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Activation of mouse melanoma cell cyclic AMP-dependentprotein kinase by melanocyte stimulating hormone
Item Type text; Thesis-Reproduction (electronic)
Authors Birch, David Edward
Publisher The University of Arizona.
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Download date 19/06/2021 14:22:18
Link to Item http://hdl.handle.net/10150/557457
http://hdl.handle.net/10150/557457
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ACTIVATION OF MOUSE MELANOMA CELL CYCLIC AMP-DEPENDENT
PROTEIN KINASE BY MELANOCYTE STIMULATING HORMONE
by
David Edward Birch
A Thesis Submitted to the Faculty of the
DEPARTMENT OF NUTRITION AND FOOD SCIENCE
In Partial Fulfillment of the Requirements For the Degree of
MASTER OF SCIENCE WITH A MAJOR IN AGRICULTURAL BIOCHEMISTRY AND NUTRITION
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 9 3 0
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STATEMENT BY AUTHOR
This thesis has been submitted in partial fu lfillm ent of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. .
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In a ll Other instances, however, permission must be obtained from the author.
SIGNED:
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
fC» ■ * ' _MAC E. HADLI
Professor of Genera jy
ADate
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ACKNOWLEDGMENTS
I wish to thank the members of my Committee, Drs. Mac E. HadTey,
William F. McCaughey and James W, Berry, for their valuable guidance
throughout my graduate career. I also wish to thank Dr. Bryan Fuller
and Ms. Joyce Lebowitz for their advice and assistance.
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TABLE OF CONTENTS
Page . '
LIST OF ILLUSTRATIONS...............> . . . . . . . . . . . . v
LIST OF TABLES ............................. .. , v . ,.......................... vi
ABSTRACT , . . V . . . . . . . v ii1. INTRODUCTION . , ............................ 1
2. MATERIALS AND METHODS . . . . . . . . 4
Compounds Studied . . . . . ................... 4Cell Culture . . . . . . . . . . . 4Preparation of Cell Fractions . . . . . . . ......................... 4Assay for Cyclic AMP-Dependent Protein Kinase
A ctivity . . . . . . . . . . . . . 5
3. RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Kinase Assay Parameters . . . . , . . ..................... 6Effects of MSH on Kinase Activity in Melanoma . . . . . 13
4. DISCUSSION .................. 16
REFERENCES CITED . . . . . . . . . . . . . . .......................... 21
tv
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LIST OF ILLUSTRATIONS
Figure Page
1. Effects of cell concentration on the kinase assay . . . 8
2. Effects of incubation time on the kinase assay . . . . 9
3. Effects of sodium fluoride on kinase.activity . . . . . 12
4. Effects Of MSH on kinase activ ity over time ................... 14
' 5, Dose-response for the effects of MSH on kinasea c t iv i t y ........................................................ 15
v
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LIST OF TABLES
Table Page
1. Effects of salt concentration on kinase activ ity . . . . 11
v i
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ABSTRACT
The purpose of this study was to determine the activ ity of
.cyclic AMP-dependent protein kinase in Cloudman S91 melanoma cell cul
tures treated with MSH (melanocyte stimulating hormone), and to relate
any changes in activ ity to other changes in these cells in response to
the hormone. To carry out the study the assay for cyclic AMP-dependent
protein kinase was adapted to the Cloudman cells. The parameters of
cell concentration, incubation time, and sodium fluoride and sodium
chloride concentrations were defined. With this assay i t was shown
that the activ ity of the kinase is higher in cells treated with MSH.
Both the MSH dose response and the time course of stimulation of the
kinase were observed to closely parallel the previously observed MSH
induced changes in cyclic AMP levels. This evidence gives support to
the theory that the effect of MSH on mouse melanoma tyrosinase ac tiv ity
is mediated by cyclic AMP and cyclic AMP-dependent protein kinase.
v if
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CHAPTER 1
INTRODUCTION
Cultured Cloudman mouse melanoma cells respond to melanocyte
stimulating hormone (MSH) with an increase in tyrosinase activ ity (23).
MSH has been shown to exert its effect on these cells through the "sec
ond messenger" cyclic AMP (23). A series of experiments have been in i
tiated in our lab to investigate the chain of biochemical events which .-
leads to the hormonally induced increase in the activ ity of tyrosinase,
the rate lim iting enzyme in the synthesis of melanin.
Kuo and Greenguard have suggested that the only receptor for
cyclic AMP and thus the only mediator of cyclic AMP action in the cell
is cyclic AMP-dependent protein kinase (17). The evidence to support
this theory is s t i l l accumulating. Hormonal stimulation of target
cells via cyclic AMP has been shown in many systems to involve activa
tion of the kinase. The effects of epinephrine and glucagon on glyco-
genolysis and of epinephrine on 1ipolysis have been shown to result
from the stimulation of cyclic AMP-dependent protein kinase (19,27).
Extensive studies of the gonadotrophic hormones have demonstrated cyc
l ic AMP-mediation of their effects on the gonads and an involvement of
the kinase (3,10). The effects of ACTH on steroidogenesis in the adre
nal cortex has also been shown to involve cyclic AMP and probably cyclic
AMP-dependent protein kinase (25).
• 1 .
-
The proposed mechanism by which cyclic AMP mediates hormonal
action involves a "cascade" of events which begins with the binding of
the hormone to a membrane receptor. A membrane-bound adenylate cyclase
system is then activated resulting in an increase in cytosolic cyclic
AMP. The increase in cyclic AMP results in the activation o f cyclic
AMP-dependent protein kinase. The kinase has been shown to be able to
phdrphorylate many ce llu lar proteins including enzymes, ribosomal pro
teins, membrane proteins, and various nuclear proteins (11,19). Phos
phorylation of any (or any combination) of these proteins could play a
role in mediating the cells ultimate response to the hormone.
Cyclic AMP-dependent protein kinase is thought to be universally
present in animal cells . The enzyme has been isolated and characterized
in many tissues and has been shown to vary l i t t l e in structure or func
tion (18,19). The kinase has two subunits and seems to exist as a
tetramer (1 ). A catalytic subunit contains the phosphotransferase
activ ity . I t catalyzes the transfer of the 5f-phosphate of ATP to ser
ine and threonine residues in the substrate protein. A regulatory sub
unit has the a b ility to either bind and inh ib it the catalytic subunit
or bind cyclic AMP. The stochiometry o f the activation of the enzyme by
cyclic AMP has been determined, to be as follows:
2cAMP + R2C2 ------ R2cAMP2 + 2C
where R is the regulatory subunit, C is the catalytic subunit and R2C2
is called the holoenzyme (1 ).
Two major forms of the holoenzyme can be isolated in most t is
sues. The forms have been designated type I and type I I in respect to
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3
the order of their elution from DEAE cellulose with a salt gradient.
The two types d iffe r in their regulatory subunit while apparently shar
ing identical catalytic subunits (19). Many differences between the
holoenzymes have been demonstrated. Other than the difference in their
a ffin ity for anion exchange medium the enzymes show differences in their
size and molecular weight, autophosphorylation, cyclic AMP a ff in ity , and
their disassociation and reassociation characteristics in respect to
substrate protein, cyclic AMP, MgATP, and salt concentration (19,4).
The ratio of type I to type IT holoenzyme varies from tissue to tissue
and in the same tissue in different species (18,19).
Cloudman mouse melanoma has been shown to exhibit the classic
response to a peptide hormone. Studies in our lab and others have shown
that MSH treatment of Cloudman cell cultures e lic its an increase in cyc
lic AMP levels in the cells (7,23) and that specific cytosolic binding
of cyclic AMP occurs (6 ), I t has also been shown that the plasma mem
brane of these cells contains adenylate, cyclase ac tiv ity which can be
stimulated by MSH (16), Finally , another lab has reported cyclic AMP-
dependent protein kinase activ ity in Cloudman melanoma and linked the
kinase activ ity to stimulation of tyrosinase (14,22). Because of. these
findings we have carried out studies to determine i f MSH stimulates the
kinase and i f the stimulation coordinates in a temporal and dose re
sponse fashion with the other components of the cascade system.
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CHAPTER 2
MATERIALS AND METHODS
Compounds Studied
The cL -MSB used in these studies was obtained from Dr. Victor
Hruby of the Department of Chemistrys University of Arizona. Fetal ca lf
serum and horse serum were obtained from Flow Labs and the p en ic illin /
streptomycin from Microbiological Associates. Adenosine 5 '-triphos
phate, adenosine 3 ',5 '-c y c lic monophosphoric acid, and ca lf thymus his-
tone type 11-AS were purchased from Sigma Chemical Company. 1-methyl-
3-isobutylxanthine was obtained from Aldrich Chemical Company. ATP- 3"-32
P, with a specific ac tiv ity of 10-40 Ci/mmol, was purchased from New
England Nuclear.
Cell Culture
Cloudmah S91 NCTC 3960 (CCL 53.1) melanoma cells were obtained
from the American Type Culture Collection Cell Repository. Cells were: 2 ■ ■ .. ■ .
grown as a monolayer in 150 cm Corning flasks in Ham's F-10 medium
fo rtif ie d with 2% feta l ca lf serum and 10% horse serum. P en ic illin /
streptomycin (100 units/m l, 100 ug/ml) was also present in the medium.
, Preparation of Cell Fractions
For experiments cells grown to confTuency were removed from
their flasks as described in the figure legends. The cells were homog
enized in 5.0 mM sodium phosphate buffer (pH 7.4) containing 1.0 mM
■' - ' . , 4 : : ;
-
EDTA, 0.5 mM 1-methyl-3-isobutylxanthine (MIX), and 5.0% glycerol. The
homogenization was carried out in a Sorval Omni-mixer at setting six
using two 30-second bursts with a one-minute cooling period between
bursts. The homogenate was centrifuged at 30,000Xg for 30 minutes at
4° C. The supernatant was assayed or stored in liquid nitrogen. Cell
counts were done with a hemocytometer.
Assay for Cyclic AMP-dependent Protein Kinase Activity
The method used was an adaptation of the method described by
Corbin and Reimann (5 ). 20 ul samples of enzyme preparation were incu
bated with 50 ul of 20 mM potassium phosphate buffer (pH 6.5) containing
6.0 mM magnesium acetate, 0.5 mM MIX, 5.0 mg/ml hi stone (type II-A S ),
0.2 mM AT^P, and 5.0 x 10"^ M cyclic AMP. One uCi of ac tiv ity was
used per 50 ul of reaction buffer for a final specific a c tiv ity , in the
reaction buffer, of 0.1 uCi/mmol ATP. Reactions were carried out for
the indicated times at 30° C and terminated by pipetting a 50 ul aliquot
onto a Whatman 3MM f i l t e r disc. The f i lte rs were washed once in cold
10% TCA for 20 minutes, twice in 5% TCA for 10 minutes, and fin a lly in
methanol for 5 minutes. The f ilte rs were dried and counted in a Beckman
LS-8000 sc in tilla tio n counter.
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CHAPTER 3
RESULTS
Kinase Assay Parameters
The assay for cAMP-dependent protein kinase has many variables
which have not been well defined for melanoma. Thus, i t was necessary
to modify the assay procedure, as described by Corbin and Reimann (5 ),
in order to optimize the assay conditions for melanoma.
The kinase assay takes advantage of the enzyme's high a ffin ity
for hi stone protein. In the assay the enzyme preparation is incubatedop
with ATP- S’ - P and histone. The phosphorylated hi stone is then precip-32itated onto f i l t e r papers and the f i l t e r papers are counted for P
32a ctiv ity . The incorporation of P into histone is taken to represent
kinase ac tiv ity . The cyclic AMP-dependence of the kinase activ ity is
determined by assaying each enzyme preparation twice, adding an excess
of cyclic AMP to one of the assays. The state of activation of the
kinase in the enzyme preparation can then be expressed as a ratio of.the
sample assayed without added cyclic AMP (jn_ £ itu ac tiv ity ) to the sample
assayed with excess cyclic AMP (100% a c tiv ity ). Cyclic AMP-dependent
protein kinase activ ity is thus described in terms of an activ ity ratio
( -cAMP/+cAMP). Using this system an enzyme preparation in which the
kinase is 100% activated w ill have an activ ity ratio of 1.0, since
addition of exogenous cyclic AMP to the reaction mixture w ill have no
added effect. I t is important to realize that other histone kinase
6
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7
activ ity might be present in the cells . For this reason the assay
described can only be considered as a qualitative indication of cyclic
AMP-dependent protein kinase.
The amount of protein kinase varies among d ifferent cells.
Therefore, i t is necessary to determine the optimum number of cells and
incubation time to be used in the assay. Figures la and lb represent
data from the same experiment in which the effects of cell concentration
in the reaction mixture were examined. Figure la demonstrates that at32concentrations below 0,05 million cells/reaction the P incorporation
is so low that the accuracy of the assay is impaired. Figure lb , which
expresses the data in terms of P incorporation/10 ce lls , shows that
with concentrations of up to 0,40 m illion cells/reaction the incorpora
tion per cell decreases but the ratio of -cAMP to +cAMP remains con
stant. The constancy of the activ ity ratio is an important considera
tion. At high concentrations of cells i t would be expected that the
reaction containing the highest a c tiv ity , the +cAMP reation, would be
more affected by substrate a v a ila b ility . I f this were the case, then
the activ ity ratio would be a r t i f ic ia l ly raised.
In figures 2a and 2b the effects of incubation time are demon
strated. Once again the same experiment is presented in two fashions.32As the incubation time increases better P incorporation per reaction
is seen. From figure 2a the incorporation in both reactions seems to be
linear with time after the f i r s t few minutes. However, figure 2b shows
that the values for the +cAMP reaction and the -CAMP reactions begin to
converge with longer incubation. Based on the above data the subsequent
-
Figure 1. Effects of cell concentration on the kinase assay,32a, data expressed in P incorporation/number of cells; b, data
expressed in protein kinase activity/number of cells . Cells were removed from their flasks by treatment with Tyrode's buffer containing EDTA. The cells were washed twice by centrifugation and suspension in Ham's medium. Cell pellets were resuspended in the homogenization buffer to give the cell concentrations/reaction indicated. The suspensions were homogenized and assayed as described in the Materials and Methods section, using an incubation time of 10 minutes. Each point represents an assay done in quadruplicate, + s. e. Assays were done with (o) and without (o) cyclic AMP. One unit of protein kinase ac tiv ity represents the incorporation into hi stone of one pmol of 32p/minute.
-
Prot
ein
Kino
.. Ac
tivity
(U
nll
./IO
'cll
.)
„p
|nc0
,(c
pM,
l0-i
,
12 -
0 .4 00 .300.10 0.20
Number of Cells (xIO- 6 )
3 0
25
20
0 .30 0 .4 00.200.10
Number of Cells
-
Figure 2. Effects of incubation time on the kinase assay.32a, data expressed in P incorporation/number of cells; b, data
expressed in protein kinase activity/number of ce lls . Cells were prepared as described in Figure 1, to give a concentration of approximately 0.10 x .10 ̂ cells/reaction. The kinase assay was carried out as described in the Materials and Methods section using the incubation times indicated. Each point represents an assay done in quadruplicate either with (©■) or without (o) cyclic AMP,+ s. e. One unit of protein kinase activity represents the incorporation into hi stone of one pmol of 32p/minute.
-
Figure 2.
Continued
Protein Kinase Activity (U n lts / I0 6 cells)
o t* o ui
cr
minutes
3 2 P Incorporated (cpm x I0 - 3 )
O r» *
-
10
experiments were carried out using cell concentrations of 0.05 to 0.20
million cells/reaction and an incubation time of 10 minutes.
To assess the effects of MSH on kinase ac tiv ity growing cells
were treated with the hormone, removed from their flask, and assayed
for cAMP-dependent kinase ac tiv ity . For these studies i t was important
that nothing in the homogenization or incubation mixtures a r t i f ic ia l ly
altered the state of activation of the enzyme. For example, i t has been
shown that sa lt concentration can have an effect on the disassociation
and reassociation of the subunits of the kinase (4 ). Type I kinase is
susceptible to dissociation by high concentrations of salt while type I I
kinase tends to reassociate in low sa lt concentrations. Thus, when t is
sues containing primarily type I kinase, such as rat heart, are assayed,
low ionic strength buffers are used. For tissues, like adipose, which
contain predominantly type I I enzyme, ionic strengths of up to 0.50 M
are used. As shown in Table 1, high salt concentration in the homogeni
zation buffer s lightly increases the activ ity of melanoma kinase under
the assay conditions employed. Since i t has been reported that mela
noma has primarily type T kinase, no salt was used when assaying for
MSH stimulation.
Because ATPase can compete with kinase for substrate, kinase
assays usually include sodium fluoride to inh ib it ATPase ac tiv ity .
However, sodium fluoride can also in h ib it kinase a c tiv ity . In our cells
the use of sodium fluoride gives no improvement over controls and
slightly inhibits the kinase activ ity at higher concentrations (see f ig
ure 3). Therefore, sodium fluoride was not used in the assays.
-
u
Table T, Effects of sa lt concentration on kinase a c tiv ity . Cells were prepared as described in the captions of Figures la and lb to give a concentration of approximately 0,15 x 106 cells/reaction. The homogenization buffer used contained the indicated concentrations of sodium chloride. The incubation time was 10 minutes. Each preparation was assayed in quadruplicate with cyclic AMP (+cAMP) or without (-cAMP). For a discussion of ac tiv ity ratio see the Results section.
NaCl (M) Activity Ratio + S.E.
0.42 + 0.03
0.08 0.41 + 0.05
0.15 0.44 > 0.05
0.30 0.64 + 0.06
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12
>*
O — < «S 5O CDISC -2
1 3CL
15
10
5
05030 4020
NaF (mM)
Figure 3, Effects of sodium fluoride on kinase a c tiv ity .
Cells were prepared as described in Figure 1 using a cell concentration of approximately 0.10 x 10 ̂ cells /reaction, and using an incubation time of 10 minutes. Sodium fluoride was present in the reaction buffer at the concentrations indicated. Each point represents an assay done in quadruplicate + s. e . , either with (o) or without (o) cyclic AMP.
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Effects of MSH on Kinase Activity in Melanoma
With a sensitive assay for cyclic AMP-dependent protein kinase
activ ity in Cloudman melanoma i t was possible to demonstrate stimula
tion of the kinase by incubation of the cells with MSH. The peak of
kinase activ ity occurs a fter the cells are incubated with MSH for 30
minutes (figure 4), Activity declines a fter 30 minutes but remains
slightly elevated above controls for as long as 90 minutes. An in
crease in ac tiv ity a fte r a 30 minute incubation can be detected in cells_ o
treated with as l i t t l e as 1 x 10" Mc L -MSH (figure 5). A separate ex--9periment which more closely examined the effects of 10 McC-MSH showed
that there is no stimulation over controls when cells are treated with
that concentration of hormone.
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14
0.5
0.4o
t r>•»>o<
0.3
o 0 .2<
-
15
1.0
0 I0~10 I0 '9 IQ-8 I O'7 I O '6
MSH Concentration (M)
Figure 5. Dose-response for the effects of MSH on kinase ac tiv ity .
Cells were treated with the indicated concentrations of MSH for 30 minutes. They were then removed from the flasks, homogenized, and assayed as described in Figure 4. Each point represents an assay done in quadruplicate, + s. e.
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CHAPTER 4
DISCUSSION .
Treatment of cultured CToudman melanoma cells with melanocyte
stimulating hormone (MSB) results in an increase in tyrosinase ac tiv ity
after a lag period of 6-9 hours (7,21), Because either dibutyryl cyclic
AMP or theophylline w ill mimic the effects, of MSH i t has been suggested
that MSB exerts its effects through cyclic AMP (21). Pawelek et a l .
have described an increase in cellu lar cyclic AMP levels in mouse mela
noma in response to MSB stimulation (23), Fuller and Viskochil have
shown that when the cells are exposed to MSB the concentration of cyclic
AMP rises to a peak approximately 2 times control by 60 minutes (7 ). .
The cyclic AMP level remains at 1.5 to 2 times control level for at
least 4 hours, Dose-response studies have shown that treatment of cellsO
with concentrations of MSB as low as 10" M w ill produce significant in
creases in cAMP (6 ),
The evidence presented in this study shows that the activation
of a cyclic AMP-dependent protein kinase in melanoma in response to MSH
stimulation closely parallels the MSB-induced change in cyclic AMP con
centrations, Korner and Pawelek have demonstrated that addition of par
t ia l ly purified cyclic AMP-dependent protein kinase preparations to the
30s000 Xg supernatant of melanoma cells w ill increase the tyrosine
hydroxylase activ ity of tyrosinase (increased melanin formation was not
seen) (14), Addition of protein kinase inhibitor blocked this
' ' ' 16
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■ 17
activation. The above data suggest a role for cyclic-AMP-dependent
protein kinase in the regulation of tyrosinase ac tiv ity .
Two theories have been proposed as to how cyclic AMP might exert
control over tyrosinase ac tiv ity . Both theories could involve cyclic
AMP-dependent protein kinase in roles similar to those which have been
attributed to the enzyme in other systems.
Ktirner and Pawelek have suggested that activation of tyrosinase
results from the kinase-dependent phosphorylation and resultant deacti
vation of a tyrosinase inh ib itor (14). They have found tyrosinase
inhibitor ac tiv ity but have not linked the activ ity to a phosphorylation
event. They suggest that the lag period which occurs between hormone
stimulation and tyrosinase activation is due to "an antagonism between
kinase-mediated phosphorylation and phosphatase-mediated dephosphoryla
tion."
On the other hand. Fuller and Viskochil suggest that the in
crease in tyrosinase activ ity is the result of de novo synthesis of the
enzyme (7 ). They have shown that stimulation of tyrosinase activ ity by
MSH, dibutyryl cyclic AMP, or theophylline can be blocked by cyclohex-
amide or acttnomycin D, With double labeling experiments they have pre
sented evidence that tyrosinase is synthesized in response to MSH
stimulation. They suggest that the lag period for induction is due to
the time needed for synthesis of the enzyme and its incorporation into
me!anosomes,
I t has been clearly demonstrated that the activ ities of several
enzymes are regulated by direct phosphorylation, A classic example is
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18
the role of phosphorylation in the control of glycogen metabolism. Cyc
l ic AMP-dependent protein kinase has been shown to inactivate glycogen
synthetase and activate phorphorylase kinase and thus promote glycogen
breakdown (26,19). Regulation by phosphorylation has also been demon
strated for hormone sensitive lipase (27), pyruvate kinase (28), tyro
sine hydroxylase (30), and glycerol phosphate acyl transferase (20).
A role for cyclic AMP-dependent protein kinase has been shown in each
of these cases.
A strong correlation has been shown between the activation of
cyclic AMP-dependent protein kinase and the induction of several enzymes
including tyrosine amino transferase (29), ornithine decarboxylase (2 ),
and tyrosine hydroxylase (9 ). The a b ility of several effectors to in
duce these enzymes corresponds closely to their a b ility to activate the
kinase. The induction of these enzymes could occur at the transcrip
tional or the translational levels. However, studies on cyclic AMP-
mediated phosphorylation of ribosomes has shown that phosphorylation
can occur but does not result in increased protein synthesis (8 ). Thus,
a transcriptional site has been suggested for the control of enzyme
production.
Many studies have addressed the possible role of cyclic AMP-
dependent protein kinase in the regulation of gene expression (fo r a
review see Jungman and Russel) (13). A cyclic AMP-mediated transloca
tion of kinase activ ity from the cytosol to the nucleus has been ob
served in several systems and cyclic AMP-mediated specific binding of
cyclic AMP-binding protein and catalytic subunit to chromatin acceptor
-
19
sites has been shown in ovarian tissue (12),' Based on this evidence a
model has emerged in which the cytosolic kinase migrates to the nucleus
and, through phosphorylation of nuclear proteins, regulates the syn
thesis of specific RNA species.
I t is clear that neither of the theories on the regulation of
tyrosinase ac tiv ity is unprecedented. However, the phosphorylation of
a tyrosinase modulator of of non-histone chromosomal proteins is not the
only means by which the kinase might exert its effects. For instance,
cyclic AMP-dependent phosphorylation of membrane proteins has been ind i
cated in the control of anion transport (24). Since tyrosinase resides
primarily in melanosomes, membrane phosphorylation could certainly play
a role in the enzyme's ac tiv ity . The number of possible models for
regulation by phosphorylation seem to be unlimited.
Krebs has described a set of c rite ria for the mediation of an
effect o f cyclic AMP by phosphorylation of a protein (15). The f ir s t
criterion has been met for melanoma: the presence of a cyclic AMP-
dependent protein kinase. The remainder, which involve the iden tifica
tion of a substrate and its activation and deactivation by phosphoryla
tion and dephosphorylation, have yet to be studied. Phosphorylation of
tyrosinase or of melanosomes has not been investigated. And though
other protein substrates have been suggested none have been isolated.
Perhaps the most interesting finding in the study of melanoma
is that of the apparent stimulation of de novo synthesis of tyrosinase
by MSH through cyclic AMP, Though the data are not conclusive, i f in
duction is involved then cultured melanoma presents an ideal system in
-
which to study one of the more interesting roles suggested for the
action of cyclic AMP-dependent protein kinase.
-
REFERENCES CITED
1. BeavOs J, A, Bechtel9 P.. J , 9 Krebs9 E, G. (1975). Mechanisms ofcontrol for cAMP-dependent protein kinase from skeletal muscle. Adv. Cyclic Nucleotide Res. 5:241-251.
2. Byus, Craig V .9 Hedge9 George A .9 Russell9 Diane H. (1977). Theinvolvement of cyclic AMP-dependent protein kinase(s) in the induction of ornithine decarboxylase in the regenerating rat liv e r and in the adrenal gland a fter unilateral adrenalectomy. Biochim. Biophy. Acta 498:39-45.
3. Catt, K. J .9 DufaUs M, L. (1976). Basic concepts of the mechanism of action of peptide hormones, Biol. Reprod. 14:1-15.
4. Corbin9 J. D .9 Keely, S, L ,s Soperling9 T. R .9 Park, C. R. (1975).Hormonal regulation of adenosine 3 ' ,5 '-monophosphate-dependent protein kinase. Adv. Cyclic Nucleotide Res. 5:265-279.
5. Corbin, J. D .9 Retmann, E. M. (1974). Assay of cyclic AMP-dependent protein kinases. Methods in enzymology (ed. by J. C.Hardman and B, W. O'Malley). Vol. 38, pp. 287-290,
6. Fuller, B. B. (1978). Biochemical mechanisms involved in themelanoma cell response to endocrine stimulation. A dissertation submitted to the faculty of the department of ce llu lar and developmental biology. The University of Arizona.
7. Fuller, B. B ., Viskochil, D, H. (1979). The role of RNA and proteisynthesis in mediating the actions of MSH on mouse melanoma cells Life Sci. 24:2405-2416,
8. Gressner, A. M., Wool, I . G. (1976). Influence o f glucagon andcyclic adenosine 3 ' ,5 ' -monophosphate on phosphorylation of rat l iv e r ribosomal protein SG, J. B iol. Chem. 251:1500-1504.
9. Guidotti, Alessandro, Costa, Erminio, (1977), Trans-synapticregulation of tyrosine 3-mono-oxygenase biosynthesis in rat adrenal medulla. Biochem. Pharmacol. 26:817-823.
10. Hunzicker-Dunn, M., Jungmann, R. A., Birnbaumer, L. (1979). Hormone action in ovarian fo llic le s : adenylyl cyclase and proteinkinase systems. Ovarian Follicular Development and Function (ed. by A. R. Midgley and W. A. Sadler). Raven Press, New York, pp. 267-303,
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11, Johnson, E, M. ( 1 9 7 7 ) Cyclic AMP̂ -dependent protein kinase andits nuclear substrate proteins. Adv. Cyclic Nucleotide Res. 8:267-309,
12, Jungmann, R. A., Lee, S ,, DeAngelo, A. B, (1975). Translocationof cytoplasmic protein kinase and cyclic adenosine monophosphate- binding protein to in tracellu lar acceptor sites. Adv, Cyclic Nucleotide Res, 5:281-306,
13, Jungmann, R. A ,, Russell, D, H. (1977), Cyclic AMP, cyclic AMP-dependent protein kinase, and the regulation of gene expression.Life Sci. 20:1787-1798,
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