS 78-kDa bands from untreated (Left) and TGF-,B- and fixed with 10% trichloroacetic acid for 30 min at 4°C. The treated (Right) cells were isolated from SDS/polyacrylamide gel slices, cells were then washed twice with 10% trichloroacetic acid and and phosphoamino acids were analyzed by two-dimensional thin-layer extracted with 0.5 M NaOH for 30 min. The extracts were electrophoresis. Positions of migration of phosphoamino acid stan- collected and assayed in a liquid scintillation spectrometer. dards are indicated. Downloaded by guest on September 24, 2021 12112 Cell Biology: Atfi et al. Proc. Nati. Acad. Sci. USA 92 (1995) Phosphoamino acid analysis of the phosphorylated p78 Similar results were obtained when the detection of type II kinase revealed mainly phosphoserine together with a small receptor, the in vitro kinase activity of type II receptor, and the amount of phosphothreonine (Fig. IC). TGF-,3 treatment in-gel kinase assay were performed with the same immuno- increased phosphorylation on both residues. Taken precipitate (data not shown). Recent studies revealed that the together, these data indicate that TGF-13 exposure increased TGF-f3 type II receptor is constitutively phosphorylated by the activity of a 78-kDa kinase and that this kinase was capable cellular kinases at multiple sites and by itself at additional sites of serine/threonine phosphorylation under in vivo conditions. and that its autophosphorylating activity is not regulated by Whether one or more protein kinases contribute to p78 activity ligand binding (9). Therefore, the 80-kDa protein recognized could not be ascertained by the assays used in these studies. by the anti-receptor II antibody might be relatively inactive Characterization of p78 Activity. TGF-,B binds to various under in-gel kinase assay conditions. membrane proteins, two of which, the TGF-,3 receptors I (53 To further investigate the relationship between receptor II kDa) and 11(80 kDa), form a signaling receptor complex (7-9). and p78, we compared the patterns of protein kinase activities Receptors I and II are also protein serine/threonine kinases in cytosolic and membrane fractions of PC3 cells by SDS/ (1). Since the molecular mass of receptor II is similar to the PAGE and in-gel kinase assay. Immunoblotting with anti- 78-kDa protein kinase examined above, we analyzed receptor receptor II antibody was used as a control for the effectiveness II kinase activity in PC3 cell extracts by SDS/PAGE and in-gel of fractionation. The p78 activity in these cells was clearly in kinase assay. Using a polyclonal antibody directed against the the membrane and cytosolic fractions (Fig. 2D). As expected, type II receptor, we were able to specifically and quantitatively receptor II was not detected in the cytosolic fraction, indicat- deplete PC3 cell extracts of receptor II (Fig. 2A) and recover ing the cytoplasmic proteins were not contaminated measur- large quantities of in vitro kinase activity in the receptor ably with membrane proteins (Fig. 2D). These data, together Il-directed immunoprecipitates (Fig. 2B). However, we failed with the lack of detectable kinase activity in anti-receptor II to detect any activity after SDS/PAGE and in-gel kinase assay immunoprecipitates, indicate that p78 is distinct from TGF-,B despite extensive overexposure of the gel (Fig. 2C). The p78 receptor II. activity was retained almost exclusively in the supernatant. p78 Is Distinct from PKC, Raf, MEKK, and RSK. The results presented above show that TGF-,B induces activation of a 78-kDa protein serine/threonine kinase in PC3 cells. It is A B important to note that the electrophoretic mobility of p78 is t3 - + - + similar to that previously reported for PKC, Raf, MEKK, and RSK. These serine/threonine kinases appear to integrate 9, - 92 W signals from multiple membrane receptors (16-19). To further 67 - 67- " investigate the nature of p78, cell lysates were prepared from 43 I4R PC3 cells and immunoprecipitated with antibodies directed 43- .04,1110. Ig against PKCs, Raf-1, MEKK, and RSK. The supernatants and 30.... 30 - immunoprecipitates were resolved on SDS/polyacrylamide gel and the gels were subsequently incubated in the presence of 3 4 radioactive ATP under conditions that promote the phospho- 1 2 3 4 1 2 rylating activity of p78 (in-gel kinase assay). As shown in Fig. D 3A), the p78 activity was almost exclusively retained in the C supernatants. In contrast, we detected no bands in the immu- -r(F -- + + noprecipitates that would be consistent with the presence of 92_ _ PKCs, Raf-1, MEKK, or RSK activities, despite the presence --- 92_- 67- 67 - _ of these kinases in the PC3 cell line (Fig. 3B). Similar results were obtained when PKCs, Raf-1, MEKK, and RSK were immunoprecipitated from PC3 cells after TGF-,3 treatment '' (data not shown). The failure to detect PKCs, Raf-l, MEKK, 30 - I I 30... _ and RSK activities was not due to poor stimulation of the cells by TGF-,B, because addition of this growth factor resulted in S S I 1 LI I 2 2 the expected increase in p78 activity. Collectively, these data FIG. 2. p78 activity is distinct from activity associated with the indicate that p78 activity is unlikely to be attributable to PKC, TGF-,B type II receptor. (A) Total cell lysates from PC3 cells were Raf, MEKK, or RSK. Additional evidence is provided by the subjected to immunoprecipitation with TGF-,3 type II receptor anti- finding that treatment of PC3 cells with TGF-,B did not induce body in the presence (lanes 3 and 4) or absence (lanes I and 2) of the detectable changes in the phosphorylation of Raf-1, MEKK, TGF-,3 type II receptor competing peptide. Immunoprecipitates (lanes and RSK, at least in the time frame we have examined. 1 and 3) and supernatants (lanes 2 and 4) were resolved by SDS/PAGE Furthermore, pretreatment of PC3 cells with the potent PKC and transferred to Immobilon-P filters; filters were probed with inhibitor staurosporine prior to the addition of TGF-,B did not anti-TGF-,B type II receptor antibody. (B) PC3 cells were treated with alter the increase in p78 activity (A.A. and S.C., unpublished 40 pM TGF-f3 for 5 min and cell lysates were subjected to immuno- data). II in the presence precipitation with TGF-,B type receptor antibody Activation in Cell Lines. p78 is (lanes 3 and 4) or absence (lanes I and 2) of the TGF-f3 type II receptor p78 TGF-4J-Responsive competing peptide. Immunoprecipitates were used for in vitro type II expressed in a wide variety of cell lineages (Fig. 4A). We receptor kinase assays as described. Products of the reactions were therefore tested the effects of TGF-,B in a variety of cell lines visualized by SDS/PAGE and autoradiography. Arrow indicates po- to determine whether the ability of TGF-j3 receptors to induce sition of TGF-,B type II receptor. (C) Cell lysates from the experiment p78 activity might potentially be linked to their ability to described in B were subjected to immunoprecipitation with TGF-f3 transduce cell growth arrest. These cell lines were treated with type II receptor antibody. Immunoprecipitates (I), supernatants (S), 40 pM TGF-,B (a dose that induced the maximal growth and unfractionated cell lysate (U) were resolved on SDS gels and inhibition in each responsive cell line used as determined in assayed for kinase activity in situ. Arrow indicates position of p78. (D) preliminary experiments) for 5 min, and p78 activity was were as described. 1) and PC3 cells fractionated Cytosolic (lane treatment induced particulate (lane 2) fractions were subjected to an in-gel kinase assay assessed as described above. TGF-/3 p78 (Left) or Western blot analysis (Rig/ht) using anti-TGF-, type II activity in PC3, DU145, HepG2, and CHO cells for which receptor antibody. Migration positions for p78 and TGF-,B receptor TGF-,B can act as a growth inhibitory factor (Fig. 4). In type II are indicated by arrows. contrast, there was no apparent induction of p78 activity when Downloaded by guest on September 24, 2021 Cell Biology: Atfi et aL Proc. Natl. Acad. Sci. USA 92 (1995) 12113

A Raf MEKK RSK 94- B .-w- 94- 67- -f*e_.I_ 67- 43- 43 30- -

30 - u s I S I s I u S I S I S I 1 2 3 4 5 6 FIG. 3. p78 activity is not immunoprecipitated by antibodies directed against PKCs, Raf-1, MEKK, and RSK. (A) Total cell lysates from PC3 cells were immunoprecipitated with antibodies directed against PKCs (a, 3, 8), Raf-1, MEKK, and RSK as indicated. Immunoprecipitates (I), supernatants (S), and unfractionated cell lysates (U) were resolved by SDS/PAGE and assayed for kinase activity in situ. Arrow indicates position of the 78-kDa protein kinase. (B) [35S]Methionine-labeled immunoprecipitates with antibodies against PKC-a (lane 1), PKC-f3 (lane 2), PKC-6 (lane 3), Raf-1 (lane 4), MEKK (lane 5), or RSK (lane 6) were resolved by SDS/PAGE and visualized by fluorography. Positions of protein standards (kDa) are indicated on the left. the assay was done with the TGF-(3 nonresponsive cell lines threonine kinase with an apparent molecular mass of 78 kDa. LNCaP, COS, HeLa, and NIH 3T3 (Fig. 4). This observation The increase in kinase activity occurs in conjunction with was particularly intriguing since previous studies indicated that phosphorylation of p78 itself, and it is possible that this NIH 3T3 cells possess TGF-(3 type I and type II receptors, as phosphorylation contributes directly to its increased enzymatic determined by cross-linking to radiolabeled TGF-,3 (23), and activity. Analogous increases in autophosphorylating activity respond to TGF-,B1, as evidenced by stimulation of fibronectin were not seen in a variety of other protein kinases detected in or a531 levels (24). We also noted a significant change the same experiments, suggesting that p78 is specifically im- in the NIH 3T3 cell morphology after TGF-f3 treatment, plicated in signal transducing machinery used by the TGF-f3 indicating that the lack of a detectable increase in p78 activity receptors. We have ruled out the possibility that induction of is not likely due to the inability of the cells to respond to p78 activity is due to a nonspecific effect by demonstrating that TGF-f. Together, these data demonstrate that TGF-,3 stim- the stimulatory effect of TGF-f3 can be blocked by addition of ulates the phosphorylating activity of p78 in different respon- polyclonal anti-TGF-,3 antibody, which competes with TGF-f3 sive cell lines despite the lack of a correlation between the receptors for TGF-f3 binding (20, 22). Consistent with the fact degree of p78 phosphorylation and the magnitude of TGF-,3- that activation of TGF-f3 receptors is required for activation of mediated growth inhibition. In this regard, one hypothesis p78, phosphorylation and activation of TGF-,B receptor I is could be that TGF-f3 receptors trigger additional signaling simultaneous with, or precedes, phosphorylation of p78 at all pathways in a cell-specific manner. TGF-,B concentrations tested (this study; see also ref. 9). The sensitivity of p78 to TGF-,3 and rapid onset after TGF-,B DISCUSSION addition make serine/threonine phosphorylation of p78 among the most sensitive and rapid responses known for In this study, we have shown that TGF-f3 rapidly induces TGF-,3, raising the possibility that activation of p78 is a critical increases in the activity of a cytoplasmic protein serine/ step downstream of TGF-,B receptors. The involvement of cyto- A PC3 DU 145 CHO LNCaP COS 3T3 Ilela

...... 92 -

67 - 1i

43 - 30 .

+ - + - + - T(GFfi + - + - + - + - + B 1209

. _...... ~~~~~.~~~~ ~ ~ 100 ,......

80i ._ 60. r=- 40. 20

0. Control PC3 DU145 ('HO HepG(2 Control L,NCap CO()S 3'13 Hela

FIG. 4. Effects of TGF-/3 on p78 activity and DNA synthesis in different cell lines. (A) PC3, DU145, NIH 3T3, CHO, COS, HeLa, HepG2, and LNCaP cells were treated with 40 pM TGF-,B for 5 min and cell lysates were resolved by SDS/PAGE and assayed for kinase activity in situ. Molecular mass markers (kDa) are indicated on the left; arrow indicates position of the 78-kDa protein kinase. (B) Cells were plated at a low cell density, grown overnight, and then treated with 40 pM TGF-f3. After 68 h, the cells were pulsed for 4 h with [3H]thymidine and the amount of incorporated radioactivity was measured by scintillation counting. Data are expressed as percentages of response elicited by 10% as in Method Section Cell and Culture FCS (control). Assays were done in triplicate, and replicates were within 10% of each other. Downloaded by guest on September 24, 2021 12114 Cell Biology: Atfi et al. Proc. Natl. Acad. Sci. USA 92 (1995) plasmic serine/threonine kinases in TGF-,B signaling had not 3T3 cells have TGF-3 receptors and, indeed, respond to been shown until now and supports the general notion that the TGF-,B, as evidenced by stimulation of fibronectin or a5f31 TGF-,B receptors signal through protein phosphorylation events. integrin levels (23, 24) and architecture (this study). Thus, A variety of observations strongly suggest that the p78 regulation of the activity of different cytoplasmic protein activity described in this report is distinct from the activity kinases may be a way to differentiate and maintain specificity associated with the TGF-,B type II receptor. First, the p78 between different signals. Irrespective of how specificity is activity was completely separable from TGF-,B receptor II by obtained, the demonstration that TGF-,B activates cytoplasmic immunoprecipitation with anti-TGF-,3 receptor II antibody. protein kinases provides an important focus for future studies Second, the p78 activity was detected in cytosolic and partic- to define signaling pathways used by receptor serine/threonine ulate fractions, whereas TGF-f3 receptor II was found only in kinases such as the TGF-,B receptors. the particulate fraction. This is consistent with the previously described properties of TGF-,3 receptor 11 (7, 8). Finally, the We thank Drs. E. Drobetsky, W. Hauck, and G. Beauregard for TGF-f3 receptor II is constitutively autophosphorylated and helpful discussion. This work was supported by the Medical Research ligand occupancy causes no appreciable change in this activity Council of Canada. S.C. received a Senior Scholarship from Le Fonds (this study; see also ref. 9). These observations make it very de la Recherche en Sante du Quebec. unlikely that p78 is TGF-,3 receptor II, although the possibility that it is a modified form of receptor II with radically altered 1. Massague, J., Attisano, L. & Wrana, J. L. (1994) Trends Cell Biol. activity cannot be eliminated until the precise structure of p78 4, 172-178. 2. Roberts, A. B. & Sporn, M. B. (1993) Growth Factors 8, 1-9. protein has been determined. 3. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M. & Many protein serine/threonine kinases of -80 kDa, such as Sporn, M. B. (1981) Proc. Natl. Acad. Sci. USA 78, 5339-5343. PKC, Raf, MEKK, and RSK, can also be stimulated by a 4. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., variety of polypeptide ligands, even though these ligands have Stern, D. F. & Sporn, M. B. (1985) Proc. Natl. Acad. Sci. USA 82, very different effects on cells (16, 17). Therefore, it is reason- 119-123. able to ask whether these kinases are related to p78. The 5. Moses, H. L., Yang, E. Y. & Pietenpol, J. A. (1990) Cell 63, evidence presented in this report indicates that the p78 kinase 245-257. activity is unlikely to be attributable to PKCs, Raf-1, MEKK, 6. Border, W. A. & Ruoslahti, E. (1992) J. Clin. Invest. 90, 1-7. and RSK. The experiment with specific antibodies has dem- 7. Massague, J. (1992) Cell 69, 1067-1070. onstrated that these protein kinases are completely separable 8. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., from p78 activity. Consistent with these observations, pub- Laiho, M., Wang, X.-F. & Massague, J. (1992) Cell 71,1003-1014. that the of 9. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F. & Massague, lished studies demonstrated PKC, target phorbol J. (1994) Nature (London) 370, 341-347. ester action, is unlikely to be involved in the response to TGF-/3 10. Franzen, P., ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., (25). This suggestion was initially based on the inhibitory effect Heldin, C.-H. & Miyazono, K. (1993) Cell 75, 681-692. of staurosporine, which had been used as a specific PKC 11. Lin, H. Y. & Lodish, H. F. (1993) Trends Cell Biol. 3, 14-19. inhibitor, and was further implied by the observations that 12. Bassing, C. H., Yingling, J. M., Howe, D. J., Wang, T., He, W. W., TGF-f3 responsiveness is retained in phorbol ester-desensitized Gustafon, M. L., Shah, P., Danahoe, P. K. & Wang, X.-F. (1994) cells (25). It is now becoming evident that phorbol ester Science 263, 87-89. activation of PKC induces of Raf-1 and 13. Ewen, M. E., Sluss, H. K., Whitehouse, L. L. & Livingston, D. M. RSK as well as activation of MEKK (26-28). The finding that (1993) Cell 74, 1009-1020. TGF-,B, in contrast to polypeptide growth factors and phorbol 14. Sherr, C. J. (1994) Cell 79, 551-555. ester (16-19), did not stimulate phosphorylation of Raf-1, 15. McCarthy, S. A. & Bicknell, R. (1992) J. Biol. Chem. 267, is from 21617-21622. MEKK, and RSK strongly indicates that p78 distinct 16. Marshall, C. J. (1995) Cell 80, 179-185. PKC, Raf-1, MEKK, or RSK. However, we cannot exclude the 17. Herskowitz, I. (1995) Cell 80, 187-197. possibility that Raf-1, MEKK, and RSK are involved in TGF-03 18. Heldin, C.-J. (1995) Cell 80, 213-223. signal transduction since, in a previous study (20), we have 19. Cohen, G. B., Ren, R. & Baltimore, D. (1995) Cell 80, 237-248. shown that TGF-f3 induces the disruption of SHC-GRB2 20. Atfi, A., Drobetsky, E., Boissonneault, M., Chapdelaine, A. & complex in PC3 cells. Chevalier, S. (1994) J. Biol. Chem. 269, 30688-30693. As mentioned in the Introduction, TGF-f3 is a multifunc- 21. Durocher, Y., Chapdelaine, A. & Chevalier, S. (1992) Biochem. tional factor that regulates many aspects of cellular functions, J. 284, 653-658. including cell proliferation, differentiation, adhesion, and mi- 22. Atfi, A., Samperez, S. & Jouan, P. (1994) Prostate 24, 149-155. gration (1, 2, 5). Functional analysis of TGF-f3 type I and type 23. Segarinin, P. R. (1990) Ann. N.Y Acad. Sci. 593, 73-90. II receptors has shown that both kinase domains are essential 24. Ignotz, R. A. & Massague, J. (1986) J. Biol. Chem. 261, 4337- the 4345. for signaling all TGF-,3 responses (9, 12). Therefore, 25. Ohtsuki, M. & Massague, J. (1992) Mol. Cell. Biol. 12, 261-265. diversity of biological responses elicited by TGF-13 must occur 26. Wood, K. W., Sarnecki, C., Roberts, T. M. & Blenis, J. (1992) at levels other than receptor activation and may be related to Cell 68, 1041-1050. differences in the substrates available to TGF-,B receptors. This 27. Thomas, S. M., DeMarco, M., D'Arcangelo, G., Halegoua, S. & mechanism is suggested by the inability of TGF-,B to induce Brugge, J. S. (1992) Cell 68, 1031-1040. p78 activity in NIH 3T3 cells, a cell line that failed to undergo 28. Howe, L. R., Leevers, S. J., Gomez, N., Nakienly, S., Cohen, P. growth arrest in response to TGF-f3 (Fig. 4). Note that NIH & Marshall, C. J. (1992) Cell 71, 335-342. Downloaded by guest on September 24, 2021