Kinase activities associated with calcium-activated neutral proteases

Vol. 120, No. 3, 1984 May 16, 1984

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 767-774

KINASE ACTIVITIES ASSOCIATED WITH CALCIUM-ACTIVATED NEUTRAL PROTEASES Un-Jin P. Zi~nerman and William W. Schlaepfer Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received March 29, 1984

SUMMARY: Studies on the phosphorylatlon of calclum-activated neutral protease (CANP) revealed the presence of klnase activities closely associated with purified CANP preparations. The kinase activity in uCANP (CANP with high affinity for calcium) was cAMP-independent whereas the kinase activity in mCANP (CANP with low affinity for calcium) was cAMP-dependent, inhibited by klnase specific inhibitor and abolished when the mCANP was preincubated in calcium. The CANP-associated klnase(s) phosphorylate uCANP and mCANP, causing modulation of their proteolytic activities.

Calclum-actlvated neutral protease (CANP) is a cytosollc enzyme widely distributed in both vertebrate and invertebrate tissues and cells (1,2).

It causes

limited proteolysls in response to a transient rise in the intracellular concentration of calcium, thereby activating (or inactivating) enzymes (3-5,6), enhancing the affinity of receptors for ligands (7-11), altering membrane plasticity (12-14), participating in the turnover of myofibrillar proteins (15-17), and causing disassembly of cytoskeletal components (18-26). Two major forms of CANP have been isolated,

uCANP has a high affinity for

calcium in that it is activated by micromolar levels of calcium, whereas mCANP has much lower affinity for calcium and requires near millimolar concentrations of calcium for enzyme activation (2,27).

Both enzyme forms share common struc-

tural and immunological (28-30) features and it has been suggested that they represent multiple forms of the same macromolecule (30). We have explored the possibility that phosphorylatlon of the enzyme could serve as a mechanism to alter the affinity of enzyme for calcium.

These studies

This work was supported by National Institutes of Realth Grant NS-15722. The abbreviations used are: Hepes,4-(2-hydroxyethyl)-l-plperazine ethanesulfonlc acid; EGTA, ethylene glycol bls ~ -amlnoetbyl ether)-N,N,N',N'-tetraacetlc acld; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresls. 0006-Z91X/84 $1.50

767

Copyr~ht © 1984byAcademic Press, Inc. Allrightsofreproductionin anyform rese~ed.

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have shown that kinase activities are closely associated with purified uCANP and mCANP preparations from rat skeletal muscle and that these kinases phosphorylate the proteolytic enzymes. MATERIALS AND METHODS Materials. Matrex Gel Blue A from Amicon Corp. was used for the Blue-dye-affinity column. Agarose-hexane-adenosine, 3':5'-cyclic phosphate from PL Biochemicals Inc. was used for the cAMP-affinity column. Protein kinase inhibitor (rabbit muscle), calmodulin (phosphodiesterase, 3':5'-cyclic nucleotlde actlvator),.~denosine 3':5'-cycllc monophosphate, Arsenazo III dye were from Sigma. (y-J P)ATP came from Amersham. Isolation of CANP from rat skeletal muscle. The procedure has been described elsewhere ~30), Brief iy, supernatant from tissue homogenate was ehromatographed in HNEED buffer (I0 mM Hepes, i0 mM NaCI, 1 mM EGTA, i mM EDTA, i mM dlthloerythrltol, pH 7.4) on a DEAE cellulose (DE52, Whatman) column and the enzyme was eluted using a 0-0.35 NaCI linear gradient, uCANP was eluted at 0,1M and mCANP at 0.3M NaCI. Each enzyme was concentrated by precipitation in 60% ammonium sulfate and was rechromatographed on a Ultrogel AcA34 column, uCANP and mCANP, purified by gel filtration, were used for subsequent affinity chromatography (see below). Purification of uCANP by casein affinity chromatography. A casein affinity column (0.8x4.3 cm; bed volume 2.5 ml) was equilibrated with calcium buffer (IO-~M in HNEED). The calcium concentration was colorimetrically monitored with Arsenazo III dye(30). Four mg of uCANP in calcium buffer were applied to the column, washed with 3xbed volume of calcium buffer, and the enzyme was eluted with 5 mM EGTA buffer in 0.8 ml-fractions. Purified enzyme, concentrated in HNEED buffer by vacuum dialysis, had a protein concentration of 0.7 mg/ml. Purificatlon of uCANP and mCANP by B!ue-dye-affinlt 7 chromatography ,. A Blue-dyeaffinity column (0.5x18 cm; bed volume 3.5mi) was equilibrated with HNEED buffer. Three mg of uCANP were applied to the column and eluted with HNEED buffer in 0.8 ml-fractions. The uCANP activity eluted at 5xbed volume. Two mg of mCANP were similarly chromatographed and eluted at 2xbed volume. The purified enzymes were concentrated, by vacuum dialysis, to 0.I mg protein/ml. Determlnation of 32p incorporation. An aliquot (80 ul) from the incubation mixture was added to an equal volume of 10% trichloroacetlc acid. The pellet from the precipitation was washed twice with 100 ul 50% ethanol, dissolved in 100 ul solubillzing buffer (8 M urea, I% SDS, I mM dithloerythritol, 0.1M Trls-PO4, pH 7.4) and analysed by SDS-polyacrylamide gel (7.5%) electrophoresls (PAGE). ~oteins were visualized by staining the gel with Coomassle brilliant blue and P incorporation was detected by autor~iography of the dried gel with Kodak Xray film (X-Omat AR). Ouantltatlon of P incorporation was carried out by counting the radioactivity of a sample (40 ul), or excised protein bands, dissolved in 3 ml aqueous counting sclntillant (ACS If, Amersham) in a ~ q u l d Scintillation ~pectrometer (Packard, Tri-Carb). A 1 nmol sample of ~ -J~P)ATP yielded 407xi0 ~ cpm. RESULTS Kinase activity in CANP preparations:

Kinase activity associated with CANP was

noted initially during attempts to phosphorylate the protease.

When mCANP, puri-

fied by gel filtration, was incubated with magnesium, cAMP, (y-32p)ATP and either a commercial kinase or supernatant from rat skeletal muscle homogenate, the trl-

768

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o

I~

10

1~

Fig. I. Autoradlograph showing caseln-afflnlty chromatography of phosphorylated uCANP. Enzyme (70 ug~in HNEED buffer was incubated with magnesium acetate (4 mM), cAMP (1 uM), (y-J=P)ATP (0.3 uM) for 30 mln. at room temperature and chromatographed on a casein-affinity column (see Methods). Lanes 16-18 represent the eluted protein.

chloroacetic acid-lnsoluble pellet contained considerably more 32p label in the control incubation, without the addition of an exogenous source of kinase. 32p labeling of CANP:

Incorporation of 32p into CANP was demonstrated by incu-

bating uCANP, purified by gel filtration, with magnesium, cAMP and ~-32p)ATP. The labeled protease was chromatographed on a casein-Sepharose 4B affinity column (see Methods).

uCANP bound to the column when applied in the presence of calcium

(I0-5M), but was eluted with excess EGTA (Sx10-3M).

SDS-PAGE and

autoradlographlc analysis of eluted proteins showed incorporation of 32p into the single uCANP band at M r 96,000 (Fig. I).

Similarly, incubation of mCANP

preparations with magnesium, cAMP and ~ -32p)ATP resulted in labeling of the mCANP band of M r 76,000 (see below). cAMP-dependence of 32p incorporation into "uCANP and mCANP:

Incubation of uCANP

and mCANP with magnesium and ~ -32p)ATP with and without cAMP resulted in the progressive incorporation of 32p into M r 96,000 (uCANP) and M r 76,000 (mCANP) bands, respectively (Table I).

The rate of phosphorylation of uCANP was not

affected by cAMP whereas that of mCANP was markedly enhanced by cAMP.

Further-

more, the rate of 32p incorporation into mCANP in the presence of cAMP is threefold greater than that of uCANP under the same conditions,

769

cAMP-dependent incor-

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Table 1.

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

32p incorporation of skeletal muscle uCANP and mCANP 32p (cpm) (t)/32p (cpm) (to) 0

1

2

cAMP

I

3.3

+ cAMP

1

- cAMP + cAMP

32p incorporation

6

18

4.7

9.0

9.3

3.8

4.6

10.6

10.1

I

2.2

-

5.4

5.7

1

3.8

-

16.7

31.9

(.in.)

(nmol/mg/min)

uCANP (Mr 96,000) -

113

mCANP (Mr 76,000)

386

poration of 32p into M r 76,000 band of mCANP is illustrated in Figure 2.

It can

also be seen that phosphorylatlon of mCANP was not calmodulln-dependent. Klnase activit~ in purified preparations of CANP:

mCANP (from gel filtration)

was purified by Blue-dye-afflnlty chromatography (see Methods) yielding a single major band of M r 76,000 by SDS-PAGE (Fig. 3 top).

Incubation of purified mCANP

with (y-32p)ATP led to cAMP-dependent Incorporatlonof 32p into the M r 76,000

Ca+caM

cAMP

Fig. 2. Autoradiograph showing time-dependent incorporation of 32p label into mCANP in the presence of Ca/calmodulin or cAMP. Enzyme (7 ug) in HNEED was incubated with magnesium acetate (4 mM) and either with calcium chloride (0.1 mM) and ca~odulin (0.25 uM), or cAMP (1 uH). The reaction was started with addition

of ~-JZ~P)ATP (0.2 uM). 770

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mCANP !00

92 bb

45

1234 Fig. 3. Effects of cAMP, klnase inhibitor, and prelncubatlon with calcium on CANP-assoclated klnase activity. (Top) Coomassle-blue stain of Blue-dye-afflnlty column purified (see Methods) mCANP (3 ug) incubated wlth magnesium acetate (4 mM) and ~ - P)ATP (0.5 uM). Lane I, without cAMP; lane 2, wlth cAMP (I uM); lane 3, with klnase inhibitor (5 ug); lane 4, with all the preceding additions, but with prelncubation (30 mln.) of enzyme with calcium (0. I mM). (Bottom) Autoradiograph of the above.

mCANP band ( F i g 3 bottom, l a n e s 1 and 2). b l o c k e d by p r o t e i n k i n a s e i n h i b i t o r

The cAMP-dependent p h o s p h o r y l a t i o n was

( F i g . 3 b o t t o m , l a n e 3).

enzyme w i t h c a l c i u m caused a u t o p r o t e o l y s i s

P r e i n c u b a t i o n of

and l a c k of p h o s p h o r y l a t t o n

( F i g . 3,

bottom, lane 4). E f f e c t s of p h o s p h o r y l a t i o n of CANP on p r o t e o l y t i c p h o s p h o r y l a t i o n of CARP a l t e r s v a t i o n was a l s o examined. the e l u t i o n column.

profile

activity:

The p o s s i b i l i t y

that

t h e l e v e l s of c a l c i u m r e q u i r e d f o r p r o t e a s e a c t i -

P h o s p h o r y l a t t o n of mCANP, f o r example, did not a l t e r

of t h e enzyme when r e c h r o m a t o g r a p h e d on a DEAE c e l l u l o s e

Both p h o s p h o r y l a t e d and n o n - p h o s p h o r y l a t e d mCARP e l u t e d a t 0.3M NaC1. 771

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If uCANP had been generated by phosphorylation, a new peak of proteolytic activity requiring less calcium for activation should have eluted at 0.1M NaCI. Similarly, substrate affinity chromatography was also used to test for possible conversion of mCANP to uCANP by phosphorylation.

In this case, mCANP

was passed through a casein affinity column at 10-5M calcium to absorb out admixed uCANP forms.

The mCANP was then phosphorylated and chromatographed on a

second casein affinity column in 10-4M calcium.

The phosphorylated mCANP, thus

purified, did not display an increase of proteolytic activity at 10-SM calcium. Comparison of phosphorylated and non-phosphorylated mCANP by casein affinity chromatography showed that phosphorylation leads to a reduction in binding of the enzyme to the column.

Furthermore, the eluted phosphorylated enzyme showed a

diminished level of specific proteolytlc activity when compared with non-phosphorylated controls. DISCUSSION While there has been increasing documentation of the widespread occurrence of CANP in different tissues and species (1,2), this is the first report of the phosphorylation of the enzyme, the co-purlflcatlon of kinase activity and the phosphorylation of the enzyme by the co-purifying kinase.

Co-puriflcation of

kinase with protease occurred using a sequence of commonly employed methods for the purification of CAN-P, including ammonium sulfate precipitation, anion exchange, gel filtration as well as affinity chromatography using Blue-dye or casein as ligand.

The same close association of kinase and protease activities

could be demonstrated during isolation of CANP of different tissues from dissimilar species, such as bovine brain (data not shown). Co-purlflcatlon of klnase and protease activities may imply the presence of both catalytic sites on the same molecule or on different molecules interacting cohesively.

Some of our present findings, in fact, suggest a high degree of

coupling between kinase and protease activities.

For example, the separation of

different forms of CANP during ion exchange chromatography is paralleled by a separation of different forms of kinase activity in both rat skeletal muscle and bovine brain.

Purification of mCANP from muscle by substrate affinity 772

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chromatography yields a single band by SDS-PAGE (Fig. 3 top), yet this preparation is enriched in both kinase as well as protease activities. Furthermore, both protease and kinase activities undergo parallel inactivation when incubated with calcium, a well-recognlzed phenomenon of autoproteolysis characteristic of the mCANP form of the protease and associated with loss of the M r 76,000 band on SDS-PAGE (data not shown). It is possible that calcium-inactivation of kinase activity could be due to proteolysis of a co-mlgrating protein or another protein band not visualized in the Coomassie-blue stained gels.

However, the calcium concentration (i mM or

less) used in our experiments is known to activate rather than inactivate other protein kinases, such as phosphorylase-b kinase (3).

Alternatively, calcium may

have an inhibitory effect on the kinase activity of mCANP.

Our preliminary stud-

ies, however, have not been able to reverse the inhibitory effects of calcium by the additions of calcium chelators.

Finally, we have been unable to dissociate

the cAMP-dependent kinase activity from the protease activity of mCANP by affinity chromatography using cAMP as a ligand (data not shown). Functional implication of the co-purlflcatlon of kinase activities with CANP will require further study.

Our data indicate that phosphorylation does not

effect the affinity of CANP for calcium but rather tends to reduce overall levels of proteolytic activity, at least with the purified enzyme forms.

It is still

unclear, however, whether phosphorylation may modulate CANP as it exists in vivo, either its activity per se or its possible interaction with the endogenous inhibitor of CANP (I).

The co-purlficatlon of kinase activity with CANP and the

demonstration that CANP can be phosphorylated represent novel findings which may serve to unravel additional control mechanism of CANP activity within the cell.

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