protein kinase c activities are increased in rat thyroid epithelial cells expressing v-ras genes

Vol. 157, No. 3, 1988

December 30, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages ]093-]]03

PROTEIN KINASE C ACTIVITIES ARE INCREASED IN RAT THYROID

EPITHELIAL CELLS EXPRESSING V-RASGI~ES

Spina, A., Chiosi, E., and Illiano, G.

3acattedra di Chimica Biologica, Dipartimento di Biochimica e

Biofisica

Uhiversit~ di Napoli, Italy

** Berlingieri, M.T., Fusco, A., and Grieco, M.

Centro di Fmdocrinologia ed Oncologia Sperimentale del C.N.R. 2 a Facolt~ di Medicina e Chirurgia

Universit~ di Napoli, Italy

Received November 2, 1988

SUMS: Both cytoplasmic and membrane-bound protein kinase C activities are increased in: Harvey-Sarcoma Virus, infected

thyroid epithelial cells. The cytoplasmic kinase C increase is

found in the chromatographic fraction eluted at lower salt

concentration (i00 mM NaCI-SI00), while the more acidic protein

fraction eluted at higher salt concentration (350 mM NaCI-S350) is virtually absent. Although the cytoplasndc SI00 fraction from the

control and ras-virus infected cells display a+~o~oarable PBt 2

binding activity ,they are different in the Ca -dependence ~d

the TPA down regulation. In addition, the membranes from the

control and ras-virus infected cells are different phosphate

acceptors in place of the HI histories. ©19s8 Academic Press, lnc.

~11tured rat thyroid epithelial cells (PC C13) retain the

typical markers of thyroid differentiation (iodide uptake,

thyreoglobulin synthesis and secretion) and depend on TSH for

growth. Upon infection by Harvey murine sarcoma virus, they become

To whom all correslmondence concerning the biochemical studies should be addressed.

To when all correspondence concerning the cellular should be addressed.

Abbreviations: TPA, 12-0-tetradecanoylphorbol 13-acetate; PBt2, phorbol-12,13 dibutyrate; TSH, thyroid stimulating hormone; PBS, phosphate saline buffer; PKC, protein kinase C; Ptd-Ser, phosphatidyl serine.

studies

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0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 157, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

transformed, loosing the markers of thyroid differentiation,

becoming independent from TSH for growth, although they are not

tumorigenic (i). The v-ras infected cells, express a pointly

mutated 21.000 daltons protein, belonging to the p21 family, which

shares some structural and functional properties with the

alfa-subunit of the hetero -trimeric G-proteins (2). In fact, it

binds and hydrolizes GTP and, like other G-proteins, it has been

proposed to play a central role on transducing external signals

toward internal amplifying systems. In the same cellular model, we

have already shown that activated p21 proteins cannot be

considered directly involved in the regulation of the adenylate

cyclase activity in v-ras-transformed cells (3) Their putative

role on regulating the hydrolysis of inositol-phospholipids has

been investigated with controversial results (4-6). On the ot~er

hand strong experimental evidences (7) have shown that

intracellular p21 protein microinjection results in a sudden

increase of diacyl-glycerol levels and prompted us to investigate

on the PKC activities in the v-ras infected cells.

Materials and Methods

Cells: PC C13 is a cell line derived from Fisher rat thyroid

glands as described by Ambesi-Impiombato et al. (8); PC C13 HaMSV

are PC C13 infected with HaMSV. The cell lines were grown in

r~dified FI2 medium supplemented with 5% calf serum (Flow Laboratories, Inc., McLean, Va.) and six growth factors (Thyrotropic hormone, hydrocortisone, insulin, transferrin,

somatostatin and glycil-histidyl-lysine, all from Sigma Chemical

Co., St.Louis, Mo.). Control experiments have been carried out

growing the infected cells in the absence of the above mentionated

six growth factors. The cells wore harvested from confluent cell

monolayers and detached with a rubber-policeman and washed two

times in phosphate buffered saline, PBS (137 mMNaCI, 2.7 mMKCI,

1.47 mM KH2P04, 8.1 mMNa2P04). Cell ~mbrane Crude Preparatzon: The cells, suspended in PBS, wore

washed three times at 5,000 rpm x 5 min in J-21 Beckman Centrifuge

with isotonic buffer, containing 10 mM ~CI 2 . F~zymic source preparation for the Kinase C activity: The cells

suspended in PBS wore washed three times by centrifugation at

5.000 rpm per 5 min. in J-21-Beckman centrifuge with the sucrose

saline buffer: 0.33 M sucrose, 20 ~4 --Tris HCI, 2 mM EDTA, 0.5 mM

EGTA, 2 ~M PMSF, 5 ug/ml leupeptin, pH 7,5. The pellet was added to the s~T~ buffer and the cells suspension was homogenized with

1094


a loose-fitting glass-glass dounce bo~)geniger (30 strokes). The

soluble fraction, obtained after centrifugation of the <:ell hor,mogenate at 15,000 x g i~er 12 rain. was retained m~d the mei~0rane

pellet was resus[~ended w~th 5 nJ of the same buffer without

sucrose mld treated for 30 lltin, with ]% triton X I00 in a ice slowly rotation. The detergent treated sus[~?nsion ~as brought to a

hor,~)geneous resus[x~.nsion by a dounce hcm~x3enization (I0 strokes)

and then recentrifuged at 12,000 x g per I0 ~tin. to obtain the

detergent-solubi]Jzed protein kinase C from the nmnff]rmle fraction.

DF2EE ce]lulose chromatograph[ (DE 52 ) : The cytosolic axed

solubilized preparation containing about i0 r~] of protein were applied on the DE 52 cromatographic col~x~Is (0.9 x 2 cm)

eqmilibrated with 20 l~,l Tris HCI pH 7.5, 0.5 Ii~d EGTA, 2 r~,l EDTA,

washed with t~) vol~u~s of the equilibrium louffer m~d eluted

step-wise ~¢ith ]00 and 350 NaCI in 20 ~I~M Tris HCI pH 7.5. All the

buffer contained 2 ~M P[,~F and 5 ug/ml leupeptin, q~le I00 and 350

~;~,] fraction containing the protein kinase C activity, were assayed

on the stone day. (9)

Assay of the kinase C: ~e incubation mixture contained in a final

vol~re of 250 ul: 25-50 ug of enz~nic source, 2@~M Tris IlCl pH

7.5, 0.75 mM calci~ml chloride, I0 ~4 magnesi~ acetate, 0.i r~M A~, 0.2 mg/~id histone H] (Boehringer), 0.05 n~]/m] ]eupept~n,

(O~p) ATP (New ~hgland Nuclear; specific activity: 500 Ci/[mDl). The kinase C activity was triggered by adding a lipid ntixttwe of

1.6 ug 1,2 diolein and 24 ug phosphatidylserine in 20 mM Tris HCl

pH 7.5. The lipid mixture was prepared with 300 ul of l0 mg

phosphatffdylserine/ml chloroform solution and i0 ul of 500 ug

dioleine/ml chlorofornl, evaporated under nitrogen gas, diluted with ffml 20 mM Tris HCI pH 7.5 and sonicated with three l-rain.

bursts with a Kontes ~iicro Cell Disrupter at high power. Lipids

are added to the reaction ntixture just prior to initiation of the reaction. The reaction was stopped by adding 1 nt] of ice-cold 25%

TCA and collected by filtration over Millii:~re filter (0.45 t~n) after 2 h at 4 ° C and washed five times with 2 ml 5% TCA. q]~e

filters were dissolved in Beckman Ready Solv ~ and couted for

radJ oactivity.

Prot~n kinase C activity is determined by subtracting the an~m]t

of P incorporated into 3~istone HI in the absence of lipid mixture from the anount of P incorporated in the presence. To~91

protein kinase C activity ~s expressed as nn~oles of P

incorporate [x~r min. per mg of protein both of cytosolic and

membrane preparation.

Protein concentration assay- The protein concentration was

deterntined according to Bradford (I0).

Results

The protein kinase activities have been investigated L~oth in tJ~e

cytoplasm and in the membrane. As indicated in .~ethods, the

soluble cytoplasmic protein kinase activities have been assayed

either in the crude supernatant or after partial purification on a

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TAHLE I

Cytosolic and membrane-bound protein kinase activities in Control (PC C13) and Harvey MSV infected PC C13 cells (PC C13 Ha MSV)

~hzymic source nmoles 32p incorporated/mg enzyme x m in.

Addition Calculated PKC activity ++

none Ca ,diacyl-glycerol phosphatidyl-serine

Crude cytosol* PC C13 0.36+.02 0.41+.021 0.05

m

PC C13 Ha MSV 0.83+.020 1.43+.027 0.60 Chromatograph.** Purified Cytosol: S i00:

PC C13 0.40+.01 0.42+.01 0.02 PC C13 Ha~IgV 0.67~,O4 1.28~.06 0.61

S 350: PC C13 0.30+.02 0.42+.02 0.12 PC C13 HaMSV I. 38~. 12 I. 38~. 03 0

C~rcmatograph. ** purified solubilized membrane proteins: M i00:

PC C13 0.67+.02 0.90+.03 0.23 PC C13 Ha~i~V 1.14~.01 1.79~.04 0.65

M 350: PC C13 1.15+.092 1.45+.08 0.30 PC C13 Ha MSV 1.26+.071 2.64+.11 1.38

The results reported, obtained fran a typical experiment carried out in triplicate, have been normalized for the cellular protein content. *,**: see Materials and Methods. S-100, S-350: Cytosolic DE52 chromatography fractions eluted respectively by 100ram and 350ram NaCI. M-100, M-350: Solubilized membrane proteins eluted through DE52 column, respectively by 100mM and 350n~ NaCI.

DE 52 column eluted by a step gradient of i00 and 350 mMNaCI. As

of the men~rane-bound kinase activities are concerned, they have

been solubilized by treating the membranes, at room temperature

for 15 min. in the presence of 0.1% Triton X I00 and partially

purified through chromatography on the anion exchange resin DE52,

as above indicated. The results are shown in the Table 1. They

indicate that a substantially higher protein kinase activity is

present in the cytosol of the v-ras infected cells, compared to

the one present in the control cells. Such activities, are of

course coni0rehensive of c-AS~ independent and dependent protein

1096


kinases, as well as of the ones which can be activated by ++

Ca -phospholipid.

As far as the PKC activity is concerned, the results reported in

Table I indicate that in the cytoplasm of the infected cells the ++

Ca -phospholipid protein kinase activities are activated in the

presence of the corresponding modulators to a larger extent in the

transformed than in the control cells.

The chromatography both of the crude supernatant and of the 1%

Triton solubilized membranes on an anion exchange resin (DE 52),

allow to collect in separate fractions, two populations of PKC

activities. The first one is eluted by 100 i~I NaCI and is more ++

sensitive to Ca -phospholipids than the second one, eluted at

the higher salt concentration (350 mH NaCI), likely because the

last one is already surrounded by a phospholipid environment. The

first kinase population is increased (about thirty times) in the

cytoplasm of the infected cells; on ~e contrary the second

population, eluted at 350 ~I NaCI concentration, is actually

undetectable in the cytoplasm of the infected cells. Finally, the

results reported in the Table I indicate that also the membrane

bound PKC activities are higher in the virus-infected cells than

in the control ones; the increase appears to be concerning both

the population eluted at i00 and 350 mMNa CI.

Ca2 + activation and the phorbol ester (PBt2) binding of the PKC

SI00 fraction isolated from the cytosol of normal and ras-virus

infected epithelial thyroid cells.

To test whether the kinase C activities associated to the

cytosolic fraction eluted by 100 mM NaCI may be regulated

differently in the two cell types, we have determinated the Ca 2+

requirements for the activation and the binding activity for the

PBt 2 The results reported in the Fig. 1 show the Ca 2+

concentration dependence of the PKC activities assayed in the

presence of standard Ptd-Ser and diacyl-glycerol concentrations,

indicating marked differences between the enzymic activities

present in the eytosol of control and transformed cells, mainly at

the extreme Ca2+ concentrations tested. In fact, at sublimiting

1097

V o l . 1 5 7 , N o . 3 , 1 9 8 8 B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

o-oPC-CI3 o--ePC-Hor v ey

c 10C - - .o_

>~ 8C

3 o

6C o .E

E

~ 2 c

"[~ CaCI 2 0.50 1.0 1.50 CoCl2

(EGTA+EDTA)(I:4)

4-t- Fig. i. Ca stimulation dose effect relationship of the PKC soluble activity eluted, with the crhomatographyc fraction S-100, from control (PC C13) and v-ras infected (PC C13 Ha 5kqV) thyroid

epithelial cells. The enzimic activity essay was carried out in presence of diacyl glycerol and phosphatidyl serine used at concentration described in Methods. ++ On the X-axis has been indicated both the absolute Ca ++ concentration and the ratio between Ca and his chelants (EGTA:EDTA, 1:4) concentration.

2+ Ca2+ Ca concentrations ( 2.5 mM corresponding to a / BGTA + EDTA

ratio of 0.5 ) the PKC activities fran the infected cell cytosol

get already a 50 % of the maximum increase and reache the maximum

at 5 ~ (Ca 2+ /EGTA = i), while a decrease from the maximum is

observe(] at satLmating Ca 2+ concentrations ( 7.5 mM, Ca 2+ /EGTA +

EDTA= 1.5). The Ca 2+ dependence of the PKC activities purified

from the cytosol of the control cells is depicted by a sigmoidal

curve, showing that the 50 % of the maximum increase is reached at

4.5 ~N4 Ca 2+, and the plateau at saturating Ca 2+ concentrations

(7.5 ~M,Ca 2+ /EGTA 1.5). The differences on the Ca 2+ dependence

could reflect structural differences between the regulatory

domains of the two enzymic populations.

As of the phorbol-dibutyrate binding activity is concerned, the

two enz~c populations (data not reported) do not show

sJgnificative differences .

Solubilized m~mbrane-bound PKC activities assayed with crude

m~mbrane preparations as phosphate acceptors.

1098


TA~,E II 32p incorporation, in crude membrane preparations, by solubilized

membrane-bound kinase C fraction, partially purified through DE52 chrcmatography and eluted with 100 or 350 ~MNaCl

Specific activity (32p ~noles incorporated/mg protein/min)

I~zymic preparation Substrates

i00 r~,,1NaCI fraction: PC C13 membranes PC Cl3HaMSVmembranes MI00 PC C13 1.48 + 0.08 3.39 + 0.23 PC CI3Ha ~v~V 2.67 + 0.018 7.28 + 0.41

350 mH NaCI fraction: M350 PC C13 1.30 + 0.28 2.20 + 0.21 PC CI31~ r.~V 17.22 + 0.58 3.46 + 0.26

The incubation mixture was performed as indicated in Material and b~thods, except that cell membranes (50-70/protein), prepared as previously indicated, replaced histone HI as phosphate acceptors.

We have carried out experiments using crude n~mbrane

preparations as phosphate acceptors, instead of Histone HI with

the purpose of testing the kinase C activities on substrates

which could be more physiological. The results are reported in

Table II. ~ley confirm that PKC activities solubilized from

transformed cell membranes are more effective than the ones from

control men,fanes on phosphorylating either type of membranes

(control or trasformed).

b~reover, the two irembrane types behave differently as phosphate

acceptors. In fact, the PKC activities solubilized from control

cell membranes, independently of which eluted fraction they are

associated with, can transfer a higher amount of -phosphates to

the membranes from transformed cells than to the ones from control

cells.

The PKC activities solubilized from infected cell membranes de

not display a univocal behaviour: the ones associated with the

fraction eluted by 100 mMNaCI work much better on the transformed

membranes too; on the contrary, the ones associated with the

fraction eluted by 350 mM NaCI work much better on the membrane

fractions prepared from the control than from the transformed

cells. Membrane protein phosphorylation depends also on several

1099


membrane factors: n~nber, exposure and accessibility of the serin

and threonin residues, c~nposition of die membrane lipids

m~choring the enz~ne and may be nmny o~ers. The combination of

all these factors must act more favor~)ly on the transforr~d

membranes with the exception of the solubi]ized PKC fraction

eluted at 350 mM NaCl. It has been described that PKC's

solubilized from the membrane have a richer lipid environn~nt

which also explains their relative independence from the

phospholipid-diaeyl glycerol activation; we suggest that the lipid

environment of the trasformated ~n~ranes, which contaminates

this enz~ic fraction, allows a more favorable interaction with

the normal rather than with the transformed membranes.

PKC activation and intracellular translocation in normal and

v-ras infected cells treated in vivo by TPA.

The well-known t~nor promoter TPA has l~en described to

activate in vitro PKC and to pronDte in vivo an intracellular

translocation of the enz~nic proteins from the cytosol to the

membrane (ii). This substance is believed to mimic diacyl glycerol

binding at the enzyme regulatory site, because of the structural

similarities between the natural and the succedaneous activator

(12). The membrane traslocation and activation of PKC lasts a

short time, variable according to the cell type investigated,

since it is shortly followed by activation of proteolytic enzymes

which destroy and down- regulate the kinase activity. Therefore,

because of the TPA regulation, the PKC decrease in the cytosol

does not parallel a corresponding increase on the membrane. This

is what we observe in the control cell, treated in vivo by i0 uM

TPA for 10 min. (Table III), while in virus- infected cells

exposed for 10m in. to TPA, the membrane bound PKC still appears

activated.

Our data do not elucidate in detail the molecular mechanisms of

differential regulation of PKC in virus-infected cells, whether

this is due to the lack of protease activation or to the

involvement of different kinase isoenzymes, which are more

resistant to protease action, or to a combination of both. In any

1100


TA~.E III

Distribution of the protein kinase C activity between Cytosol and Membrane, both in Control and Harvey MSV infected cells, in vivo treated

by 10ul TPA for 10min

% of kinase C activity after i0 mM TPA i0 min presence versus ~O as vehicle

Crude Cytosol O/tosolic fractions DE52

i0~I NaCI 350mM NaCI

PC C13 PC-Ha PC C13 PC-}~ PC C13 PC-[~

~,~mbrane fractions DE52

i00~4 NaCI 35@~M NaCl

PC C13 PC-Ha PC C13 PC-Ha

60% 76% 66% 68% 68% 65% 111% 201% 68% 151% The results are expressed as the percentage of the kinase C activity present in the cytosol and in men~ranes of the corresponding control cells. For other experimental procedures see 5k~terials and Methods.

case the consequences must be physiologically relevant for the

cells.

Discussion

Our results indicate that higher levels of PKC are present "in

toto" in the v-ras transformed rat thyroid epithelial cells. These

could be related to the expression of the mutated p21 proteins .In

fact it has been shown that p21 proteins are involved in the

diacyl-glycerol production (7), which is known to be anDng the

"physiological activators" of these enzymic activities.

Our results also indicate qualitative differences concerning the

PKC of the normal and v-ras-transformed cells which are difficult

to directly correlate with the v-ras gene expressed proteins. In

fact the results shown in the Table I indicate that the more

acidic enzymic population, associated to the fraction eluted by

350 mMNaCl,is almost undetectable on the cytoplasm of transfonmed

cells. Several possibilities could explain this observation: in

the cytoplasm of transforrmed cells a same_ protein could he present

in a less phosphorylated form and this, in turn, because both of

less active auto or hetero-phosphorylating activities and of

more active phosphatase ones. ~]t we have to take to account that

1101


at least five isoenzyme forms of PKC have been described and

cloned from several cell systems (13). The differential gene

expression of one isoenzyme compared to others might also explain

this result.

The PKC in the v-ras transformed cells displays a higher

sensitivity to Ca 2+ activation (Fig.l). This suggests some

structural differences between the regulatory d~nains of the

enzymic proteins. Moreover, other differences that we think cannot

be directly correlated to the viral p21 proteins,are indicated by

the results of the experiments in vivo with TPA: t~y indicate

that PKC's from treated cells are less promptly down regulated,

after the TPA activation and translocation, likely through the

activation of proteolytic processes. Whatever the molecular

mechanisms underlying this phenomenon in the transformed cells

(less active proteases,structural differences in the enz~nic

proteins which make them more resistant to proteolytic digestion

or a combination of both) they cannot be directly related to the

viral p21 proteins.

It has been already described that ras-virus infection and

transformation is characterized both by the overexpression of

point mutated p21 proteins and by the activation of phospholipase

C (and than PKC) (14). Our finding of an increased PKC activity in

ras-virus transformed epithelial cells is to us very interesting

in consideration of the biological peculiarities of t~

investigated model (PC C13 Ha MSV): the ras-virus infected thyroid

epithelial cells do not respond any more to TSH (8) and have lost

all the biological responses (iodin uptake, thyroglobulin

synthesis...) triggered by TSH except the growing capacity; this

might be related to the fact that PC C13 Ha MSV cells have the

Protein Kinase C system in the "on" state too. On the other hand,

it is well known that TSH interaction with sensitive epithelial

thyroid cells results in the activation of a variety of amplifying

systems, one of them leading to P}~ activation (15-16): our

finding reinforces the hypothesis that PKC activation is a

critical moment in the TSH action on thyroidal cell growth.

1102


Acknowledgment

This research was supported by grants from C.N.R.N. 87.01491.44

("Progetto Finalizzato Oncologia").

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protein kinase c activities are increased in rat thyroid epithelial cells expressing v-ras genes

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