protein kinase c from small intestine epithelial cells

Vol. 139, No. 3, 1986

September 30, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 875-882

PROTEIN KINASE C FROM SMALL INTESTINE EPITHELIAL CELLS

Gloria Velasco, Carmen F. Iglesias, Pedro Domfnguez, Francisco Barros,

Santiago Gasc6n, and Pedro S. Lazo

Departamento Interfacultativo de Bioquimica,

Universidad de 0viedo, 33071 0viedo, Spain

Received August i, 1986

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The eytosolie fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+was 10-7M. The phorbol ester TPA substituted for diacylglyeerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes The presence of PKC in sma%l intestine epithelial cells might have important implications in the C# + mediated control of ionic transport in this tissue. © 1986 Academic Press, Inc.

Protein kinase C is a part of a signal transduction mechanism,

controlling a variety of cellular processes. This signal transduction

mechanism is stimulated by a wide variety of extracellular messengers, in-

cluding musearinie eholinergic and~-adrenergie stimulators, peptide hormones

and growth factors which control both short-term and long-term processes,

such as secretion, smooth muscle contraction and cell proliferation (i). When

cells are stimulated, PKC is transiently activated by diaeylglyeerol, which

is produced in the membrane during the signal induced turnover of inositol

phospholipids (I,2).PKC was first found in a cytosolie fraction from rat

brain and subsequently in other mammalian tissues (3-5).

Intestinal secretion is a complex process in which Na +, CI- and

water fluxes are mainly implicated. The process is considered to be under

Abbreviations: PKC: protein kinase C;BB membrane: brush border membrane; BL membrane: basolateral membrane; PS: phosphatidylserine; PI: phosphatidylinositol; TPA: 12-tetradeeanoylphorbol-13 acetate; EGTA: ethylene glycol bis ( 8-aminoethylether)-N,N,N',N' tetraaeetie acid; EDTA: ethylene- diaminetetraeetie acid; PhMeS02 F: phenyl methylsulfonyl fluoride; Me2 SO: dimethyl sulfoxide.

875

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Vol. 139, No. 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dual control of cyclic AMP and Ca 2+ , through changes in their intracellular

concentrations (6,7), which, according to the current hypothesis of the

biological effects of these second messengers, would act to regulate the

activity of protein kinases. Knowing that agonists which activate the PI-PKC

transduction mechanism in other systems stimulate intestinal secretion (8)

and in the absence of data about PKC in intestinal epithelium it seemed

pertinent to investigate the presence of this enzyme in intestinal epithelial

cells. We now report experiments showing for the first time that, indeed, PKC

is present in enterocytes from both the rat and the rabbit. The kinetic

characteristics and requirements of the enzyme have been studied using a

partially purified preparation obtained from the cytosolic fraction. Purified

brush border and basal lateral membranes from rabbit small intestine have

been used to study the distribution of the enzyme.

MATERIALS AND METHODS

Materials. IY~2PIATP was obtained from New England Nuclear. PS was obtained from Supelco. All the other phospholipids, neutral lipids, histone (Sigma Type III-S from calf Thymus) ATP and TPA were purchased from Sigma Chemical Co. DE52 was from Whatman. Separation of PKC. Rat enterocytes were obtained from jejunum and ileum by treatment of the everted intestine with hyaluronidase as described (9). The cells were resuspended in 20 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA, 1 mM EGTA and 50 mM B-mercaptoethanol and were homogenized in a Potter-Elvehjem with 20 strokes at 2400 rpm. The cell extract was centrifuged for 60 min at i00.000 x g. The supernatant of this centrifugation is referred to as cytosolic fraction. The cytosolic fraction was routinely chroma- tographied as follows: 1-2 ml of cytosolie fraction was applied onto 0.5-1 ml columns of DE52 which had been previously equilibrated with homogeneization buffer. After washing with 8 ml of the same buffer, the activity was eluted with i ml each of 0.i, 0.2 and 0.4 M NaCi. Conductivity measurements of the eluates gave salt concentrations of 15-20 mM, 40-50 mM and 110-140 mM respectively in various experiments. PKC activity was confined to the 40-50 mM salt eluate. Preparation of purified membranes from rabbit small intestine. White New Zealand rabbits were used. Brush border (i0) and basal lateral membranes (ii) were prepared from mucosal scrappings as previously described. The purity of membranes was assessed by the specific activity of sucrase (BB) and Na+/K + ATPase (BL). Protein kinase C assay. PKC was assayed by measuring the incorporation of $ 2 p from IY- 32PIATP into HI historic. The assay system (50 ~I) contained 30 mM Tris-HCl buffer, pH 6.8, i0 mM Mg acetate, 0,5 mg/ml HI histone, 0,4 mM ECTA, 0,4 mM EDTA, i mM CaCI2 unless otherwise indicated, the appropriate amounts of phospholipids and neutral lipids and the enzyme (5-10 ~ g of protein). The reaction was started by addition of I¥-32pIATP (0, 5-IXI02 epm/pmol to a concentration of 130 ~M and maintained for I0 min at 37~ C. The reaction was terminated by addition of i ml of ice cold i0 mM phosphate, 10% trichloroacetic acid. Then the tubes were soaked in a water bath at 80eC for 5 min. Phosphorylation products were collected in Whatman GF/C filters, the filters were washed twice with 5 ml of 5% trichloroaeetie acid, dried and the radioactivity determinated by liquid scintillation. Phospholipids and neutral lipids were dissolved in chloroform, mixed appropriately, dried under nitrogen and resuspended in 20 mM Tris-HCl buffer, pH 7.5, using a 150 W MSE Ultrason sonieator for 30 sec. The sonieated samples were stored at room temperature in the dark and used within two weeks. When membranes were used,

876


they were previously solubilized in 20 mM Tris-HCl buffer, pH 7.5, containing 1 mM EDTA, 1 mM EGTA, 50 mM B -mercaptoethanol and 0,5 % Triton X-IO0. After 1 hour extraction at O~C, the sample was centrifuged for 60 min at i00.000 x g and the supernatant applied onto DE52 columns which were eluted as indicated

above. Determination of free Ca 2+ concentrations. The concentrations of ionic species in Ca 2t-EGTA/EDTA buffers were calculated with a computer program according to Fabiato and Fabiato (12). Logarithms of the apparent association constants at pH 6.8 for CaEGTA, CaEDTA, CaATP, MgEGTA, MgEDTA and MgATP were 6.06, 7.15, 3.50, 1.62, 5.14 and 4.03 respectively.

Other determinations. Sucrase and Na+/K+ATPase were assayed as described (i0). Protein concentration was determined according to Bradford (13) using

bovine serum albumin as standard.

RESULTS

Identification and separation of protein kinase C

Our preliminary studies showed that the cytosolic fraction from

enterocytes contained a Ca 2+dependent protein kinase which was activated up

to 4-fold in the presence of 0.5 mM free Ca 2+. However, the activity was not

enhanced when, in addition to Ca 2+, phospholipids were present in the assay

mixture.

DEAE cellulose (DE52) chromatography using a linear gradient of

NaCI resolved protein kinase activity into two fractions. One fraction,

representing up to 80 % total activity eluted at 110-140 mM salt. This

activity was Ca and phospholipid independent. Another fraction representing

20-40% of the total activity eluted at 40-60 mM salt and appeared as PKC( not

shown). Since it is known that proteases can cause the conversion of PKC into

a Ca 2+ independent, irreversibly activated, enzyme (14). We tested whether

the appearence of the fraction eluting from DE52 columns at high salt

concentrations could be prevented by inhibiting proteolysis. When cells were

homogeneized in the presence of 400 KIU/ml aprotinin, 5 mM PhMeSO2F and 0.i

mg/ml of soybean trypsin inhibitor, no significant difference was found in

the proportion of protein kinase activity eluting at 50 mM and 130 mM salt.

In view of these results, for rutine assays, PKC was eluted stepwise as

indicated in Methods.

While the cytosolie fraction could be frozen at -70~C and thawed

without lost of activity,PKC in DE52 eluates was completely inactivated after

thawing. Therefore, the cytosolic fractions were prepared, stored at -70~C

and, when required, aliquots were thawed, ehromatographed on DE52 columns and

assayed immediately for PKC activity.

Characterization of protein kinase C

Phosphatidylserine has been found to be the most common

phospholipid eofactor of PKC (15,16). With PKC from intestinal epithelium, a

maximum of six fold stimulation was observed with 80-100 ~g/ml of PS. The

addition of a low concentration (5-10 ~g/ml) of the neutral lipid diolein

enhanced the activation of the enzyme (Fig. IB). The Ka for Ca 2+ in the

877

Vol. 139, No 3, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[=

ft .

/ o ~ °

o

o / /

10 f o - -

/

6 o / "

/ 2 j o / O f O

PSlpg/ml) Free caz+(M)

B

Figure I. Effect of PS,diolein and Ca 2+ on PKC. A) Dependence of PKC activity on PS concentration. Reaction mixtures containing the indicated amounts of PS were prepared as described in Methods, and used for the assay of PKC in the presence of 0.2 mM free Ca 2+ and in the absence of diacy-lglycerol. B) Dependence of PKC activity on Ca 2+ concentration: PKC was assayed at the indicated free Ca 2 + concentrations in the presence of 25 ~g/ml of PS (o) or in the presence of 25 ~g/ml of PS and i0 pg/ml of diolein (@). Activation by diolein alone was negligible.

presence of phospholipid and diolein was IO-7M. Of a number of phospholipids

tested, only PS and PI stimulated the enzyme activity (Table I) althought the

activation by PI was only 20% of the obtained in the presence of P$. The

specificity of the fatty acid moiety of diacylglycerol has been suggested to

be critical for the activation of the enzyme. In our hands, the presence of

unsaturated fatty acid residues in the diacylglycerol molecule was not

Table I Lipid specificity for activation of protein kinase C. PKC was assayed as described in Methods. To test phospholipid specificity the reaction was carried out in the presence of 25 ~g/ml phospholipid and i0 ~g/ml of diolein. To test neutral lipid specificity the reaction was carried out in the presence of 25 pg/ml of PS and i0 pg/ml of the diacylglycerol. Free C~ + concentration was always 0.2 mM. Data are from a representative experiment and represent average values from duplicate determinations.

PKC activity % of maximal

pmol/min activation

A) Phospholipid added

None Phosphatidylserine DL-~-phosphatidylcholine(dipalmitoyl) L-a-phosphatidylcholine(dioleyl) L-a-phosphatidylethanolamine(dipalmitoyl) Phosphatidylinositol (from soybean)

b) Diacylglycerol added None Dipalmitin (16:0) ~ Distearin (18:0) ~ Dimyristin (14:0)* 1,2 Diolein (18:1)

0.6 8.0 1 O0 0.9 4 0.9 4

1.2 8 2.0 19

0.6 6.1 69 7.5 86 8.6 i00 8.0 92

Mixture composed of 50% 1,2 and 50% 1,3 isomers.

878


"E

o_

2

/ /

/

2-51 / /~o~- -o s

05

o 5 lb " 2b ~ 15 I~ Diotein(IJg/ml] TPA(ng/rnl)

Figure 2. Dependence of PKC activity on diolein concentration (A) and TPA concentration (B). A) Lipid mixtures containing PS and various concentrations of diolein were prepared as indicated in Methods. PS concentration in the assay was 25 P g/ml and free Ca 2+ concentration was 0.2 mM. B) PS concentration was 25 ~g/ml. Free Ca 2 +concentration was 0.2 mM. TPA was added immediatelly before starting the reaction from i00 fold concentrated solutions in 90% methanol, 10% Me2SO. (A) indicates the activity in the absence of phospholipids. (~) indicates the ~ctivity in the presence of 25 ~g/ml PS, i0 pg/ml diolein and 0.2 mM free Ca ~+. In the absence of PS, TPA did not cause any activation.

essential for the activation of PKC. Thus, dimyristine (14:0) and diolein

(18:1) were equally effective as stimulators of the enzyme. Also,distearin

activated 86% and dipalmitin 69% with respect to the maximal stimulation (14

fold) obtained in the presence of dimyristin (Table I). Thus, the same

activation was observed in the presence of 1,2 diolein and in the presence of

a mixture composed of 85% of the 1,3 isomer and 15% of the 1,2 isomer of

diolein (not shown).

Tumour promoting phorbol esters activate PKC (17). The phorbol

ester TPA activated PKC from intestinal epithelium in the presence of

micromolar Ca 2+ and PS, thus substituting for diacylglycerol, althought TPA

was approximately three orders of magnitude more potent as an activator.

Thus, in the presence of PS(25 ~g/ml ) and excess free Ca2+(0.2 mM), diolein

activated at concentrations ranging from 1 to i0 pg/ml(Fig. 2A); TPA

stimulated the enzyme at concentrations ranging from 1 to 5 ng/ml (Fig. 2B).

Using saturating concentrations of diolein (I0 Dg/ml) and TPA (i0 ng/ml) in

the presence of 25 ~g/ml of PS, similar Ca 2+ titration curves were obtained

(not shown).

Localization of protein kinase C

PKC has been implicated in a variety of membrane associated

processes and, moreover, it is thought that the translocation of the enzyme

from the cytosol to the plasma membrane might have important implications

(18-20). Therefore, it was of interest to know if PKC associates to a

specific membrane in the case of a polar cell such as the enterocyte. For

this purpose we have used purified BB and BL membranes obtained from rabbit

879


Table II Distribution of PKC in enterocyte membranes as compared to marker enzymes. The specific activity of the enzymes in the indicated fractions is compared.Values represent the mean value of at least three separate preparations. PKC activity is expressed as pmoles of phosphate incorporated per min per mg of membrane protein. Suerase and Na+/K + ATPase activities are expresed as nmoles of substrate hydrolyzed per min per mg of protein, n.d.= not determined.

Fraction Protein Kinase C Sucrase Na+/K+ATPase Homogenate n.d. 200 5 Crude plasma membranes 159 300 15 Brush border membranes 83 3,100 9 Basal lateral membranes 352 50 68

enterocytes. We had previously determined that rabbit enterocytes contained

PKC and that the enzyme behaved as the rat enzyme with respect to

phospholipid dependence and chromatographic elution from DE52 columns.

PKC activity can be detected in the cytosol and in a crude

membrane fraction, although the majority was found in the cytosol. Thus, the

percentage of total activity in the membrane fraction ranged from 21 to 33%

in various preparations while the percentage in the soluble fraction ranged

from 67 to 79% (similar results were obtained in the rat). When PKC was

determined in purified membranes, it was found(Table II) that the specific

activity of BL membranes (352 pmol/min/mg membrane protein) was four-fold

higher than the specific activity of the BB membranes (83 pmol/min/mg

membrane protein). This resembles the behaviour of Na + /K + ATPase. These

membranes were prepared in the absence of C 2+ and studies are in progress to

determine if the presence of Ca 2+ influences the fraction of the enzyme which

is membrane associated or the type of membrane to which the enzyme binds,

since it is known that Ca2+promotes the binding of PKC to membranes (20).

DISCUSSION

We have identified a Ca2+-dependent protein kinase in enterocytes

which appears to be PKC. In the presence of phospholipids, diacylglycerols

containing either saturated of unsaturated fatty acids in their hydrophobic

moieties, were equally effective as activators of the enzyme. This constrasts

with the enzyme from rat cerebral cytosol which was stimulated by

diacylglycerols containing unsaturated fatty acid, at least at position 2

(21), and it is similar to the enzyme from renal microvillus membranes which

was stimulated by diolein and dipalmitin (22). The concept that the

identified activity is PKC is also substantiated by the fact that tumour

promoting phorbol ester TPA activated the enzyme at very low concentrations

in the presence of phospholipids and Ca 2+.Cytosolic fractions contained a C~ +

dependent, phospholipid independent activity and only after DE52

880


chromatography was an absolute requirement for both Ca 2+ and phospholipids

demonstrated. The presence of lipids could also account for the greater

stability to freezing of the crude enzyme in the homogenate.

It has been suggested that translocation of PKC from cytoplasm to

the plasma membrane is implicated in activation of the enzyme (18-20). In

tissues such as intestinal or renal epithelium, where membrane transport is

an important physiological activity, the localization of PKC should be

important. Our results indicate that in intestinal epithelium PKC can be

located in cellular membranes, particularly in BL membranes. This contrasts

with the reported localization in rabbit renal microvillus membranes (22).

Since most of the agents that control intestinal ionic transport act on the

BL membrane of this epithelium, it is important to know their effect on the

distribution of PKC and the mechanisms by which the enzyme is activated.

The presence of PKC in intestinal cells might have important

implications for the control of ion transport in this tissue. Intestinal

secretion is known to be regulated by the intracellular concentration of

cyclic AMP and Ca2 + (6,7). It is interesting that those seeretagogues which

act by increasing the level of Ca 2+ are known to be stimulators of the PI-PKC

transduction system in other tissues (1,2). Thus, the presence of PKC in

enterocytes suggest that this transduction mechanism is operating in the

control of intestinal ionic transport. Two recent reports show that phorbol

esters can activate ion secretion in the intestine (23,24). On the other

hand, it is unknown at present if the seeretagogues which act to increase the

intracellular concentration of Ca 2+, stimulate PI turnover in the intestinal

epithelium.

ACKNOWLEDGEMENTS: We wish to thank Dr. Javier Vald@s, from the Department of Mathematics of this University, for his help in setting the computer program, and Drs. C@sar de Haro and Fernando Pel~ez, from the Centro de Biologla Molecular, Madrid, for their advice in our initial studies. This work was supported in part by grants from the F.I.S. and the C.A.I.C.Y.T. G. Velasco is recipient of a fellowship from the F.I.S.

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protein kinase c from small intestine epithelial cells

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