Calcitonin inhibits the phosphorylation of various proteins in rat brain synaptic membranes

Vol. 130, No. 2, 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

July 31, 1985 Pages 669-676

CALCITONIN INHIBITS THE PHOSPHORYLATION OF VARIOUS PROTEINS IN RAT BRAIN SYNAPTIC MEMBRANES

'Jitendra Patel, 'Andrea Fabbri, 3Candace Pert, 2Lucio Gnessi, 'France Fraioli, and 1Robert McDevitt

1 Biological Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20205

2 V Clinica Medica, Universita llLa Sapienza", Rome, Italy 3

Section on Brain Biochemistry. National Institute of Mental Health, Bethesda, Maryland 20205

Received June 20, 1985

The present report examines the effect of different calcitonins and analogs on the in vitro phosphorylation of brain synaptic membrane proteins. -- The findings demonstrate that calcitonin is a potent inhibitor of several brain synaptic proteins and that salmon and eel calcitonins are considerably more potent than thyrocalcitonin in eliciting this effect. Deletion of leucine from position 16 in salmon calcitonin sequence resulted in a drastic loss of inhibitory activity, indicating the importance for a hydrophobic residue at position 16 in the intact calcitonin molecule. The mechanism of calcitonin inhibition of protein phosphorylation was likely due to the blockade of stimulation of protein kinases by calmodulin. 0 1985 Academic Press, Inc.

A number of recent observations suggest that the hormone calcitonin

(CT) has effects other than that of regulating calcium metabolism in the

bone. Of particular interest are the effects of calcitonin on the CNS

function. The central effects of calcitonin include production of analgesia

(121, modulation of prolactin release (3,4) and the inhibition of eating

and drinking behavior (5,6,7).

Brain from several species has been found to contain significant levels

of calcitonin like material and its distribution appears to be heterogenous

(8,9,10,11). Furthermore, specific and saturable binding sites for CT have

been demonstrated in neuronal tissue from several species (11,12,13,14).

Autoradiographic studies show that the CT binding sites are mainly localized

in the hypothalamic region, the brain stem nuclei and the dorsal horn of the

spinal cord (15,16,17).

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Prompted by the observation that CT can interact with calmodulin in a

Cat+ dependent fashion (18), we have examined the effect of calcitonin on

the calmodulin stimulated phosphorylation in brain membranes. We report

herein that CT is a potent inhibitor of calmodulin stimulated protein phos-

phorylation of certain proteins and demonstrate that this is a specific

effect requiring the intact CT molecule.

MATERIALS AND METHODS

Salmon(s), human(h) and eel calcitonin and desleu 16 sCT calcitonin were from Armour Pharmaceutical Co. (Kankakee, Illinois). 32P-ATP was from New England Neuclear. Reagents for gel electrophoresis were from Bio-Rad Lab. Calmodu- lin (CDR) and S-100 protein were purchased from Calbiochem.

Phosphorylation: Phosphorylation was performed as previously described (19). Briefly, synaptic membranes (0.75 mg/ml) were incubated in a final volume of 0.1 ml containing 50mM Tris-HCl (pH 7.4), 1OmM MgC12 and, where stated, 50pM CaC12 50ug/ml calmodulin, 50ug/ml S-100 protein, and/or various concentrations of calcitonin. This reaction mixture was preincubated for 3 min. at 3ooc. Phosphorylation was initiated with the addition of 2.5 Ci 32P-ATP (5pM, final concentration) and terminated after 30s with the 50~1 stopping solution containing 75mM Tris-HCl, 75mM glycine, 1.5% sodium dodecyl sulfate (SDS), 15% glycerol, 1.5% mercaptoethanol, and 0.005% bromophenol blue, at pH 8.7. The resultant mixture was heated for 3 min. at 100°C, allowed to stand at room temperature overnight and an aliquot used for gel electrophoresis. Tissue Preparation: Male rats (NIH OM Strain, 125-1503) were decapitated and the whole brain removed, homogenized in 0.32M Sucrose and fractionated to obtain the crude synaptic membrane preparation (P-2 fraction) according to the published procedure (20).

Polyacrylamide gel electrophoresis: A discontinous 10% polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (21). Autoradiography of the polyacrylamide gels and the quantitative analyses of the autoradiographs thus obtained was performed as previously described (19).

Protein assays: Protein was quantified as described by Bradford (22).

RESULTS

Addition of 0.006 to 0.2 mg/ml sCT to the synaptic membranes in the

presence of 50 M calcium produced a marked and dose dependent inhibition of

phosphorylation of several proteins with molecular weights 180, 160, 140, 87

63 and 51 kilodaltons (Fig. 1). With, the exception of the 87k protein, the

phosphorylation of these protein was enhanced by calmodulin- The phosphory-

lation of the 87k protein was actually inhibited by calmodulin as previously

reported.

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LANE 1 2 3 4 5 6 7

FIGURE 1 Autoradiograph illustrating dose dependent inhibition of protein phosphorylation by sCT. Lane 1, no sCT; Lane 2, 0.2 mg/ml; Lane 3, 0.1 mg/ml; Lane 4, 0.05 mg/ml; Lane 5, 0.025 q/ml; Lane 6, 0.013 mg/ml, Lane 7, 0.006 mg/ml. The molecular weights of relevant proteins are indicated.

Phosphorylation of several proteins was observed to be either calcium

independent and/or cyclic AHP dependent. Llowever, this phosphorylation was

found not to be significantly effected by sCT (Fig. 2). Addition of

exogenous calmodulin was found to attenuate the inhibition produced by sCT

(Pig. 2A) and at higher concentrations of calmodulin the effect of sCT could

be completely blocked (data not shown). In contrast, the calmodulin inhibi-

tion of the 87k phosphorylation was blocked by sCT that caused an apparent

stimulation of this phosphoprotein (Fig. IA).

The potency of sCT to inhibit the phosphorylation of 180,160,140,87,63

and 51 kilodaltons proteins is shown in Fig. 3 and demonstrates differential

sensitivity of these phosphoproteins to sCT inhibition. The 63 and 50k phos-

phoproteins were the most sensitive to inhibition by sCT. The effect of sCT

on the 87k phosphoprotein was found to be biphasic; sCT at lower concentra-

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Mg2+ ’ + + + + + + + + + ci32+ + + + + sCT + + + + cm + + CAMP + +

FIGURE 2 Autoradiographs of a representative experiment showing the effect of sCT on endogenous synaptic membrane protein phosphorylation in the presence of magnesium (Mg*), Calcium (La*), Cyclic AMP (CAMP) or calmodulin (CDX). The incubation concentration of these agents and other conditions were as indicated in the text. The molecular weights of the relevant proteins are indicated. The separation gel was 10% acrylamide.

tion enhanced the 87k phosphorylation, but at higher concentration attenuated

its phosphorylation.

A number of other calcitonins were tested for their activity to inhihit

the phosphorylation of the 63k protein (Fig. 4). The eel CT was equipotent

to sCT (with an IC5C of about 0.019 mg/ml or 5.@pM), while the human CT

was more than 6-fold less potent than sCT (with IC50 of about 0.118 mg/ml

or 35~11). Deletion of leucine from position 16 in sCT sequence, as in leu 16

sCT, resulted in a drastic loss of activity (IC50 of about 0.1 mg/ml or 30pM).

DISCUSSION

The present report describes a novel effect of CT. We have noted

that CT inhibits the phosphorylation of a number of proteins with molecular

weights 180,160,140,87,63 and 51 kilodaltons present in the crude synaptic

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0.025 0.05 0.075 0.1

sCT lmgiml)

FIGURE 3 The dose response of the inhibition by sCT of the phosphorylation of various proteins indicated in Fig. 1 and the text. The crude synaptic membrane were incubated at 30°C for 30 sec. with various concentrations of sCT. The level of phosphorylation of each protein was obtained by densitometric analyses of the autoradiographs. Each point is the mean of two deter- minations each in duplicate which varied less than 10%.

(0) 87K; (A) 16OK; (0) 180K; m) 140K; (a) 63K; to) 50K

membrane fraction (Fig. 1 and 2). These proteins appear to be phosphorylated

by calmodulin dependent protein kinase as the addition of exogenous calmodulin

enhances its phosphorylation (Fig. 2A), and calmodulin antagonists such as

trifluoperazine, W-7 and calmidazolium (R24571) inhibit the phosphorylation of

these proteins (data not shown).

The phosphorylation of the 63 and 50k protein appeared to be relatively

more sensitive to inhibition by sCT than other proteins. These two phospho-

proteins may represent the autophosphorylated subunits of calcium/calmodulin

dependent protein kinase (23). Such autophosphorylation may play a role in

the regulation of kinase activity (24). Phosphorylation of the 87K protein

was apparently enchanced by CT. The phosphorylation of this 87k protein is

believed to be mediated by phospholipid-sensitive calcium-dependent protein

kinase (25) and has been shown to be inhibited by calmodulin (19,26).

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I I I I

0.025 0.05 0.075 0.1

VARIOUS CALCITONINS (mg/ml)

FIGURE 4 The potency of sCT, (A), eel CT (Oi, hCT (u and des leu 16sCT (0), on the phosphorylation of 63K protein. The crude synaptic membrane preparation were incubated with various concentrations of the specified calcitonin at 30C for 30 sec. The level of phosphory- latfon of the 74K protein was obtained by densitometric analyses of the autoradiographs. Each point is the mean of two determina- tions each in duplicate which varied less than 10%.

These effects of CT may be as a result of a specific interaction of the

peptide with calmodulin. Such an interaction has been reported previously (18)

and may contribute to antagonize the actions of calmodulin. It is unlikely

that CT mediates its action by sequestering calcium required for the calmodu-

lin activity since the effect of CT on protein phosphorylation could not be

reversed by excess calcium (data not shown).

sCT and eel CT were more potent as inhibitors of phosphorylation than

hCT. The difference in the relative potencies between sCT and hCT we observed

here are consistent with those reported for the central biological actions of

actions of this peptide. These actions include, the anorectic activity (6-7),

inhibition of calcium uptake (27), inhibition of calcitonin binding (13,17)

and stimulation of adenylate cyclase (28). sCT and eel CT are very similar in

their amino acid sequence but differ from hCT in sixteen of the thirty-two

amino acid residues and show a much higher helical structure potential, mainly

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Vol. 130, No. 2. 1985 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

due to the central portion of the peptides (29). This difference seems to be

of crucial importance in explaining the higher potency of ultimobranchial

calcitonins compared to thyrocalcitonin. It has been suggested that a

positive correlation exists between helical structure potential, receptor

affinity and biological potency of CT peptides (29,30,31). In support of this

hypothesis, we found that desleu 16 sCT, a sCT analogue with Leucine deletion

at position 16 and a probable reduction in helicity (29), showed a much lower

activity in inhibiting brain synpatic phosphorylation (IC50 = 0.019 versus

0.1 mg/ml). DesLeu16 - sCT has also been reported to possess less

biological activity and a much lower affinity for the brain receptor than sCT

(17,28).

The potency of CT noted here is about loo-fold less than those noted for

sCT binding (11,13,17) and behavioral effects of the hormone (5,6,7) but are

well within the concentration range at which sCT inhibits calcium uptake by

neuronal tissue (6,27). Considering that calmodulin and protein phosphory-

lation have been implicated in the regulation of calcium mobilization (32,33,

34), it is conceivable that CT may inhibit calcium uptake by interfering

with calmodulin dependent protein phosphorylation.

In conclusion, the finding that calcitonin is a potent inhibitor of

phosphorylation of selective synaptic membrane proteins could provide a

possible biochemical basis for some of the neural actions of this peptide.

ACKNOWLEDGMENTS

The authors are deeply indebted to Dr. R.C. Orlowsky for the generous gift of CTs peptides and analogs. We are also indebted to Dr. P.J. Marangos for helpful and insightful discussions and Ms. Coopersmith for typing this manuscript.

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Calcitonin inhibits the phosphorylation of various proteins in rat brain synaptic membranes

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