Protein Kinase C-
Activates Tyrosinase by Phosphorylating
Serine Residues in Its Cytoplasmic Domain*
Hee-Young
Park
§,
J. Manuel
Perez¶,
Richard
Laursen¶
,
Masahiro
Hara
, and
Barbara A.
Gilchrest
From the
Department of Dermatology, Boston University
School of Medicine and the ¶ Department of Chemistry, Boston
University, Boston, Massachusetts 02118
 |
ABSTRACT |
We have previously shown that protein kinase
C-
(PKC-
) 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-
into
nonpigmented human melanoma cells lacking PKC-
lead to the phosphorylation and activation of tyrosinase. Preincubation of intact
melanosomes with purified active PKC-
in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-
but
not PKC-
was closely associated with tyrosinase on the outer surface
of melanosomes. Western blot analysis confirmed the association of
PKC-
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-
. 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-
and then subjected to trypsin digestion revealed that both
serine residues are phosphorylated by PKC-
. We conclude that PKC-
activates tyrosinase directly by phosphorylating serine residues at
positions 505 and 509 in the cytoplasmic domain of this
melanosome-associated protein.
 |
INTRODUCTION |
Melanin production is principally responsible for skin color and
plays an important role in prevention of sun-induced skin injury (1).
Melanin is produced in melanocytes, neural crest-derived cells residing
at the basal layer of epidermis; tyrosine and
L-dihydroxyphenylalanine (L-dopa) serve as
precursors for this complex biopolymer (1, 2). These precursor amino
acids are oxidized by a rate-limiting enzyme tyrosinase (EC 1.14.18.1)
(3, 4), and subsequent reactions involving tyrosinase and
tyrosinase-related proteins result in deposition of melanin pigment in
specialized organelles termed melanosomes (1).
Mammalian tyrosinase is a copper-dependent glycoprotein
spanning the melanosomal membrane with a molecular mass ranging from 60 to 75 kDa, depending on its state of glycosylation (4). Human
tyrosinase has been cloned and sequenced (5), and the deduced amino
acid sequence reveals that tyrosinase is composed of 511 amino acids
with >90% of the enzyme, the part containing the copper-binding sites
(5), localized inside the melanosome. The remainder of the tyrosinase
protein consists of a short amino acid sequence that spans the membrane
of melanosome, followed by a tail of approximately 30 amino acids that
reside in the melanocyte cytoplasm. Therefore, tyrosinase can be
divided into inner, transmembrane, and cytoplasmic domains. Patients
with oculocutaneous albinism, who lack tyrosinase activity, frequently
display mutations in the copper binding region of tyrosinase, as well
as other regions in the inner domain (6, 7). Point mutations at codons
encoding for amino acids at positions 489 or 501 that result in
premature termination of tyrosinase and loss of 21 or 22 amino acids at the C terminus lead to oculocutaneous albinism (6), suggesting that the
cytoplasmic domain is similarly critical to melanogenic function. A
second role for the cytoplasmic domain in proper subcellular trafficking of tyrosinase has been inferred from the observation that
the platinum mutation in mice, which results in deletion of the 27 terminal amino acids of the cytoplasmic domain, causes tyrosinase to be
incorporated into the plasma membrane rather than the melanosomal
membrane (8-11). However, a recent report has suggested that only six
amino acids residing near the melanosomal membrane within the
cytoplasmic domain are responsible for the subcellular trafficking
(11).
Although the biochemistry of melanogenesis has been extensively
characterized, molecular mechanisms involved in regulation of
tyrosinase activity are still poorly understood. In murine melanoma
cells, agents that increase the intracellular level of cAMP, such as
-melanocyte stimulating hormone (
-MSH), dibutyryl cAMP, and
forskolin, are known to be potent inducers of pigmentation (12-17).
These agents increase tyrosinase mRNA, protein, and activity (13-15), as well transcription of tyrosinase (12). Inhibitors and
activators of tyrosinase have also been postulated (16, 17), although
neither an inhibitor nor an activator has yet been identified. Because
tyrosinase is a glycoprotein, inhibition of glycosylation was
specifically postulated to play a physiologic role in modulating
tyrosinase activity (18).
In human melanocytes, even less is known about regulation of tyrosinase
activity. Unlike murine melanoma cells, the regulation of tyrosinase
activity appears to occur mostly at the posttranslational level. For
example, the level of tyrosinase mRNA does not correlate with the
total melanin level in cultured human melanocytes (19, 20), and human
melanoma cells often fail to produce melanin despite having abundant
tyrosinase protein (21, 22). However, the posttranslational mechanism
by which tyrosinase activity is putatively regulated is not well
understood. Again, an inhibitor of tyrosinase in human skin has been
suggested (23), but it is yet undocumented.
Recently, our laboratory has demonstrated that protein kinase C (PKC)
plays an important role in regulating both human and murine
pigmentation (21, 24-26). Diacylglycerol, the known physiologic activator of PKC that is often cleaved from cell membrane lipids when
ligands bind their cell surface receptors (27) or following ultraviolet
irradiation (28, 29), increased melanin content in cultured human
melanocytes, an effect blocked by PKC inhibitors (25). In murine
melanoma cells, depletion of PKC completely blocked
-MSH induced
pigmentation (24); in intact guinea pig skin, topical application of
diacylglycerols known to activate PKC caused tanning, whereas
application of analogues lacking PKC activity did not (26). Subsequent
experiments revealed that the
-isoform of PKC specifically regulates
human pigmentation, in that human melanoma cells expressing a normal
level of tyrosinase but lacking PKC-
were amelanotic and that
transfection with PKC-
activated tyrosinase in these cells (21).
PKC is a serine/threonine kinase for which one of the
best-characterized intracellular substrates is myristoylated
alanine-rich C kinase substrate (30). Although tyrosinase contains
possible phosphorylation sites for a serine/threonine kinase, it has
never been determined whether phosphorylation is a mechanism by which tyrosinase activity is regulated. In the present study, we demonstrate that PKC-
acts directly on human tyrosinase by phosphorylating serine residues in its cytoplasmic domain.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Dulbecco's modified Eagle's medium (DMEM), L15,
nonessential amino acids, glutamine, medium 199, and trypsin were
purchased from Life Technologies, Inc. Recombinant basic fibroblast
growth factor was purchased from Amgen, triiodothyronine from
Collaborative Research, and hydrocortisone from Calbiochem. Phorbol
12,13-dibutyrate (PDBu),
12-O-tetradecanoylphorbol-13-acetate (TPA), insulin,
transferrin, purified tyrosinase, acidic phosphatase, synthetic
melanin, and phosphoserine, phosphothreonine, and phosphotyrosine
standards were from Sigma. Choleragen was from List Biologicals. Nylon
membranes were from Amersham Pharmacia Biotech. PKC antibodies were
from Seigakagu International. Bovine calf serum and fetal calf serum were from HyClone Laboratories, Inc. All radiolabeled compounds were
purchased from New England Nuclear. Cellulose thin-layer plates were
purchased from Merck. Young rabbit brains were purchased from
Pel-Freeze. Polyvinyl difluoride (PVDF) membranes were from Bio-Rad.
Colloidal gold-labeled IgG was from Janssen Life Science.
Cells and Media--
Neonatal foreskins were used to culture
human melanocytes as described previously (21). In brief, the epidermis
was separated from the dermis after overnight incubation in 0.25%
trypsin at 4 °C. Primary cultures were then established in medium
199 containing 1.8 mM Ca+2 and supplemented
with 10
9 M triiodothyronine, 10 µg/ml
insulin, 1.4 × 10
6 M hydrocortisone,
10
9 M choleragen, 10 ng/ml basic fibroblast
growth factor, and 5-10% fetal bovine serum. All postprimary cultures
were maintained in a low calcium (0.03 mM) version of this
medium known to selectively support melanocyte growth (19). Cells at
first to third passages were routinely used. The MM4 human melanoma
cells were obtained from Dr. U. Stierner (Gothenburg, Sweden) and
maintained in DMEM (55%), L15 (27%), fetal bovine serum (15%),
nonessential amino acids, 1% glutamine (2 mM), and insulin
(10 µg/ml) (19).
Generation of Stably Transfected NP-MM4 Cells with PKC-
cDNA--
PKC-
cDNA cloned into a SV40 promoter-driven
expression vector (31) was kindly provided by Dr. Shegio Ohno (Tokyo,
Japan) and was subcloned into a cytomegalovirus promoter-driven
expression vector to enhance its expression in human melanoma cells.
Selection of the transfected cells was made by co-transfection with
SV2Neo cDNA, a neomycin resistance gene. As a control,
MM4 cells were transfected with only SV2Neo were used. All
cultures were maintained in medium containing G418 (100-500
µg/ml).
In Vivo Phosphorylation of Tyrosinase--
In vivo
phosphorylation of tyrosinase was performed according to the method of
Simek et al. (32). Cultured melanocytes were preincubated
for 2 h to overnight with culture medium devoid of phosphate,
containing 50 µM sodium vanadate, no serum, and
[32P]orthophosphate (8500-9120 Ci/mmol). Then, cells
were treated with TPA (10
7 M) for 90 min to
activate PKC and lysed in a buffer containing 20 mM Tris
(pH 7.5), 200 mM NaCl, 1% Triton X-100, 2 mM
EDTA, 2 mM EGTA, 0.5 µg/ml aprotinin, and 1 mM PMSF. The lysate was spun at 800 × g
for 15 min, and tyrosinase was immunoprecipitated from the supernatant.
Immunoprecipitation of Tyrosinase--
Immunoprecipitation of
tyrosinase was performed according to Jimenez et al. (33).
[32 P]Phosphate labeled extracts were incubated with
-PEP7-polyclonal antibody against tyrosinase (33) for 60 min at
37 °C with occasional shaking. 100 µl of 10% protein A-Sepharose
was added, and the incubation was continued for 30 min at 23 °C with
constant mixing; the immune complexes were washed five times with 0.1%
Nonidet-P40 in phosphate-buffered saline and once in water. The
specifically immunoprecipitated proteins were eluted and separated by
Laemmli SDS-polycrylamide gel electrophoresis (SDS-PAGE) (34).
PKC Activity--
Soluble cell lysates, containing both
cytosolic- and membrane-associated PKC, were prepared as described
previously (21). Calcium- and phospholipid-stimulated PKC activity was
determined using 50 µl of eluent (2-5 µg of protein),
phosphatidylserine, and PDBu to activate PKC, and histone type III-S as
substrate, as described previously (21). A 10-min incubation at
37 °C was routinely used. For each determination, a control reaction
was performed in the absence of phosphatidylserine and PDBu. Activity was expressed as cpm of incorporated radioactivity/µg of protein/min in the presence of phosphatidylserine and PDBu, minus the radioactivity incorporated in the absence of phosphatidylserine and PDBu (background kinase activity). The background was generally less than 5-10% of the
total incorporated radioactivity.
Western Blot Analysis--
Immunoblot analysis was performed for
PKC as described (21). 1-2 µg of partially enriched lysate for
purified PKC was routinely used. Protein samples were subjected to
7.5% SDS-PAGE and transferred to a nitrocellulose membrane
electrophoretically. The membrane was preincubated in 100% blotto (5 g
of nonfat dry milk in 100 ml of phosphate-buffered saline) for 3 h
at room temperature with shaking, followed by an overnight incubation
with antiserum (0.5-1 µg/ml in 10% blotto) at 4 °C. At the end
of the incubation, the membrane was washed extensively with
phosphate-buffered saline containing 0.5% Tween-20 and processed using
the ECL kit. The membrane was then exposed to Eastman Kodak Co. X-OMAT film.
Purification of PKC--
PKC was purified from young rabbit
brain by a modification of procedures previously described (35). In
brief, 40-50 g of wet tissue was homogenized and centrifuged to
generate crude extract. Crude extract was applied to a DEAE-cellulose
column, and proteins were eluted by a linear gradient of 0-300
mM NaCl. Fractions containing PKC activity were pooled and
applied to the 5-ml phenyl-Sepharose CL-4B column. The active fractions
were pooled and applied directly to a 1-ml protamine-agarose column.
Active fractions were pooled and 10% glycerol was added and applied to
hydroxylapatite column. Fractions of 1.5 ml were collected and assayed
for PKC activity. Active fractions corresponding to the three peaks of
-,
-, and
-isoforms were analyzed by immunoblot analysis using
monoclonal antibodies specific to each isoform. The active fractions
corresponding to the
-isoform were further analyzed by immunoblot
analysis using monoclonal antibodies against
-,
-, and
-isoforms to rule out the possibility that this fraction was
contaminated by other isoforms.
Melanosome Isolation--
Melanosomes were purified by a
modification of the method of Seiji et al. (36) from
cultured human melanocytes. Cultures were washed with
phosphate-buffered saline, scraped, pelleted, and resuspended in 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA. Then they were disrupted by freeze-thaw followed
by nitrogen cavitation. The unbroken cells were removed by
centrifugation at 1000 × g for 5 min; cavitation was
repeated on the pellet; and the resulting supernatants were pooled,
layered over an equal volume of 30% sucrose in 20 mM
Tris-HCl (pH 7.2), 150 mM NaCl, 1 mM EDTA, and
centrifuged in a swinging bucket rotor at 50,000 × g
for 30 min. Electron microscopy was routinely performed on the purified
melanosomes to assure the intactness of this organelle.
Phosphoamino Acid Analysis by Thin Layer
Electrophoresis--
Tyrosinase was phosphorylated in the presence of
10
7 M TPA and
[32P]orthophosphate and subsequently immunoprecipitated
as described above. The SDS gel containing the immunoprecipitated
tyrosinase was electroblotted onto a PVDF membrane and exposed to
autoradiography to identify radiolabeled tyrosinase. The tyrosinase
band was cut out and diced into small pieces that were incubated with
5.9 M hydrochloric acid for 1 h at 110 °C. The
sample was evaporated and redissolved in electrophoresis buffer (formic
acid/acetic acid (2.5:7.8, pH 1.9)) before phosphoamino acid analysis
by thin-layer electrophoresis. The product of acid hydrolysis was
spiked with phosphoserine, phosphothreonine, and phosphotyrosine
standards and separated on a cellulose thin-layer plate using
electrophoresis at pH 1.9 in the first dimension and at pH 3.5 in the
second dimension. After the plate was dry, radioactivity was detected
by autoradiography, and the location of the phosphoamino acid standards
was found by ninhydrin staining (0.33% ninhydrin in acetic
acid/pyridine/water (1:5:5)).
Immunoelectron Microscopy--
Immunoelectron microscopy was
performed by first fixing melanocytes with 0.5% glutaraldehyde in 0.9 mg of sodium barbital-sodium acetate buffer (pH 7.4) in 0.07 M potassium chloride for 15 min at room temperature. Then
cells were fixed for 15 min at room temperature with 0.5% osmium
tetroxide-1.5% potassium ferrocyanide solution in the same buffer. The
samples were then dehydrated in a graded series of ethanols starting
with 70% and then embedded in a 1:1 mixture of Araldite 502 and
dodecenyl succinic anhydride. After polymerization at 60 °C for
18 h, thin sections were cut and then mounted on nickel grids.
Immunostaining was performed directly on mounted sections. Grids were
placed for 30 min in normal goat serum diluted 1:30 with 0.1 M Tris buffer (pH 7.4) containing 1.0% bovine serum
albumin (BSA)1 in a humidified environment at room
temperature. The sections were then stained with the primary antibody
by inverting the grids onto droplets of the antibody diluted in 0.1 M Tris buffer (pH 7.4) containing 0.1% BSA for 18 h
in a humidified environment at room temperature. The specimens were
then rinsed two times each with 0.05 M Tris buffer (pH 7.4)
containing 0.2% BSA. Colloidal gold-labeled IgG secondary antibody
staining solution was prepared by diluting 15 µl of 5- or
10-nm-diameter gold-labeled goat anti-rabbit IgG with 90 µl of 0.05 M Tris buffer (pH 8.3) containing 0.1% BSA and clarified
by centrifugation for 9 min at 4 °C in an Eppendorf microcentrifuge.
Grids were then placed in the immunogold staining solution for 1 h
at room temperature, rinsed once each with 0.05 M Tris
buffer (pH 7.4) containing 0.2% BSA and 0.05 M Tris buffer (pH 7.4), and then rinsed two times with water. All specimens were
counterstained with uranyl acetate and lead citrate and examined using
a Philips 300 electron microscope.
Peptide Synthesis--
Peptides were generated by solid phase
synthesis using Fmoc-protected amino acids (37) and starting with
Fmoc-L-Leu-PAC-resin (0.38mmol/g). Serine phosphate was
introduced as Fmoc-O-(monobenzylphosphono)-serine. The amino
acid coupling time, normally 1 h, was doubled for
Fmoc-O-(monobenzylphosphono)-serine. After
completion of the peptide chain and removal of the
N-terminal Fmoc group with piperidine (20% in dimethylformamide), the
peptide was cleaved and deprotected with reagent R (90%
trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, and 2%
anisole). The synthetic peptides were purified by preparative reversed
phase HPLC using a Vydac C-18 column (22 × 250 mm; 300-Å pore
size). Purity and integrity of the phosphopeptides were assessed by
analytical reversed phase HPLC and matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry.
Two-dimensional Phosphopeptide Mapping--
The lyophilized HPLC
fraction containing both the mono- and diphosphopeptides was
redissolved in electrophoresis buffer (formic acid/acetic acid
(2.5:7.8, pH 1.9)) and separated on a cellulose thin layer plate using
electrophoresis at pH 1.9 in the first dimension and chromatography in
1-butanol/pyridine/acetic acid/water (6.4:5:1:4) in the second (38).
After the plate was dry, radioactivity was detected by autoradiography,
either on x-ray film or on a PhosphorImager; the synthetic peptides
were localized by ninhydrin staining. This mapping procedure is known
to be highly exact, in that only peptides of identical size and charge
will co-migrate (39).
 |
RESULTS |
Tyrosinase Is Phosphorylated by PKC-
--
Because PKC-
was previously shown to activate tyrosinase in human melanocytes (21),
we sought to determine whether the activation involves phosphorylation
of tyrosinase by PKC-
. Paired cultures of human melanocytes were
preincubated with phosphate-free DMEM in the presence of
[32P]orthophosphate (8500-9120 Ci/mmol) for 90 min; one
culture received TPA (1 × 10
7 M) for 90 min to activate PKC, and the other culture remained untreated. At the
end of the incubation period, cells were harvested, and tyrosinase was
immunoprecipitated using a polyclonal antibody specific for tyrosinase
(33). Untreated melanocytes showed a low level of
[32P]phosphate incorporation into tyrosinase, whereas
melanocytes treated with TPA showed increased
[32P]phosphate incorporation (Fig.
1). These results indicate that tyrosinase is a phosphoprotein that is phosphorylated through the
PKC-dependent pathway and are consistent with our previous report that a portion of melanocyte PKC is active under basal culture
conditions (21).

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Fig. 1.
In vivo phosphorylation of
tyrosinase in melanocytes. Paired cultures of subconfluent
melanocytes were treated with either 1 × 10 7
M TPA or vehicle alone (Me2SO) for 90 min in
presence of [32P]orthophosphate in phosphate-free medium.
Cells were harvested and tyrosinase was immunoprecipitated as described
under "Experimental Procedures." Incorporation of radiolabeled
phosphate into tyrosinase was determined by separating total protein by
SDS-PAGE (1-2 × 106cpm/lane), followed by
autoradiography. One representative experiment from five separate
experiments is presented.
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|
We have previously shown that nonpigmented MM4 human melanoma cells
(NP-MM4) lack PKC-
but contain tyrosinase and PKC-
protein at
levels comparable to those of the pigment-producing parental melanoma
line or normal human melanocytes (21). We have also previously shown
that introduction of PKC-
into these amelanotic MM4 cells by
transient transfection activates tyrosinase (21), although no mechanism
was established. To examine whether tyrosinase is phosphorylated
specifically by the PKC-
, NP-MM4 cells were permanently transfected
with PKC-
and SV2Neo cDNAs or SV2Neo cDNA alone. NP-MM4 cells permanently transfected with PKC-
cDNA (PKC-
NP-MM4) expressed a readily detectable level of the
-isoform (Fig. 2A), and
their tyrosinase was active (Fig. 2B), as previously reported for transiently transfected NP-MM4 cells (21). NP-MM4 cells
transfected only with SV2Neo cDNA (control NP-MM4) had
undetectable levels of both PKC-
protein and tyrosinase
activity.

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Fig. 2.
Expression of PKC-
and activation of tyrosinase. To determine whether PKC-
is required for tyrosinase phosphorylation, NP-MM4 cells were
permanently transfected with the expression vector containing PKC-
cDNA and SV2Neo or with SV2Neo alone as
control. A, paired cultures of control and
PKC- -transfected cells were harvested, and the levels of
PKC- protein were determined by Western blot analysis. B,
tyrosinase activity was determined by Pomerantz assay.
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|
Paired cultures of control NP-MM4 cells and PKC-
NP-MM4 cells were
then preincubated in phosphate-free DMEM in the presence of
[32P]orthophosphate for 90 min, followed by treatment
with TPA (1 × 10
7 M) for an additional
90 min. Tyrosinase was immunoprecipitated from both cell types.
Incorporation of [32P]phosphate into tyrosinase was
readily detected in TPA-treated PKC-
NP-MM4 cells, whereas
[32P]phosphate was undetectable in the TPA-treated
control NP-MM4 cells (Fig.
3A). However, phosphorylation
of myristoylated alanine-rich C kinase substrate was apparent in
TPA-treated control NP-MM4 cells examined in parallel, indicating that
the other PKC isoforms present in control NP-MM4 cells are activable
(Fig. 3B). These results strongly suggest that tyrosinase
phosphorylation is specifically mediated by PKC-
.

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Fig. 3.
Phosphorylation of tyrosinase in control and
PKC- transfected NP-MM4 cells.
A, a set of subconfluent control NP-MM4 and
PKC- -transfected NP-MM4 cultures were treated with 1 × 10 7 M TPA or vehicle alone
(Me2SO) for 90 min in the presence of
[32P]orthophosphate. Cells were harvested and
tyrosinase-immunoprecipitated (1-2 × 106cpm/lane),
and incorporation of radiolabeled phosphate was visualized by
autoradiography. Both cells types are known to have comparable levels
of tyrosinase (21). One representative experiment from three
independent experiments is presented. B, to eliminate the
possibility that PKC is not properly activated in control-NP-MM4 cells,
these cells were treated with either TPA or vehicle alone for 90 min in
the presence of [32P]orthophosphate. The 80-kDa
myristoylated alanine-rich C kinase substrate (30) was
immunoprecipitated using an antibody specific for this substrate
protein and separated in a 7.5% SDS-PAGE (0.5-1 × 106cpm/lane). A phosphorylated band of the expected size
was readily detected.
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|
Phosphorylation of Tyrosinase by PKC-
Activates the Enzyme in
Vitro--
To further confirm that phosphorylation of tyrosinase by
PKC-
directly activates the enzyme, purified PKC-
was incubated with purified human melanosomes, the organelle in which tyrosinase is
imbedded as a transmembrane protein and in which melanogenesis occurs.
PKC-
was purified from mouse brain, and the identity of PKC-
was
confirmed by Western blot analysis using a monoclonal antibody specific
for PKC-
. Monoclonal antibodies against PKC-
and PKC-
were
also used to determine possible contamination of the preparation by
these isoforms, but they were undetectable in the purified PKC-
fraction (Fig. 4A). Tyrosinase
activity was increased 3-fold when melanosomes were incubated with
PKC-
in the presence of phospholipid and TPA (Fig. 4B). A
constitutive level of tyrosinase activity was observed in the absence
of those activators, again consistent with prior in vivo
tyrosinase activation by constitutively active PKC-
in human
melanocytes (21). Combined with the in vivo phosphorylation
results, this result specifically implicates PKC-
in direct
activation of the enzyme by phosphorylation.

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Fig. 4.
Up-regulation of tyrosinase activity by
PKC- in vitro. To examine
whether PKC- can directly phosphorylate tyrosinase, purified
mushroom tyrosinase was incubated with purified PKC- in
vitro. A, the purity of PKC- isolated from mouse
brain was confirmed by performing immunoblot analysis using antibodies
against the -, -, and -isoforms (labeled at top) of
PKC. B, purified melanosomes were preincubated with purified
PKC- (5 ng) in the presence of phospholipid and TPA, required for
PKC- activation, or in the absence of phospholipid and TPA, a
condition inadequate for PKC- activation. Tyrosinase activity in
each condition was determined using Pomerantz assay as described under
"Experimental Procedures."
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PKC-
Co-Localizes with Tyrosinase at the Melanosomal
Membrane--
It has been shown in many other cell types that
compartmentalization of a specific PKC isoform is associated with a
unique biological function (40, 41). To determine whether PKC-
was physically associated with tyrosinase at the site of melanin synthesis, the melanosomes, we performed immunoelectron microscopy. Paired cultures of melanocytes were immunostained with either tyrosinase and
PKC-
antibodies, tagged with gold beads of different size, or as a
control with tyrosinase and PKC-
antibodies. The antibodies were
labeled with 10-nm diameter gold beads for tyrosinase and 5-µm beads
for PKC-
or PKC-
, as described under "Experimental Procedures." Tyrosinase was restricted to melanosomes, as expected, and 10-nm gold beads were observed on the cytoplasmic side of the
melanosomal membrane, as also expected, given that the tyrosinase antibody employed is directed against the cytoplasmic domain of the
enzyme (33) (Fig. 5A).
Interestingly, PKC-
was localized to the cytoplasmic side of the
melanosomal membrane, and always close to tyrosinase, but PKC-
was
instead observed diffusely in the melanocyte cytoplasm, unrelated to
melanosomes (Fig. 5A). This suggests that PKC-
compartmentalizes at the melanosome, thus determining its specific
function in regulating tyrosinase activity.

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Fig. 5.
PKC- co-localizes
with tyrosinase on the outer membrane of melanosomes.
A, to determine whether PKC- specifically co-localizes
with melanosomes, immunoelectron microscopy was performed as described
under "Experimental Procedures." Antibody to PKC- , known to be
expressed at comparable levels to PKC- (21), was studied as a
control. Tyrosinase antibody was labeled with 10-nm immunogold beads,
and each of the PKC antibodies was labeled with 5-nm immunogold beads.
Fields representative of the more than 20 examined for each PKC isoform
are shown. B, melanosomes were purified from cultured human
melanocytes and divided into two aliquots: one untreated and the other
treated with trypsin. Proteins were then separated by SDS-PAGE, and
immunoblot analysis was performed using an antibody specific for
PKC- . Whole melanocyte lysate (lane 1, control) showed a
readily detectable level of PKC- , as did untreated melanosomes
(lane 2). In contrast, melanosomes treated with trypsin
(lane 3) had no detectable PKC- .
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|
Because the electron density of melanosomes might obscure the presence
of immunogold staining within the organelle, we could not determine
from above experiments whether PKC-
is also present inside
melanosomes. Melanosomes were therefore purified from intact melanocytes and separated into two groups. One was untreated, and the
other was treated with trypsin to remove any membrane-associated proteins, including PKC-
. Whole melanocyte lysate, loaded as a
positive control, contained readily detectable levels of PKC-
, as
did untreated melanosomes, whereas trypsin-treated melanosome preparation had no detectable PKC-
(Fig. 5B). These
results suggest that PKC-
is not present inside melanosomes, but
instead closely associates with the cytoplasmic surface of melanosomes,
consistent with the electron micrographs and with current literature
indicating that PKC-
is a cytoplasmic protein (27).
PKC-
Phosphorylates the Cytoplasmic Domain of
Tyrosinase--
Tyrosinase has a large inner domain, residing within
melanosome, a transmembrane domain, and a cytoplasmic domain that
extends approximately 30 amino acids beyond the melanosomal membrane
(5). The published amino acid sequence of human tyrosinase reveals a total of 60 serine and threonine residues, but only two serines and
no threonines in the cytoplasmic domain (5). Because our data confirmed
that PKC-
in melanocytes is a cytoplasmic protein, as in other cell
types (27, 41), we hypothesized that PKC-
phosphorylates only the
cytoplasmic domain of tyrosinase. To test this hypothesis, paired
cultures of melanocytes were preincubated with
[32P]orthophosphate, and PKC was activated to allow
phosphorylation of tyrosinase. Melanosomes were purified and divided
into two groups, one of which was exposed to trypsin for 2 h to
digest off the cytoplasmic domain of tyrosinase (4), known to have multiple trypsin sensitive arginine and lysine sites (42) at amino acid
positions of 483-484, 485-486, 491-492, 493-494, and 500-501,
beyond the melanosomal membrane. Trypsin-treated and control
melanosomes were then immunoprecipitated using PEP-5, a polyclonal
antibody specific for the inner domain of tyrosinase (33).
Immunoprecipitated proteins were run on a gel and
[32P]phosphate label incorporation was readily detected
in untreated full-length tyrosinase, as expected, but not in samples
exposed to trypsin prior to immunoprecipitation (Fig.
6A), indicating that the
remaining intramelanosomal portion of tyrosinase is not phosphorylated.
When the supernatant of the trypsin-treated melanosome was concentrated
and run on a 17% gel, a radiolabeled cytoplasmic peptide, consistent
with the largest predicted tryptic digestion fragment from the
cytoplasmic domain of tyrosinase, was recovered (Fig. 6B).
Other smaller predicted fragments were not detected, as expected,
because they were too small to be retained in the gel. To be certain
that there was tyrosinase in all immunoprecipitated samples, Western
blot analysis was performed in parallel. Equal amounts of tyrosinase
protein reactive with antibody directed against epitopes in the inner
domain of tyrosinase were present in both preparations (Fig.
6C), excluding the possibility that lack of label was due to
complete loss of trypsin-treated tyrosinase during immunoprecipitation.
Taken together, these results indicate that PKC-
activates
tyrosinase by phosphorylating the cytoplasmic domain of the enzyme.

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Fig. 6.
PKC- phosphorylates
the cytoplasmic domain of tyrosinase. To determine whether PKC-
phosphorylates only the cytoplasmic domain of tyrosinase, melanocyte
cultures were preincubated with [32P]orthophosphate, PKC
was activated with TPA, and melanosomes known to contain tyrosinase as
a transmembrane protein were purified. A, purified
melanosomes were divided into two groups: one group remained untreated
as a control, and the other group was treated with 0.25% trypsin for
60 min at 37 °C to release the cytoplasmic domain of tyrosinase.
Subsequently, tyrosinase from trypsin-treated and untreated melanosomes
was immunoprecipitated using antibodies directed against the inner
domain (33) or the full-length tyrosinase (33), respectively.
Incorporation of [32P]orthophosphate was visualized using
autoradiography. B, the supernatant from the melanosomes
treated with trypsin was concentrated and separated by 17% SDS-PAGE,
and the fragment corresponding to the
[32P]orthophosphate-labeled cytoplasmic domain of
tyrosinase was recovered, as demonstrated by autoradiography of the
gel. Smaller tryptic fragments of the cytoplasmic domain (<28 amino
acids) could not be recovered because they ran with the dye, which was
run off the gel. C, in parallel cultures, melanocytes were
treated identically except in the absence of
[32P]orthophosphate, and immunoprecipitated
trypsin-treated or untreated tyrosinase was transferred to a
nitrocellulose membrane. The membrane was then reacted with an antibody
against the inner domain and visualized by ECL. Equal amounts of
tyrosinase were detected in trypsin-treated and untreated
samples.
|
|
Phosphoamino Acid Analysis of Human Tyrosinase--
The deduced
amino acid sequence of human tyrosinase contains two serines but no
threonines in the cytoplasmic domain (5). To confirm the expectation
that only serine residues are therefore phosphorylated by PKC,
phosphoamino acid analysis of immunoprecipitated radiolabeled
tyrosinase was performed. Tyrosinase was phosphorylated in
vivo by preincubating melanocytes with
[32P]orthophosphate, followed by activation of PKC with
TPA. Subsequently, [32P]tyrosinase was
immunoprecipitated, separated by SDS-PAGE, and transferred to a PVDF
membrane. Immunoprecipitated tyrosinase was further identified by
incubating the PVDF membrane with L-dopa, which is
incorporated by active tyrosinase into melanin and then deposited on
the PVDF membrane. The protein band corresponding to human tyrosinase
was then cut out and hydrolyzed to individual amino acids. The
resulting mixture of amino acids was separated using two-dimensional
thin-layer electrophoresis and subjected to autoradiography. The
location of the radioactive spots was compared with those of the
unlabeled phosphoserine, phosphothreonine, and phosphotyrosine
standards. All of the radiolabeled phosphate was associated with
phosphoserine (Fig. 7). Radiolabeled
phosphate associated with phosphothreonine or phosphotyrosine was not
detected. In combination, these data demonstrate that tyrosinase is
phosphorylated by PKC-
and that only serine residues on the
cytoplamic domain are phosphorylated.

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Fig. 7.
Phosphoamino acid analysis of
immunoprecipitated tyrosinase. Because the cytoplasmic domain of
tyrosinase contains only serine and no threonine residues, phosphoamino
acid analysis was performed to determine whether only serines are
phosphorylated by PKC- . Subconfluent cultures of melanocytes were
treated with TPA in presence of [32P]orthophosphate to
phosphorylate and radiolabel tyrosinase. Tyrosinase was then
immunoprecipitated and transferred to a PVDF membrane. Tyrosinase was
then visualized by reacting the PVDF membrane with L-dopa.
The band containing the radiolabeled tyrosinase was cut out and
incubated with 5.9 M hydrochloric acid for 1 h at
110 °C. The sample was evaporated, mixed with phosphoamino acid
standards, and subjected to two-dimensional thin-layer electrophoresis.
Radioactivity was detected by autoradiography, whereas the location of
the phosphoamino acid standards was found by ninhydrin staining.
(pS, phosphoserine; pT, phosphothreonine;
pY, phosphotyrosine). This figure is representative of five
analyses.
|
|
Both Serine Residues on the Cytoplasmic Domain Are
Phosphorylated--
To determine whether one or both serines on the
cytoplasmic domain are phosphorylated, radiolabeled tyrosinase was
first partially purified from human melanosomes. Tyrosinase was
extracted from melanosomes and separated from other melanosomal
proteins by nondenaturing gel electrophoresis. The protein was then
identified by the appearance of a brown-colored band after the in
situ tyrosinase activity assay (33). Because the product of
L-dopa oxidation by tyrosinase is highly hydrophobic and
might interfere with the digestion of tyrosinase or with recovery of
the tryptic peptides from the gel, the sample was run in multiple
lanes, one of which was excised and stained with L-dopa.
The remaining portion of the gel was subjected to autoradiography, and
the area corresponding to the stained section of the L-dopa
treated lane was excised and treated directly with trypsin. Of the
predicted tryptic fragments of the human tyrosinase cytoplasmic domain
(5), Glu-Asp-Tyr-His-Ser-Leu-Tyr-Gln-Ser-His-Leu contains both of the
serines. All three possible phosphoserine-containing peptides, one
phosphorylated on both serines and two phosphorylated on each one of
the serines, were synthesized and used as phosphorylated standards in
subsequent co-migration experiments. To remove salts, unincorporated
[32P]orthophosphate, and other unrelated peptides, the
tryptic digest was first subjected to an HPLC purification step.
Nonlabeled synthetic phosphopeptides were then added to the digest as
markers, because the quantity of 32P-labeled peptides was
too low to detect by UV absorption. In the HPLC elution profile of the
32P-labeled tyrosinase tryptic peptide mixture, the more
polar diphosphopeptide eluted earlier than the monophosphopeptides,
which were not resolved. Because the radioactivity of individual
fractions was so low and to avoid handling losses, the fractions
containing the carrier phosphopeptides were collected as a single
fraction and subjected to two-dimensional peptide mapping.
The mixture was first separated by thin layer electrophoresis on
cellulose in formic acid/acetic acid/water (pH 1.9), followed by
thin-layer chromatography at right angles. The thin-layer chromatology plate was dried and exposed to x-ray film. Ninhydrin staining of the
plate revealed two spots, corresponding to the diphosphopeptide and the
faster moving unresolved mixture of monophosphopeptides (Fig.
8A). Autoradiography revealed
an area of radioactivity coincident with the diphosphopeptide (Fig.
8B). No radioactivity was associated with the
monophopeptides. The radioactive signal associated with the
diphosphopeptide was weak, requiring long exposures to detect. Nevertheless, these results demonstrate that tryptic digestion of
in vivo 32P-labeled tyrosinase produces a
peptide that co-migrates with the synthetic tyrosinase-related peptide,
Glu-Asp-Tyr-His-Ser(P)-Leu-Tyr-Gln-Ser(P)-His-Leu, in three separation
procedures: HPLC, thin layer electrophoresis, and thin-layer
chromatography. Together with earlier results, this confirms that both
serine residues of tyrosinase, at amino acid positions of 505 and 509, are phosphorylated by PKC-
.

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Fig. 8.
Two-dimensional phosphopeptide mapping of
HPLC-purified carrier phosphopeptides. The fraction containing
both mono- and diphosphopeptide standards was collected and subjected
to two-dimensional mapping on a cellulose plate using electrophoresis
at pH 1.9 in the first dimension and chromatography in
1-butanol/pyridine/acetic acid/water (6.4:5:1:4) in the second. The
plate was then air dried. A, the synthetic phosphopeptide
standards were detected by ninhydrin staining. B,
32P-labeled peptides were detected by autoradiography. The
short arrow indicates the position of the ninhydrin-stained
diphosphopeptide standard that corresponds to a radioactive spot on the
autoradiography. C, the in vitro phosphorylation
product was spotted on a cellulose plate along with the peptide
standard TP 501-511 and subjected to thin layer electrophoresis at pH
1.9. Only one radioactive spot was detected by autoradiography. The
dashed ovals represent the locations of two ninhydrin spots
too faint to be seen in a photograph, superimposed on the
autoradiogram, one corresponding to TP 501-511 and the other to the
phosphorylated TP 501-511 peptide.
|
|
To demonstrate that the cytoplasmic domain of tyrosinase can act as a
substrate for PKC-
, a peptide containing the putative phosphorylation sites in tyrosinase
(Glu-Asp-Tyr-His-Ser-Leu-Tyr-Gln-Ser-His-Leu, termed TP 501-511), was
synthesized and incubated with [32P]ATP in the presence
of active recombinant PKC-
. The peptide was then eluted from the
phosphocellulose membrane with a 50 mM ammonium bicarbonate
solution and analyzed by thin layer electrophoresis. The eluent was
spiked with TP 501-511 and a portion of the eluent (approximately 10 µl) was subjected to one-dimensional thin layer electrophoresis (pH
1.9). It was reasoned that if the peptide (TP 501-511) had become
phosphorylated, it would migrate more slowly toward the anode than the
nonphosphorylated peptide on thin layer electrophoresis. Fig.
8C shows the electrophoretic mobility of the peptide TP
501-511 phosphorylated in vitro with [32P]ATP. Autoradiography of the plate reveals only a
single radioactive spot, whereas ninhydrin staining yieldeds two
spots (Fig. 8C, dashed ovals), one corresponding to TP
501-511 and the other co-migrating with the radioactive spot seen by
autoradiography. This radioactive spot migrated more slowly toward the
anode than TP 501-511, consistent with incorporation of phosphate
groups into the synthetic peptide.
 |
DISCUSSION |
It has been surprisingly difficult to decipher the regulation of
human melanogenesis despite rapid evolution of adequate culture methods
for human melanocytes over the past two decades. The confounding role
of phorbol esters, initially added to media to stimulate melanocyte
growth in the absence of then-unidentified key mitogens (43), but
eventually discovered to severely down-regulate tyrosinase activity
through PKC depletion (21), has only recently been appreciated. In
addition, many potent inducers of murine pigmentation, such as
-MSH,
either fail to induce human pigmentation or are far less effective, a
situation also likely to be attributable at least in part to media
artifacts, such as reliance on choleragen or other agents to elevate
basal cAMP levels (21, 43). For example, after several negative or
ambiguous studies, it was finally reported that under defined culture
conditions,
-MSH can induce tyrosinase mRNA, protein, and
activity in human melanocytes (44, 45), but its overall contribution to
regulation of human melanogenesis is appears nevertheless to be far
less than in the murine system. Importantly, posttranslational
modification appears to play a more critical role in regulating
tyrosinase activity in human than in murine melanocytes, with mRNA
and protein levels poorly correlated with the enzyme activity (19, 20),
and amelanotic human melanoma cells often expressing ample tyrosinase
protein (21, 22).
PKC-
is the first documented control point for human tyrosinase
activity. The protein level of PKC-
correlates with total pigment
content in melanocytes and activation of PKC leads to increased
tyrosinase activity, whereas depletion of PKC decreases tyrosinase
activity (21). PKC-
is required to activate tyrosinase, in that when
the lysate from amelanotic human melanoma NP-MM4 cells, which lack
PKC-
expression but have abundant tyrosinase protein, was mixed with
the lysate of normal melanocytes, tyrosinase in the NP-MM4 cells was
activated. This activation was abolished when the melanocyte lysate was
immunoprecipitated using a specific monoclonal antibody to remove
PKC-
before being mixed with the NP-MM4 cell lysate. In addition,
transient expression of PKC-
in NP-MM4 cells activates tyrosinase
(21). A key role for PKC-
is not unique to human melanogenesis but
also has been documented in the murine system. Pigmented S91 murine
melanoma cells, known to progressively lose their ability to pigment
both under basal conditions and after
-MSH stimulation if serially
passaged (46), in parallel lose the expression of PKC-
, whereas the
expression of PKC-
and tyrosinase remains unaffected (46).
Furthermore, the induction of murine pigmentation by
-MSH or other
cAMP elevating agents, such as isobutyl methyl zanthine or dibutyryl
cAMP, requires PKC in that when PKC is depleted, induction of
pigmentation by these agents is completely blocked (24). In human
melanocytes, we have further recently shown that PKC-
is
rate-limiting for cAMP-mediated elevation of tyrosinase activity in
that cAMP-induced tyrosinase activation does not correlate temporally
with increased tyrosinase protein, but rather parallels the later
cAMP-mediated induction of PKC-
(47).
In cultured human melanocytes, we and others have reported that at
least the
-,
-,
-, and
-isoforms of PKC are expressed (48,
49). The present immunoelectron microscopy studies show that the
-isoform co-localizes with tyrosinase at the surface of melanosomes,
whereas the
-isoform is found diffusely in the cytoplasm and not
specifically near melanosomes. These results suggest that
compartmentalization of PKC-
with melanosomes may determine its
ability to phosphorylate tyrosinase. The mechanism by which certain PKC
isoforms may adopt a nonrandom localization within cells is
incompletely elucidated. However, specific receptors for activated
protein kinase C have been identified, suggesting that PKC isoform
specific receptor for activated C kinase proteins may govern the
intercellular distribution of PKC isoforms (50). It would thus be
interesting to determine whether melanosomes express a PKC-
specific
receptor activated C kinase that is melanosome-associated.
Our current and previously published work (21) establishes that PKC-
activates tyrosinase activity in human melanocytes. The data presented
here further indicate that PKC-
-mediated phosphorylation exclusively
involves the cytoplasmic domain of tyrosinase and that both serine
residues in this domain are phosphorylated. Radioactivity from the
tryptic digest of 32P-labeled tyrosinase co-migrates
with the synthetic diphosphopeptide Glu-Asp-Tyr-His-Ser(P)-Leu-Tyr-Gln-Ser(P)-His-Leu, corresponding to
amino acids 501-511, using separation methods based on hydrophobicity (reversed phase HPLC), charge and size (thin layer electrophoresis), and polarity (thin layer chromatography). Furthermore, synthetic standard peptides corresponding to this portion of tyrosinase are
phosphorylated by PKC-
in vitro, even though the
conventional substrate sequence for PKC (51) is lacking, further
supporting this conclusion.
Tyrosinase-related proteins 1 and 2 (TRP1 and TRP2), two proteins known
to be important for melanogenesis, share 70-80% nucleotide sequence
homology and 40-45% amino acid identity with tyrosinase (52). Like
tyrosinase, they are transmembrane melanosomal proteins, with
cytoplasmic domains of about 26-28 amino acids. In addition, TRP1 has
one serine at amino acid position 502 and one threonine at amino acid
position 516, and TRP2 has two serines at amino acid positions 510 and
511 and three threonines at amino acid positions 503, 508, and 516, (52). They are therefore potential PKC substrates, although they have
never been reported to be phosphoproteins. However, TRP1 and TRP2 do
not share any amino acid homology with tyrosinase in their cytoplasmic
domains (5, 52), and therefore, the tryptic fragments generated from
the cytoplasmic domains of TRP1 and TRP2 are vastly different from
those of tyrosinase. Hence, the tryptic fragments of TRP1 and TRP2
would not co-migrate with the synthetic mono- and diphosphopeptide
standards that were used for mapping experiments in this report,
excluding the possibility that our phosphorylation results reflect TRP1
and/or TRP2 contamination. It has also been demonstrated that only
peptides of identical size and charge will co-migrate in the thin-layer
chromatography employed in these experiments (39).
A central role for PKC-
in melanogenesis does not diminish the
importance of other signal transduction molecules.
cAMP-dependent protein kinase (PKA), the principal
serine/threonine kinase mediating the actions of cAMP, also activates
tyrosinase either directly or indirectly through phosphorylation.
Addition of purified PKA to murine S91 cell lysate activates tyrosinase
(17), presumably through phosphorylation of a PKA substrate protein,
but in one early study (15), when these cells were treated with
-MSH
in the presence of [32P]orthophosphate, tyrosinase
apparently was not phosphorylated. Indeed, only recently was an
obligate role for PKA in
-MSH-induced murine melanogenesis firmly
established (53). As noted above, the role of the PKA pathway in human
melanogenesis is even less clear and appears intimately connected to
the PKC pathway (53).
The critical role of the cytoplasmic domain of tyrosinase in its
melanogenic activity established by the present experiments is also
strongly suggested by mutations identified in patients with
oculocutaneous albinism. Insertion of single nucleotides at codons
determining the amino acid at position 489 or 501 causes premature
termination of tyrosinase, deleting 20-22 amino acids from the
cytoplasmic domain, including the serines at 505 and 509 (6). This
abolishes tyrosinase activity, although the intramelanosomal domain of
the tyrosinase protein is present and normal. Site-directed mutagenesis
to alter or eliminate one or both of the serines in the cytoplasmic
domain, followed by expression of the mutated DNA in pigment cells
lacking wild-type tyrosinase, would rigorously demonstrate that
phosphorylation of these residues is required for protein activity, but
unfortunately, substantial technical difficulties in transfecting human
melanocytes would need to be overcome before such experiments could be
performed. In mice harboring the platinum mutation, in which the last
27 amino acids of tyrosinase are absent, pigment production is greatly
reduced and, in addition, tyrosinase is directed to the cytoplasmic
membrane (8-10). However, a more recent study has suggested that amino
acids at positions 494-498 of the human tyrosinase are responsible for
the subcellular trafficking (11). These findings and our report suggest
that the cytoplasmic domain of tyrosinase is critical for both
subcellular trafficking and the activation of the protein.
Our results strongly suggest that direct phosphorylation of tyrosinase
by PKC-
leads to its activation. When purified human melanosomes are
incubated with purified activated PKC-
, tyrosinase activity
increases. This activation is thus likely due to the direct interaction
between tyrosinase and PKC-
. Demonstration of direct activation of
purified human tyrosinase by PKC-
was not attempted due to the
difficulty of obtaining a sufficient amount of highly purified enzyme
from poorly proliferative human melanocytes, particularly in light of
its close association in the melanosome membrane with highly homologous
TRP1 and TRP2 (52) and the hydrophobic polymer melanin. Moreover,
PKC-
-mediated phosphorylation of isolated human tyrosinase might
well not lead to the same activation as when the protein is residing in
the melanosome membrane, in a specific steric relationship to other cellular constituents complicating interpretation of negative results.
We have shown that phosphorylation occurs on the two serine residues in
the cytoplasmic domain of tyrosinase, but the precise mechanism by
which this phosphorylation activates tyrosinase has yet to be
determined. Recent studies suggest that tyrosinase might interact with
other melanogenic enzymes, such as TRP1 and TRP2, in the melanosomal
membrane (54, 55), and tyrosinase activity was reported to be
diminished when the enzyme complexes with TRP1 and TRP2 (55). Moreover,
it has recently been suggested that a point mutation in the TRP1 gene
that changes the coat color in mice disrupts the interaction of TRP1
with tyrosinase (54), suggesting that TRP1 may function as the
long-sought tyrosinase inhibitor. In this context it would be of
interest to determine whether phosphorylation of tyrosinase, and
possibly also TRP1, by PKC-
might sterically hinder their
association in the melanosome.
Our approach to identifying the exact phosphorylation sites using
co-purification of 32P-labeled tryptic peptides along with
unlabeled synthetic carrier phosphopeptides is novel and may have
general applicability for analyzing phosphoproteins for which the amino
acid sequence is known. It has the advantage of not requiring
sophisticated instrumentation, as do Edman degradation, mass
spectroscopy, and extensive purification of the protein. A mixture of
nonlabeled, predicted synthetic tryptic phosphopeptides added as
carriers to a tryptic digest of the 32P-labeled protein
allows purification to homogeneity of the peptide of interest by
several separation methods, ensuring that the specific carrier peptide
and the labeled peptide are identical. Moreover, the target protein
does not have to be particularly pure, in that contaminating
radiolabeled peptides derived from unrelated proteins will eventually
be separated from the carrier peptides. In addition, intact cells can
be used to phosphorylate the substrate of interest, increasing the
probability that the observed phosphorylation sites are physiologically
relevant. This is especially critical when portions of the substrate
protein are inaccessible to kinase in vivo due to physical
sequestration. For example, in vitro phosphorylation of
isolated recombinant tyrosinase might be misleading because phosphorylation might occur on amino acids in the more than 90% of the
tyrosinase protein normally sequestered within the melanosome from
cytoplasmic kinases such as PKC-
.
In summary, we have demonstrated that tyrosinase is directly activated
by PKC-
-mediated phosphorylation, and that the phosphorylation occurs on the two serine residues in the C-terminal cytoplasmic domain
of tyrosinase. This first detailed insight into the regulation of human
melanogenesis should increase understanding of normal and pathologic
pigmentation in human skin and hair, as well as facilitate its manipulation.
 |
ACKNOWLEDGEMENTS |
We are grateful to Mr. Vladmir Russakovsky
for technical assistance and to Dr. Vincent Hearing at National
Institutes of Health for providing the tyrosinase antibodies.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the
Charlotte Geyer Foundation and NCI, National Institutes of Health,
Grant RO1 CA 72763 (to H. Y. P.) and by a National Institutes of
Health Molecular Biophysics Training Grant (to J. M. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Dermatology,
J206, Boston University School of Medicine, 80 East Concord St.,
Boston, MA 02118. Tel.: 617-638-5527; Fax: 617-638-5515; E-mail:
hypark{at}acs.bu.edu.
Present address: Solid Phase Sciences Corporation, 550 Boston
Ave., Medford, MA 02155.
 |
ABBREVIATIONS |
The abbreviations used are:
BSA, bovine serum
albumin;
L-dopa, L-dihydroxyphenylalanine;
PKC, protein kinase C;
NP-MM4, nonpigmented MM4 human melanoma;
PDBu, phorbol 12,13-dibutyrate;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
DMEM, Dulbecco's
modified essential medium;
-MSH,
-melanocyte stimulating hormone;
PKA, cAMP dependent protein kinase;
HPLC, high performance
liquid chromatography;
PAGE, polyacrylamide gel electrophoresis;
PVDF, polyvinyl difluoride;
Fmoc, fluorenylmethyloxycarbonyl.
 |
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