Vol. 6, 291-302, March 1995 Cell Growth & Differentiation 291 Identification of p90RSK as the Probable CREB-Ser’33 Kinase in Human Melanocytes Markus B#{246}hm, Gisela Moellmann, Elaine Cheng, Mayra Alvarez-Franco, Susanne Wagner, Paolo Sassone-Corsi, and Ruth Halaban2 Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06520-8059 EM. B., G. M., E. C., M. A-F., R. H.]; University of Massachusetts Medical Center, Program in Molecular Medicine, Worcester, Massachusetts 01 605 [S. WI; and Laboratoire de G#{233}n#{233}tique Mol#{233}culaire des Eucaryotes du CNRS, Unite 184 de Biologie Mol#{233}culaire et de Genie G#{233}n#{233}tique de l’INSERM, Institut de Chimie Biologique, Facult#{233} de M#{233}decine,67404 lllkirch, CU de Strasbourg, France [P. S-C.l Abstrad Normal human melanocytes proliferate in vitro only in response to costimulation by at least two seleded peptide growth fadors. In the presence of only one mitogen, melanocytes become quiescent or die. These mitogens also enhance expression of differentiated fundions, since in their presence the proliferating melanocytes become progressively more pigmented. To assess the intermediates participating in this dual response, we have determined the adivated state of several known ligand-induced signal transducers. We demonstrate that hepatocyte growth fador/scatter fador, mast/stem-cell growth fador, basic fibroblast growth fador, and endothelin-1 induce phosphorylation of Ser133 within the KID domain of the cAMP-responsive element binding protein, a modification necessary for transcriptional adivation of all members of this family of transcription fadors, including also cAMP-responsive element modulator’r and adivating transcription fador 1 . The costimulation with synergistic growth fadors prolonged the phosphorylated state and adivity of the mitogen-adivated protein kinase 2 cascade. cAMP-responsive element binding protein phosphorylation in response to synergistic growth fadors was due probably to the adivation of p9ORS and, to a lesser extent, to p7O. Our findings support the concept that signals initiated at the cell surface converge on regulatory proteins that sustain both cell division and differentiation. lntrodudion In culture, mebanocytes respond to the synergistic action of a restricted number of mitogenic peptides which, in combination with each other or with TPA3 or dbcAMP, stimulate not only proliferation but also pigmentation (me- viewed in Refs. 1-3). These mitogens include several FGFs, HGF/SF, and MGF (also known as KIT ligand, stem cell growth factor, and steel factor), all of which stimulate me- ceptom tyrosine kinases, and the neuropeptides ET-l and ET-2 (4, 5), the latter activating G-pmotein-coupled receptors (reviewed in Ref. 6). Some of these growth factors control the fate of melano- cytes during embryogenesis. For example, inactivation mu- tations in either MGF or its receptor KIT lead to piebaldism (reviewed in Ref. 1), and bFGF promotes differentiation of avian neural crest-derived cells into a melanocytic lineage (7, 8). These growth factors also have a mole in postnatal melanocyte biology, for all have been detected in human skin, with bFGF being the most abundant one expressed in basal keratinocytes and present also in the extracellular matrix (9-1 1). In the mouse, MGF is required for melano- cyte proliferation during the regenerative cycle of hair fob- bides (12). Involvement of growth factors in suntanning is suggested by the observation that UVB irradiation raises ET-l in cultured human keratinocytes to bioactively detect- able levels (4), increases secretion of HGF/SF and MGF by fibmoblasts, and stimulates the release of bFGF from kemati- nocytes and fibrobbasts (2, 1 3). Furthermore, the restricted distribution of human epidemmal melanocytes to the base of the epidermis could be due to the predominant elaboration of melanocyte growth factors by basal keratinocytes (14). Our aim has been to elucidate the mechanism(s) by which the synergistic growth factors converge to effect or enhance proliferation and differentiation in human mela- nocytes. Since melanocyte proliferation and differentiation are positively regulated by agents that increase cAMP (1 5-1 7), we have focused on the transcription factor CREB, which is known to be activated by cAMP, as the possible mediator of the differentiation signal and on the serine/ threonine MAPK2/ERK2, a target of many diverse ligand- initiated activations (reviewed Refs. 1 8-21). We show that one of the enzymes activated by synergistic pairs of growth factors is a Mr l00,000 CREB/CREMT/ATF1 kinase that phosphorybates the transcription regulator CREB on Sem1 This Mr 1 00,000 protein is probably p90RS(, whose activity is stimulated by the same growth factors that enhance the phosphorybation/activation of the MAPK2 cascade through activation of RAF1 and MEK (MAPK2 kinase). Received 10/25/94; revised 1/6/95; accepted 1/9/95. 1 This study was supported by USPHS Grants CA44542 (to R. H.), AR39848 (to R. H.), and AR41 942 (Yale Skin Diseases Research Center; to R. E. Tigelaar, principal investigator); by the Charlotte Geyer Foundation (to R. H.), and by Grant Bo 1075/2-i (to M. B.) from the Deutsche Forschungsge- meinschaft. 2 To whom requests for reprints should be addressed, at Department of Dermatology, Yale University School of Medicine, P.O. Box 208059, New Haven, CT 06520-8059. 3 The abbreviations used are: TPA, 1 2-O-tetradecanoyl phorbol-i 3-acetate; dbCAMP, dibutyryl cyclic AMP; bFGF, basic fibroblast growth factor; HGF/ SF, hepatocyte growth factor/scatter factor; MGF, mast/stem cell growth factor; ET, endothelin; CRE, cAMP-responsive element; CREB, CRE binding protein; CREM, CRE modulator; MAPK2/ERK2, microtubule-associated or mitogen-activated protein kinase 2/extracellular signal-regulated kinase 2; PDT, population doubling time; ATF1 , activating transcription factor 1; CaMK, calciunVcalmodulin kinase; PKA, protein kinase A; apCREB, a-phos- pho-CREB; TFP, trifluoroperazine; GST-CREB, glutathione S-transferase- CREB fusion protein; aPY, anti-phosphotyrosine antibodies; IBMx, isobutyl- methyl xanthine; KID, kinase induced domain; MBP, myelin basic protein; MEK, MAPK kinase; MEKK, MEK kinase; PKC, protein kinase C; PKI, protein kinase A inhibitor; RSK or 56K, ribosomal 56 kinase; ECL, enhanced chemiluminescence.