Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.10.3.1 (tyrosinase)
9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

When we studied the effects of polyphenols from apple fruits on melanogenesis in B16 mouse melanoma cell lines, phloridzin had dose-dependent progressive effects on melanogenesis between 10 and 500 micrograms/ml without inhibiting cell growth. At a concentration of 500 micrograms/ml, phloridzin increased the melanin content in the cells to 181% of that in control cells. In contrast, phloretin, the aglycon of phloridzin, did not activate melanogenesis in the cells and was cytotoxic at a concentration of 5 micrograms/ml. Phloridzin increased the activity of tyrosinase to 223% of that in control cells. Furthermore, phloridzin inhibited the activity of protein kinase C (PKC), which is recognized to regulate tyrosinase activity. The inhibition of PKC activity continued for 120 min from the addition of phloridzin. Therefore, we estimated that the activation of melanogenesis by phloridzin resulted from the increase of tyrosinase activity caused by the inhibition of PKC activity.
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PMID:Progressive effects of phloridzin on melanogenesis in B16 mouse melanoma cells. 943 75

In B16 melanoma cells, cAMP-induced melanogenesis is inhibited by the tumor promoting phorbol ester, TPA. However, the role of PKC activation or depletion in the inhibition of melanogenesis by TPA remains controversial. In this report, using specific PKC inhibitors, we demonstrated that PKC inhibition does not impair cAMP-induced melanin synthesis and tyrosinase expression. Further, the inhibition of melanogenesis by TPA results from a decrease of the tyrosinase promoter transcriptional activity and this effect is mimicked by over-expression of a constitutively active form of PKC alpha. These findings clearly demonstrate that PKC activation accounts for the inhibition of melanin synthesis by TPA. Additional experiments were undertaken to elucidate the mechanism by which TPA inhibits the tyrosinase gene transcription. Deletions and mutation in the tyrosinase promoter showed that TPA acts on a M-box which is involved in tissue-specific expression and regulation by cAMP of the tyrosinase gene. We showed that TPA decreases the binding of microphthalmia, a basic helix-loop-helix transcription factor, to the M-box. Since microphthalmia, strongly stimulates the transcriptional activity of the promoter we propose that TPA, through PKC activation, decreases microphthalmia binding to the M-box of the tyrosinase promoter, thereby leading to a reduced tyrosinase expression and melanogenesis inhibition.
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PMID:In B16 melanoma cells, the inhibition of melanogenesis by TPA results from PKC activation and diminution of microphthalmia binding to the M-box of the tyrosinase promoter. 958 14

Treatment with 5 microM of saikosaponin (SS) b2 for 30 days was found to induce differentiation of B16 melanoma cells, with potentiation of expressions of melanogenesis and tyrosinase. To explore the mechanism of this effect, we observed the cell growth, cell cycle and morphology, and found that SSb2 did not affect any of these parameters. That is, SSb2 induced the differentiation of B16 melanoma cells without growth inhibition or cytotoxicity under conditions of low dose and long-term treatment. Phorbol ester, a protein kinase C (PKC) activator, markedly inhibited the expressions of melanogenesis and tyrosinase in both the control B16 melanoma cells and the long-term treated B16 melanoma cells. Down-regulation of the PKC activity may be involved in the effects of SSb2.
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PMID:Saikosaponin b2 induces differentiation without growth inhibition in cultured B16 melanoma cells. 987 67

We have previously shown that protein kinase C-beta (PKC-beta) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-beta but not PKC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-beta. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-beta and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-beta. We conclude that PKC-beta activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.
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PMID:Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain. 1034 9

In this study, we used melb-a melanoblasts as a model to study mechanisms involved in stimulating melanocyte function in vitiliginous skin following exposure to 8-methoxypsoralen (8MOP). Melanin content and tyrosinase activity increased 3- and 7-fold, respectively, in melanoblasts treated with 8MOP for 6 d compared with untreated controls. The intracellular signal pathways involved in 8MOP-induced effects on melanoblasts were investigated, particularly the roles of protein kinase A and protein kinase C. Forskolin, a protein kinase A activator, mimicked and enhanced the 8MOP stimulation of melanoblast pigmentation whereas a protein kinase C activator, 1-oleoyl-2-acetylglycerol, had no effect, indicating that the protein kinase A pathway is involved rather than the protein kinase C pathway. Those observations were confirmed using inhibitors of the protein kinase A or protein kinase C pathways. Western blot and semiquantitative reverse transcriptase polymerase chain reaction were performed to assess the protein and mRNA expression levels of microphthalmia-associated transcription factor and tyrosinase in melanoblasts treated with 8MOP for 3 h, 6 h, 1 d, 3 d, or 6 d. Incubation with 8MOP stimulated microphthalmia-associated transcription factor protein and mRNA levels within 3 h, but, in contrast, tyrosinase mRNA and protein levels did not increase following 8MOP treatment until 1 d after treatment. The proteasome inhibitor lactacystin blocked the proteasome-mediated proteolysis of tyrosinase, and its effect on proteasomal function was enhanced by 8MOP. Taken together, these results show that 8MOP functions by initially stimulating levels of microphthalmia-associated transcription factor expression via activation of the protein kinase A pathway, which thereby stimulates tyrosinase expression and function and eventually leads to dramatic increases in melanin production by melanoblasts.
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PMID:Stimulation of melanoblast pigmentation by 8-methoxypsoralen:the involvement of microphthalmia-associated transcription factor, the protein kinase a signal pathway, and proteasome-mediated degradation. 1248 19

Tyrosinase, the key enzyme in melanogenesis, is activated when protein kinase C-beta (PKC-beta) phosphorylates the serine residues at amino acid positions 505 and 509. To further elucidate the mechanism by which phosphorylation of tyrosinase by PKC-beta leads to the activation of tyrosinase, a possible complex formation between phosphorylated tyrosinase and tyrosinase related protein-1 (TRP-1), a melanogenic protein suggested to influence tyrosinase activity, was investigated. Non-denaturing gel electrophoresis of melanocyte lysate revealed two molecular weight forms of TRP-1 and a monoclonal antibody against TRP-1 co-immunoprecipitated tyrosinase and TRP-1, suggesting that TRP-1 may be complexed with tyrosinase. Activation of PKC by treating melanocytes with phorbol 12,13-dibutyrate (PDBu) increased the level of tyrosinase co-immunoprecipitated with TRP-1; whereas a selective PKC inhibitor bisindolylmaleimide inhibited PDBu-induced increase in the level of tyrosinase co-immunoprecipitated with TRP-1. These results suggest that phosphorylation of tyrosinase by PKC-beta induces a complex formation between tyrosinase and TRP-1.
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PMID:Protein kinase C-beta-mediated complex formation between tyrosinase and TRP-1. 1462 73

To determine whether inhibition of PKC-beta activity decreases pigmentation, paired cultures of primary human melanocytes were first pretreated with bisindolylmaleimide (Bis), a selective PKC inhibitor, or vehicle alone for 30 min, and then treated with TPA for an additional 90 min to activate PKC in the presence of Bis. Bis blocked the expected induction of tyrosinase activity by activation of PKC. Addition of a peptide corresponding to amino acids 501-511 of tyrosinase containing its PKC-beta phosphorylation site, a presumptive PKC-beta pseudosubstrate, gave similar results. To determine whether Bis reduces pigmentation in vivo, the backs of four shaved and depilated pigmented guinea pigs were UV irradiated with a solar simulator for 2 wk excluding weekends. Compared to vehicle alone, Bis (300 microM), applied twice daily to paired sites for various periods encompassing the irradiation period, decreased tanning. Bis also, although less strikingly, reduced basal epidermal melanin when topically applied twice daily, 5 d per wk, for 3 wk to shaved and depilated unirradiated skin. Moreover, topical application of Bis (100 microM) once daily for 9 d to the freshly depilated backs of 8-wk-old mice markedly lightened the color of regrowing hair. These results demonstrate that inhibiting PKC activity in vivo selectively blocks tanning and reduces basal pigmentation in the epidermis and in anagen hair shafts.
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PMID:Topical application of a protein kinase C inhibitor reduces skin and hair pigmentation. 1496 4

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 micro M). The intracellular Ca2+ chelator BAPTA/AM (1 micro M) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 micro M protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.
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PMID:The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells. 1506 19

The involvement of phospholipase D (PLD) in the regulation of melanogenesis was examined. Treatment of B16 mouse melanoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the activation of PLD and a decrease in melanin content. 1-Butanol, but not 2-butanol, completely blocked the TPA-induced inhibition of melanogenesis, suggesting the involvement of PLD in this event. Reverse transcription-PCR and immunoblot analyses revealed the existence of both PLD isozymes, PLD1 and PLD2, in B16 cells. When PLD1 or PLD2 was introduced into those cells by an adenoviral gene-transfer technique, both PLD1 and PLD2 were activated by TPA. When PLD1 and PLD2 were overexpressed, PLD2 potently caused a decrease in melanin content, whereas the effect of PLD1 expression on melanin content was minimal. Over-expression of PLD2 itself did not affect protein kinase C activity, as assessed by the intracellular distribution and levels of expression of each isoform expressed in B16 cells. The effects of TPA on the down-regulation of basal or alpha-melanocyte-stimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activity-negative mutant, LN-PLD2, but not by LN-PLD1. Further, the PLD2-induced decrease in melanin content was accompanied by a decrease in the amount and activity of tyrosinase, a key enzyme in melanogenesis, whereas the mRNA level of tyrosinase was unchanged by the over-expression of PLD2. Moreover, treatment with proteasome inhibitors completely blocked the PLD2-induced down-regulation of melanogenesis. Taken together, the present results indicate that the TPA-induced down-regulation of melanogenesis is mediated by PLD2 but not by PLD1 through the ubiquitin proteasome-mediated degradation of tyrosinase. This suggests that PLD2 may play an important role in regulating pigmentation in vivo.
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PMID:Down-regulation of melanogenesis by phospholipase D2 through ubiquitin proteasome-mediated degradation of tyrosinase. 1506 2

Elevated expression of stress proteins can be a characteristic of human cancer and may be involved in the development of resistance to some types of chemotherapeutic agent. In this paper, the effect of physiological stress conditions, such as glucose deprivation, was investigated in overexpressing nPKCdelta murine melanoma BL6 (BL6T) cells. Glucose stress conditions decreased the proliferative capacity, increasing the percentage of BL6T cells in the G0/G1 phase of the cell cycle. Furthermore, under such conditions, nPKCdelta, whose subcellular localization is cell cycle dependent, showed a cytoplasmic and perinuclear localization by immunohistochemistry, this being typical for cells in G0/G1 phase. Moreover, these cells expressed GRP-78, a known stress protein. On the other hand, glucose depletion enhanced intracellular melanin as well as tyrosinase activity and expression. In summary, these data demonstrate that stress conditions can modify the biological characteristics of BL6T cells, and therefore can select a quiescent cellular population.
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PMID:Effect of glucose stress conditions in BL6T murine melanoma cells. 1545 89


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