Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.10.3.1 (tyrosinase)
 9,065 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have investigated the role of protein kinase C (PKC) in human melanogenesis. The level of PKC activity paralleled the total melanin content in cultured newborn melanocytes. Activation of PKC by treatment with 5 x 10(-7) M phorbol dibutyrate acutely caused a doubling in the activity of tyrosinase, the rate-limiting enzyme in melanogenesis, known to correlate directly with melanin synthesis in these cells. When PKC was depleted to 5-10% of initial levels, there was a 40-50% parallel reduction in tyrosinase activity; and regeneration of PKC activity was associated with the recovery of tyrosinase activity. By Northern blot analysis, the alpha and beta but not the gamma isoforms were detectable in melanocytes. By Western blot analysis, the racially determined pigment level in cultured melanocytes correlated with PKC-beta protein expression. In a pigmented human melanoma line (P-MM4, 20-30 ng melanin/micrograms protein)and its nonpigmented subclone (NP-MM4, undetectable melanin), PKC-alpha mRNA was expressed in both, whereas PKC-beta mRNA was detectable only in P-MM4 cells. Tyrosinase protein level was comparable in both cell lines. When NP-MM4 cell lysate was incubated with melanocyte lysate known to contain PKC-beta, tyrosinase activity per microgram of melanocyte protein in the combined lysate increased, consistent with activation of the previously inactive tyrosinase of NP-MM4 origin. Moreover, NP-MM4 cells transiently transfected with PKC-beta cDNA increased tyrosinase activity from undetectable to detectable levels. These combined data show that PKC-beta regulates human melanogenesis by activating tyrosinase.
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PMID:The beta isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells. 768 20

Protein kinase C (PKC) is a multigene family of at least 12 isoforms involved in the transduction of extracellular signals. We investigated whether PKC-alpha, a major isoform known to be relatively abundant in brain tissue, is increased in human melanocytes relative to keratinocytes in vitro and in situ. Immunohistochemical staining for PKC-alpha in frozen neonatal human foreskin exhibited intermittent 2-3 + staining along the basal cell layer consistent with melanocytes, and 0-1 + staining of keratinocytes (on a scale of 0-3). Microscopic densitometry of the intermittent cellular staining was at least 3-fold greater than that of adjacent keratinocyte cell cytoplasm. Sequential frozen sections revealed similar intermittent cell staining with PKC-alpha and Mel-5 (tyrosinase related protein-1), known to specifically react with melanocytes. Northern blot analysis with a specific cDNA probe for PKC-alpha showed strong PKC-alpha mRNA expression in cultured melanocytes, whereas PKC-alpha mRNA in cultured non-stratifying keratinocytes was expressed at low levels. Western blot analysis revealed a prominent PKC-alpha band at approximately 80 kDa in melanocytes as opposed to a weak band in keratinocytes. Densitometry of the northern and western blots revealed that melanocytes had at least 10-fold more PKC-alpha mRNA and approximately 6-fold more PKC-alpha protein expression than keratinocytes. Total PKC activity measured in vitro revealed that melanocytes had 5-fold more activity than keratinocytes. The marked difference in melanocyte and keratinocyte expression of PKC-alpha provides further evidence for cell type specificity in the balance of PKC-alpha expression and may implicate differential PKC isoform signaling pathways in neuro-ectodermally derived cells.
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PMID:In situ and in vitro expression of protein kinase C alpha in human melanocytes. 952 31

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

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, alpha, beta, delta, epsilon and zeta isoforms of PKC are expressed, but uniquely PKC-beta activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-beta phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-beta, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-beta, revealed abundant PKC-beta, whereas PKC-alpha was not detected when immunoblot analysis was performed using antibody against PKC-alpha. Activation of PKC in melanocytes increased the level of PKC-beta co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-beta in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-beta and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-beta on the melanosome membrane, allowing PKC-beta to phosphorylate tyrosinase.
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PMID:The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-beta on melanosomes. 1525 33