Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells (cysteine proteasesyER chaperonesyproteasome)

The loss of tyrosinase, the key enzyme in melanin synthesis, has been implicated in the dedifferentiation of malignant melanocytes. The presence of tyrosinase transcripts and antigenic peptides in melanoma tumors prompted us to investigate whether the basis for the loss of the enzyme was proteolytic degradation. Toward this aim, we followed the kinetics of synthesis, degradation, processing, chaperone binding, inhibitor sensitivity, and subcellular localization of tyrosinase in normal and malignant melanocytes. We found that, in amelanotic melanoma cell lines, tyrosinase failed to reach the melanosome, the organelle for melanin synthesis, because it was retained in the endoplasmic reticulum (ER) and then degraded. Tyrosinase appeared mostly as a 70-kDa core-glycosylated, endoglycosidase H-sensitive, immature form bound to the ER chaperone calnexin and had a life-span of only 25% of normal. Maturation and transit from the ER to the Golgi compartment was facilitated by lowering the temperature of incubation to 31°C. Several proteasome inhibitors caused the accumulation of an '60-kDa tyrosinase doublet that was more prominent in malignant than in normal melanocytes and promoted, to various degrees, the maturation of tyrosinase in melanoma cells and the translocation of the enzyme to melanosomes. The appearance of ubiquitinated tyrosinase after treatment of normal melanocytes with N-acetyl-L-leucinyl-L-leucinal-L-norleucinal reinforced our notion that some tyrosinase is normally degraded by proteasomes. Proteolysis of tyrosinase by proteasomes is consistent with the production of antigenic tyrosinase peptides that are presented to the immune system by major histocompatibility complex class I molecules. Loss of pigmentation is observed in human melanomas in situ and in metastatic melanoma cells established in culture. The several studies designed to elucidate the basis for this phenotype have been focused on tyrosinase, the key enzyme of melanogenesis. In those in which both protein and mRNA levels were examined, a posttranscriptional regulation was implicated because, despite low or undetectable tyrosinase protein levels, tyrosinase mRNA was detected easily (refs. 1–4 and unpublished results). The latter was found in solid tumors, in cells in culture, and in blood-borne melanoma cells (5–7). The possibility that melanocyte-specific proteins were synthesized but later degraded was supported by the numerous reports identifying peptides derived from such melanogenic proteins as tyrosinase, TRP1ygp75, and gp100yPmel 17 that serve as tumor antigens recognized by T cells of melanoma patients (see, for example, refs. 8 and 9 reviewed in ref. 10). Tyrosinase (70–80 kDa) is a type I membrane glycoprotein whose cDNA predicts a peptide of '58 kDa, a 28-amino acid cytosolic tail, and five putative N-glycosylation sites (refs. 2 and 11 and for review see refs. 12 and 13). Like other membrane glycoproteins, tyrosinase is processed in the endoplasmic reticulum (ER) by resident chaperones and enzymes (14, 15). Reductions in melanoma tyrosinase levels could be mediated by the quality control system of the ER because selective retention in the ER and subsequent degradation by the proteasome complex occurs in several genetic diseases. An example is the cystic fibrosis transmembrane conductance regulator in which a mutation blocks proper processing and transport to the cell membrane (refs. 16 and 17 and see reviews in refs. 18 and 19). Proteasomal proteolysis is implicated also in the generation of peptides for presentation by major histocompatibility complex class I molecules (20, 21), so we set out to examine whether tyrosinase in amelanotic melanoma cells was also subject to degradation by proteasomes. MATERIALS AND METHODS Cell Culture. Neonatal and adult normal human melanocytes were maintained in growth factor supplemented PC-1 or Ham’s F-10 medium (22). Adult donors were 17 to 35 years old (details in the legend to Fig. 1). Metastatic melanoma cells were maintained in Ham’s F-10 medium containing 5% fetal calf serum and 5% newborn calf serum and no additional growth factors. Two of the melanoma cell strains persistently were amelanotic (YUSIT1 and YUSAC2), and two, for yet unknown reasons, fluctuated between being amelanotic and slightly melanotic (501 mel and YUGEN8). These four strains represented phenotypes seen in at least 10 other melanoma cell lines (1, 3, 23, 24). Despite having low levels of tyrosinase protein, they maintained tyrosinase mRNA levels similar to those of normal melanocytes (unpublished results) and expressed other melanogenic proteins essential for the formation of melanin (data not shown). Inhibitors. Protease inhibitors LLnL (N-acetyl-L-leucinyl-Lleucinal-L-norleucinal) and E64 (L-trans-epoxysuccinic acid; both from Boehringer Mannheim) and MG132 (N-carbobenzoxyl-Leu-Leu-leucinal; Calbiochem) were dissolved in dimethyl sulfoxide (DMSO; 100 mM stock solutionyeach) and used as indicated. Lactacystin (provided by E. J. Corey, Department of Chemistry, Harvard University, Cambridge, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y946210-6$2.00y0 Abbreviations: Endo H, endoglycosidase H; ER, endoplasmic reticulum; LLnL, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal; DMSO, dimethyl sulfoxide. †To whom reprint requests should be addressed. e-mail: ruth. [email protected].