Erk 1 / 2 kinase and p 21 CIP 1 / WAF 1 activation after UVC radiation in normal human melanocytes and melanoma cells

As a survival factor for melanocytes lineage cells, MiTF plays multiple roles in development and melanomagenesis. What role MiTF plays in the DNA damage response is currently unknown. In this report we observed that MiTF was phosphorylated at serine 73 after UVC radiation, which was followed by proteasome-mediated degradation. Unlike after c-Kit stimulation, inhibiting p90RSK-1 did not abolish the band shift of MiTF protein, nor did it abolish the UVC-mediated MiTF degradation, suggesting that phosphorylation on serine 73 by Erk1/2 is a key event after UVC. Furthermore, the MiTF-S73A mutant (Serine 73 changed to Alanine via site-directed mutagenesis) was unable to degrade and was continuously expressed after UVC exposure. Compared to A375 melanoma cells expressing wildtype MiTF (MiTF-WT), cells expressing MiTF-S73A mutant showed less p21 accumulation and a delayed p21 recovery after UVC. Consequently, cells expressing MiTF-WT showed a temporary G1 arrest after UVC, but cells expressing MiTF-S73A mutant or lack of MiTF expression did not. Finally, cell lines with high levels of MiTF expression showed higher resistance to UVC-induced cell death than those with low-level MiTF. These data suggest that MiTF mediates a survival signal linking Erk1/2 activation and p21 regulation via phosphorylation on serine 73, which facilitates cell cycle arrest. In addition, our data also showed that exposure to different wavelengths of UV light elicited different signal pathways involving MiTF. Background MiTF plays a critical role in melanocyte lineage differentiation and survival [1], as well as melanomagenesis [2]. The MiTF gene is amplified in about 20% of melanomas and is capable of transforming normal melanocytes in certain genetic environments, therefore it has been suggested that MiTF can function as an oncogene [3,4]. However, re-expression of MiTF in BRAF-expressing human melanocytes inhibited cell proliferation, suggesting that MiTF represses cell cycle progression [5]. This is consistent with reports showing that MiTF activates the cyclin-dependent kinase inhibitors p21 and p16 [6,7]. More and more evidence indicates that MiTF plays multiple roles in melanomagenesis including stimulating angiogenesis via activating Hif1a [8], enhancing cell proliferation via activating transcription of Bcl-2 and CDK2 [9,10], preventing apoptosis via activating melanoma inhibitor of apoptosis (ML-IAP) [11], inhibiting invasion via activating DIAPH-1 [12], and promoting survival after elevation of cellular reactive oxygen species via activating Ape/Ref-1 [13]. A recent study using mouse melanocytes with various MiTF doses indicated that MiTF dose was a primary determinant for murine melanocytes survival after UVR [14]; however, the mechanism(s) by which this occurred was not clear. A genetic hallmark of human melanoma is mutually exclusive mutations of BRAF and NRAS, which are found in more than 90% of tumors [15]. Oncogenic BRAF or NRAS mutations activate cell proliferation pathway through downstream mitogen-activated kinases Mek1/2 and extracellular signal-regulated kinase (Erk1/ 2) [16]. BRAF or NRAS activation leads to Mek1/2 activation which in turn activates Erk1/2 which directly phosphorylates MiTF at serine 73 [17,18]. Activated Erk1/2 can further activate its downstream kinase p90RSK1 which can also phosphorylate MiTF at serine 409 [19]. Phosphorylation at both sites triggered by c-Kit * Correspondence: liufe@uci.edu Department of Medicine, University of California-Irvine School of Medicine, Orange, CA 92868, USA Full list of author information is available at the end of the article Liu et al. Molecular Cancer 2010, 9:214 http://www.molecular-cancer.com/content/9/1/214 © 2010 Liu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. stimulation leads to a signal cascade for pigment cell development [19]. This dual phosphorylation results in a transient increase of MiTF trans-activation activity and a subsequent degradation; however, the biological consequence of this transient activation and degradation is not clear. Recently in vivo studies indicated that mutation at serine 73 completely rescued mouse coat color [20], suggesting this mutation may have other functions than melanocyte development, among which participating in the DNA damage response is one of the possibilities [21]. Whether MiTF plays a role in DNA damage response has not been previously reported and is the subject of this study. In this study, we report that the DNA damaging agent UVC radiation leads to Erk1/2 mediated phosphorylation of MiTF at serine 73, which in turn leads to proteasomemediated MiTF degradation. Erk1/2 phosphorylation of MiTF played a critical role in activating p21 transcription and a temporary G1 cell cycle arrest, which enhanced cell survival after UVC radiation. These results suggest a novel function of MiTF in linking Erk1/2 activation and p21 regulation after UVC radiation in normal human melanocytes and melanoma cells.