Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12110)-12114, December 1995 Cell Biology Activation of a serine/threonine kinase signaling pathway by transforming growth factor 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 Biochemistry 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). Phosphorylation 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 protein (p78) serine/threonine kinase as evidenced by A fundamental aspect of the antiproliferative action of an in-gel kinase assay. Ligand-induced activation of the kinase TGF-,3 is its ability to promote cell cycle 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 cyclins and kinases 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 cyclin- including protein kinase C, Raf, mitogen-activated protein dependent kinases, including cdks and cdc2 (13, 15). While kinase kinase kinase, and ribosomal S6 kinase. 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 tyrosine 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 proteins 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 protein phosphorylation 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 kidney 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 cell growth 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 cancer, and other human disorders (2, 6). kinase activity, indicating that this enzyme may be involved in TGF-f3 signals through a heteromeric complex between the TGF-,B signal transduction 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 gene 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, protein kinase C; 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 autophosphorylation. 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.