Activation of mouse melanoma cell cyclic AMP-dependentprotein kinase by melanocyte stimulating hormone

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Authors Birch, David Edward

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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

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

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.

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

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

LIST OF TABLES

Table Page

1. Effects of salt concentration on kinase activ ity . . . . 11

v i

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

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

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.

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.

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

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

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.

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.

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.

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

■ 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

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,

21

22

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,

14, Korner, A,, Pawelek, J, (1977), Activation of melanoma tyrosinaseby a cyclic AMP-dependent protein kinase in a cell free system. Nature 267:444-447,

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