Phosphoserine in peptide substrates can specify casein kinase II action

Vol. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 190-196

October 15, 1990

PHOSPHOSERINE

IN

T.W.

PEPTIDE KINASE Hrubey§

SUBSTRATES II A C T I O N and

P.J.

CAN

SPECIFY

CASEIN

Roach

D e p a r t m e n t of B i o c h e m i s t r y and M o l e c u l a r B i o l o g y I n d i a n a U n i v e r s i t y School of M e d i c i n e I n d i a n a p o l i s , IN 46223 Received August 24, 1990 SUMMARY. Casein kinase II is a u b i q u i t o u s serine/threonine protein kinase which utilizes acidic amino acid residues as recognition determinants in its s u b s t r a t e s , the motif -S/T-X-XD/Ebeing particularly important. To test whether a phosphoserine residue can act as a s u b s t r a t e determinant, a p e p t i d e was s y n t h e s i z e d , c o n t a i n i n g the s e q u e n c e - S - X - X - S , w h i c h was not phosphorylated by casein kinase II. However, upon p h o s p h o r y l a t i o n at the +3 p o s i t i o n , the p e p t i d e b e c a m e a s u b s t r a t e for c a s e i n k i n a s e II. With another peptide, a positive influence of m o r e d i s t a l p h o s p h o r y l a t i o n s was found. The r e s u l t s i n d i c a t e t h e p o t e n t i a l for c a s e i n k i n a s e II to p a r t i c i p a t e in h i e r a r c h a l p h o s p h o r y l a t i o n schemes. ©1990AcademlcPress,Inc.

Recent studies have established that phosphorylated amino a c i d r e s i d u e s in s u b s t r a t e s m a y act as r e c o g n i t i o n f a c t o r s for certain protein kinases (1-4). These protein kinases, glycogen synthase kinase-3 (GSK-3) a n d c a s e i n k i n a s e I, a r e m e m b e r s of a small f a m i l y of e n z y m e s w h i c h in g e n e r a l i d e n t i f y t h e i r s u b s t r a t e s by the presence of a c i d i c a m i n o a c i d r e s i d u e s n e a r t h e t a r g e t s e r i n e or t h r e o n i n e . G S K - 3 a n d c a s e i n k i n a s e I are i m p l i c a t e d in hierarchal phosphorylation mechanisms in which an initial p h o s p h o r y l a t i o n event d e t e r m i n e s s u b s e q u e n t p h o s p h o r y l a t i o n s . Another protein kinase whose substrate sites have been d e f i n e d in t e r m s of a c i d i c r e s i d u e s is c a s e i n k i n a s e II (5-7), a u b i q u i t o u s e n z y m e in e u k a r y o t i c systems, w h i c h m a y be r e g u l a t e d by extracellular signalling (eg. 8,9) . This protein kinase phosphorylates a l a r g e n u m b e r of p r o t e i n substrates (5-7) a n d t h e r e h a v e b e e n s u b s t a n t i a l e f f o r t s to d e f i n e s u b s t r a t e s for the e n z y m e b a s e d on the d i s p o s i t i o n of G l u a n d A s p r e s i d u e s in the v i c i n i t y of t h e p h o s p h o r y l a t i o n site (10-15). An A s p or Glu in the +3 p o s i t i o n has b e e n d e e m e d e s s e n t i a l for c a s e i n k i n a s e II action (13-15). A p o s s i b l e role for p h o s p h a t e g r o u p s in c a s e i n

§ Present address: College of Pharmacy, Butler University, 4600 Sunset Avenue, Indianapolis, IN 46208. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

190

Vol. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

k i n a s e II s u b s t r a t e s has a l s o b e e n i n f e r r e d f r o m r e c e n t s t u d i e s (16-18). In t h e p r e s e n t work, we u t i l i z e d enzymatic m e a n s to insert phosphate groups i n t o the +3 p o s i t i o n of a s y n t h e t i c p e p t i d e w h i c h was not i t s e l f a s u b s t r a t e for c a s e i n k i n a s e II. The e n z y m a t i c i n s e r t i o n of t h e s e p h o s p h a t e s created a substrate p e p t i d e for the p r o t e i n kinase. The results s h o w e d a d d i t i o n a l l y that more distal COOH-terminal phosphorylations could also i n f l u e n c e casein kinase II action.

MATERIALS

AND

METHODS

Peptides.

The p e p t i d e R R R D D D S D D D was the g e n e r o u s g i f t of Dr. Thomas Stephens, Eli Lilly and C o m p a n y R e s e a r c h L a b o r a t o r i e s . All other peptides utilized were synthesized on a phenylacetamidomethyl resin (0 . 61 m m o l / g ) using an A p p l i e d B i o s y s t e m s M o d e l 4 3 1 A s y n t h e s i z e r run w i t h the small scale (0.i mmol) Boc program. A r g i n i n e side chains were p r o t e c t e d by the Mts group. The c o m p l e t e d p e p t i d e s were c l e a v e d f r o m the resin w i t h HF/10% a n i s o l e at 0 o for 45 min, e x t r a c t e d into dilute a c e t i c acid and lyophilized. Peptides were purified by r e v e r s e p h a s e h i g h pressure liquid chromatography (HPLC) using an a c e t o n i t r i l e g r a d i e n t in 0.1% t r i f l u o r o a c e t i c acid. H o m o g e n e o u s fractions, as d e t e r m i n e d b y a n a l y t i c a l HPLC w i t h the same system, were c o m b i n e d a n d lyophilized. The sequence of the p e p t i d e s was c o n f i r m e d using a P o r t o n Instruments Model 1090 sequencer.

Enzymes. C a s e i n k i n a s e II a n d g l y c o g e n s y n t h a s e k i n a s e 3 were purified as p r e v i o u s l y described (19,3). Catalytic s u b u n i t of c A M P - d e p e n d e n t p r o t e i n k i n a s e was the g e n e r o u s gift of Dr. Edwin Krebs, University of W a s h i n g t o n . The s p e c i f i c cAMP-dependent protein kinase inhibitor PKI-5-24-amide was purchased from Peninsula Laboratories (Belmont, CA).

Phosphorylation

reactions. The p e p t i d e s w e r e t y p i c a l l y a s s a y e d in 35 m M T r i s - H C I pH 7.2, 1 m M EDTA, 0.22 m M EGTA, O.4 m M [32p]ATP (0.5-1.0 cpm/fmol), 6.0 mM magnesium acetate, I0 mM 6mercaptoethanol, a n d p r o t e i n k i n a s e in a t o t a l v o l u m e of 15~i. For d e t e r m i n a t i o n of k i n e t i c p a r a m e t e r s , p e p t i d e s u b s t r a t e s were v a r i e d in the range 5-200 ~M. The r e a c t i o n s were t e r m i n a t e d by the a d d i t i o n of E D T A and a d e n o s i n e to final c o n c e n t r a t i o n s of 27.5 m M a n d 3.0 mM, r e s p e c t i v e l y . A l i q u o t s were s p o t t e d on 2.0 X 2.0 cm squares of W h a t m a n P81 f i l t e r paper, washed with constant s t i r r i n g four times w i t h 75 m M p h o s p h o r i c acid, a n d i m m e d i a t e l y washed once with 100% e t h a n o l . Label incorporated i n t o the p e p t i d e s was d e t e r m i n e d in 0.5% d i p h e n y l o x a z o l e in toluene, u s i n g a l i q u i d s c i n t i l l a t i o n counter. Phosphopeptides. D i f f e r e n t p h o s p h o r y l a t e d forms of the p e p t i d e s EEESEAEPAPSRRGSARR and EEESEASPAPSRRGSARR were prepared as follows. L a r g e scale p h o s p h o r y l a t i o n reactions (i m g peptide) were run, i n c u b a t i n g w i t h c A M P - d e p e n d e n t k i n a s e first. [32p]ATP was r e m o v e d by p a s s a g e over D o w e x 1 X 8 e q u i l i b r a t e d in 30% acetic acid. The p h o s p h o r y l a t e d forms of the p e p t i d e s w e r e s e p a r a t e d f r o m the m i x t u r e b y a f f i n i t y c h r o m a t o g r a p h y using iminodiacetic a c i d - e p o x y a c t i v a t e d S e p h a r o s e 6B (Sigma, St. Louis) c h a r g e d with ferric chloride (20). After drying, portions of each monophosphopeptide w e r e p h o s p h o r y l a t e d w i t h m u l t i p l e a d d i t i o n s of GSK-3 and i n c u b a t i o n at 30°C overnight. The d i p h o s p h o r y l a t e d form of EEESEAEPAPSRRGSARR and the triphosphorylated form of 191

V o l . 1 7 2 , N o . 1, 1 9 9 0

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

EEESEASPAPSRRGSARR (as i d e n t i f i e d using isoelectric focussing) were separated from these reaction mixtures using reverse phase HPLC and two different solvent systems. Separation was f i r s t a c c o m p l i s h e d u s i n g a 0-70% a c e t o n i t r i l e g r a d i e n t in 25 m M MES pH 6.0, then using a 0-70% acetonitrile gradient in 0.1% trifluoroacetic acid. The yields and concentrations of t h e phosphorylated forms of t h e peptides were determined using C h e r e n k o v counting.

Other procedures. Isoelectric focusing was performed using previously described procedures (i) . The k i n e t i c c o n s t a n t s for t h e v a r i o u s p e p t i d e s were d e t e r m i n e d f r o m L i n e w e a v e r - B u r k p l o t s of t h e k i n e t i c d a t a for e a c h p e p t i d e . Protein concentrations were determined by the method of B r a d f o r d (21). Other chemical reagents were obtained in t h e h i g h e s t quality available from c o m m e r c i a l vendors. RESULTS The r a t i o n a l e b e h i n d t h e d e s i g n of s y n t h e t i c p e p t i d e s for t h i s s t u d y was to c r e a t e a p h o s p h o s e r i n e at the +3 p o s i t i o n w i t h respect to a potential c a s e i n k i n a s e II site. Since chemical s y n t h e s i s of p h o s p h o s e r i n e c o n t a i n i n g p e p t i d e s is not yet routine, our strategy was to build a protein kinase site at t h e +3 position. A s u c c e s s f u l d e s i g n was b a s e d on t h e s e q u e n c e of a p h o s p h o r y l a t e d r e g i o n of the G - c o m p o n e n t of p r o t e i n p h o s p h a t a s e - i (Fig. i) . The synthetic peptide with this sequence was an e x c e l l e n t s u b s t r a t e for GSK-3 a f t e r p r i o r p h o s p h o r y l a t i o n by cAMPdependent protein kinase (2). We s y n t h e s i z e d a v a r i a n t of this p e p t i d e w i t h an N H 2 - t e r m i n a l extension containing a potential c a s e i n k i n a s e II a n d w i t h a G S K - 3 site at t h e +3 a m i n o a c i d (Fig. I). B a s i c r e s i d u e s w e r e a d d e d at the c a r b o x y l t e r m i n u s to e n a b l e the p e p t i d e to b i n d to PSI p a p e r (see " M a t e r i a l s a n d M e t h o d s " ) . A c o n t r o l p e p t i d e was s y n t h e s i z e d c o n t a i n i n g g l u t a m a t e at t h e +3 amino acid position from the casein kinase II site. As a reference, we also analyzed phosphorylation of the peptide

cAMP PK GSK-3 p

P

P

I i I KPGFSPQPSRRGSESSEEVYV P

P

P

I I l EEESEASPAPSRRGSARR potential CKII site

Figure i. Peptides based on the sequence of the G-component of protein phosphatase i. The upper peptide is identical in sequence to the G-component protein and contains phosphorylation sites for the indicated protein kinases. The lower peptide is based on the same sequence, but additionally contains a potential site for phosphorylation by casein kinase II (CKII) after prior phosphorylation by cAMP-dependent protein kinase (cAMP PK) and glycogen synthase kinase-3 (GSK-3) .

192

V01. 172, No. 1, 1 9 9 0

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I.

Kinetic Parameters for the Phosphorylation of Synthetic Peptides by Casein Kinase II

Peptide

Vma x

Km

Vma x /Km

(mM) (nmoles/min) 97.2

RRRDDDSDDD

58.2

1.67

EEESEAEPAP SRRGSARR EEESEAEPAPSRRGS (P) ARR EEESEAEPAPS (P) RRGS (P) ARR

29.8 9.7 38.4

122.3 20.9 49.3

EEESEASPAPSRRGSARR EEESEASPAPSRRGS (P) ARR EEE SEAS (P) PAP S (P )RRGS (P )ARR

<0.1 <0.1 2.04

38.6

synthetic

substrate

RRRDDDSDDD, which k i n a s e II (14).

is

an

excellent

0.244 0.464 0.780

0.0528

for

casein

The peptides EEESEAEPAPSRRGSARR and EEESEASPAPSRRGSARR were phosphorylated as d e s c r i b e d under "Materials and Methods" with c A M P - d e p e n d e n t k i n a s e , G S K - 3 , or a s e q u e n t i a l c o m b i n a t i o n of t h o s e kinases. 24-amide

When cAMP-dependent protein kinase was inhibitor peptide was added to a final

5.0 ~ g / m l

before

addition

work on the related excellent substrates

of o t h e r

kinases

(22).

utilized, the concentration As

5of

expected

from

G-component peptide (2), b o t h p e p t i d e s for c A M P - d e p e n d e n t k i n a s e . The peptides

were were

n o t s u b s t r a t e s for G S K - 3 u n t i l a f t e r t h e y h a d b e e n p h o s p h o r y l a t e d by cAMP-dependent kinase. Analysis by isoelectric focussing indicated that phosphorylation of the peptide EEESEAEPAPSRRGS(P)ARR by GSK-3 generated a single new phosphorylated single

species

phosphate

group

corresponding while

to

the

phosphorylation

introduction of

the

of

6" o

5 4

8.

O

3

O

o_

8 f_

1

0

20

40

60

Time (mm) Figure 2. Time course of the phosphorylation of the various phosphorylated forms of the synthetic peptides by casein kinase II. The incorporation of phosphate into the diphosphorylated (O), monophosphorylated (O), and unphosphorylated (~) forms of the peptide EEESEAEPAPSRRGSARR and into the triphosphorylated (D) and monophosphorylated (A) forms of the peptide EEESEASPAPSRRGSARR were monitored as a function of time. The concentration of the peptides was 20.0 ~M.

193

a

peptide

Vol. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EEESEASPAPSRRGS(P)ARR resulted also in a m o r e a c i d i c species correlating with modification at t w o sites (not shown). Again, these results are consistent with earlier studies (1,2). By v a r y i n g the p r o t e i n k i n a s e s used, we were thus able to g e n e r a t e a set of f o u r d i f f e r e n t phosphopeptides (Table I), which were p u r i f i e d as d e s c r i b e d u n d e r " M a t e r i a l s and M e t h o d s " . The peptides and phosphopeptides were then tested as s u b s t r a t e s for c a s e i n k i n a s e II. A r e p r e s e n t a t i v e t i m e c o u r s e of phosphorylation of t h e p e p t i d e s is s h o w n in Fig. 2. Table I compares the kinetic constants for casein kinase II phosphorylation of t h e same peptides. The unphosphorylated peptide EEESEAEPAPSRRGSARR was a s u b s t r a t e for c a s e i n k i n a s e II. The monophosphorylated f o r m of t h e p e p t i d e , generated after phosphorylation by cAMP-dependent p r o t e i n kinase, was a b e t t e r s u b s t r a t e t h a n the u n p h o s p h o r y l a t e d form, in terms of K m or Vmax/K m (Table I) . The d i p h o s p h o r y l a t e d f o r m of the p e p t i d e , f o r m e d by phosphorylation with both cAMP-dependent protein kinase and glycogen synthase kinase-3, had a higher Km than the monophosphorylated form, but h a d the h i g h e s t V m a x of a n y of the t h r e e forms of this peptide, m a k i n g it o v e r a l l of the t h r e e in terms of V m a x / K m values.

the b e s t

substrate

The p e p t i d e E E E S E A S P A P S R R G S A R R , w i t h Ser in the +3 p o s i t i o n , was not an e f f e c t i v e s u b s t r a t e for c a s e i n k i n a s e II n o r was the same peptide after phosphorylation by cAMP-dependent protein k i n a s e alone. However, phosphorylation of t h e p e p t i d e b y b o t h c A M P - d e p e n d e n t p r o t e i n k i n a s e a n d GSK-3 d i d c r e a t e a s u b s t r a t e for c a s e i n II. The K m v a l u e of 39 ~ M was e x c e l l e n t b u t the V m a x was low.

Comparing

V m a x / K m values,

the

triphosphorylated

form

of the

peptide EEESEASPAPSRRGSARR was a b o u t an o r d e r of m a g n i t u d e less e f f e c t i v e t h a n any of the forms of the p e p t i d e w i t h Glu at the +3 position.

DISCUSSION The results of this investigation demonstrate that phosphoserine r e s i d u e s c a n act as s p e c i f i c i t y determinants for c a s e i n k i n a s e II. A t t a c h m e n t of a p h o s p h a t e g r o u p to a Ser in the +3 p o s i t i o n of a p e p t i d e not recognized by casein kinase II generated a peptide substrate with respectable kinetics of phosphorylation a n d a v e r y f a v o r a b l e Km of 39 ~M. The c o n t r o l p e p t i d e w i t h the - S - X - X - E - m o t i f a c t u a l l y h a d a s i g n i f i c a n t l y h i g h e r Km but its i m p r o v e d Vma x m a d e it a s u b s t a n t i a l l y better substrate b a s e d on Vmax/Km. The Km for o n e of t h e b e s t k n o w n substrates, RRRDDDSDDD, is a r o u n d 60 ~ M (14; T a b l e I) . The w o r k of P i n n a a n d c o l l e a g u e s (16,17) h a d p r o v i d e d t h e f i r s t e v i d e n c e for a r o l e of p h o s p h a t e g r o u p s in c a s e i n k i n a s e II a c t i o n b y showing that partial dephosphorylation of certain multiply p h o s p h o r y l a t e d s u b s t r a t e s i n c r e a s e d the rate of p h o s p h o r y l a t i o n by

194

VoI. 172, No. 1, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

c a s e i n k i n a s e II. L i c h f i e l d et al. (18) s h o w e d t h a t c h e m i c a l l y s y n t h e s i z e d R R R E E E S A A S ( P ) A was p h o s p h o r y l a t e d by c a s e i n k i n a s e II w i t h a Km of 0.57 mM, 10-fold g r e a t e r than for our p e p t i d e w i t h the same - S - X - X - S ( ~ ) motif. since the p e p t i d e s d i f f e r t h o u g h that p h o s p h o s e r i n e II action with low Km.

Rationalizing this d i f f e r e n c e is h a r d in other respects. It is s i g n i f i c a n t is c a p a b l e of s p e c i f y i n g c a s e i n kinase

The e f f e c t of p l a c i n g a p h o s p h o s e r i n e at the +3 p o s i t i o n is consistent with the previous work suggesting that an a c i d i c residue at t h i s location is e s s e n t i a l and demonstrates that p h o s p h o s e r i n e can p a r t l y s u b s t i t u t e Glu or Asp in this role. The n a t u r e of o t h e r r e s i d u e s s u r r o u n d i n g the p h o s p h o r y l a t i o n site is a l s o k n o w n to i n f l u e n c e casein k i n a s e II a c t i o n (10-15). In our studies, t h e c o n t r o l p e p t i d e was i m p r o v e d as a s u b s t r a t e after p h o s p h o r y l a t i o n s quite distal to the casein kinase II site, at the +7 or at t h e +7 a n d +ii p o s i t i o n s . These locations are m o r e remote than residues usually implicated in c a s e i n kinase II recognition a n d m a y not be i n v o l v e d directly in the e n z y m e substrate interactions. One p o s s i b i l i t y is that these p h o s p h a t e s m a y " s h i e l d " the p o s i t i v e c h a r g e s on the A r g r e s i d u e s at +8, +9, +13 and +14 which could c o n c e i v a b l y act as n e g a t i v e d e t e r m i n a n t s . The a b i l i t y of p h o s p h o s e r i n e to d i r e c t casein kinase II a c t i o n s u g g e s t s that some p h y s i o l o g i c a l s u b s t r a t e s of the enzyme might require prior phosphorylation. In our w o r k we have shown that the p r e s e n c e of a GSK-3 site at the +3 p o s i t i o n is c o m p a t i b l e w i t h c a s e i n k i n a s e II action. The same m a y be true of sites for o t h e r p r o t e i n k i n a s e s a l t h o u g h sites for e n z y m e s r e q u i r i n g NH2terminal basic residues would likely introduce strong negative d e t e r m i n a n t s for c a s e i n k i n a s e II. Of course, p h o s p h o r y l a t i o n at other positions could also influence c a s e i n k i n a s e II. Casein k i n a s e II joins GSK-3 and c a s e i n kinase I in b e i n g able to act as s e c o n d a r y p r o t e i n k i n a s e s in h i e r a r c h a l p h o s p h o r y l a t i o n schemes, mechanisms whereby initial phosphorylations provoke subsequent ones. C a s e i n k i n a s e II w o u l d thus be a rare e x a m p l e of p r o t e i n kinase able to serve as either a p r i m a r y or a s e c o n d a r y kinase.

ACKNOWLEDGMENTS

T h i s w o r k was s u p p o r t e d by N I H g r a n t DK27221. r e c i p i e n t of a fellowship from t r a i n i n g grant T32 HL07595.

TWH

was

REFERENCES

I. 2.

Fiol, C. J., M a h r e n h o l z , A. M., Wang, Y., Roeske, R. W., and Roach, P. J. (1987) J. Biol. Chem. 262, 14042-14048. Fiol, C. J., Haseman, J. H., Wang, Y., Roach, P. J., Roeske, R. W., K o w a l c z u k , M. a n d D e P a o l i - R o a c h , A. A. (1988) Arch. Biochem. Biophys. 267, 797-802. 195

VoI. 172, No. 1, 1990

3. 4. 5. 6.

7.

8. 9. I0 II 12 13 14 15

16. 17= 18.

19. 20. 21. 22.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fiol, C. J., Wang, A., Roeske, R. Wo, a n d Roach, P. J. (1990) J. Biol. Chem. 265, 6061-6065. Flotow, H. and Roach, P. J. (1989) J. Biol. Chem. 264, 91269128. Edelman, A. M., B l u m e n t h a l , D. K., a n d Krebs, E. G. (1987) Ann. Rev° Biochem. 56, 567-613. Tuazon, P. T. a n d Traugh, J. A. (1990) A d v a n c e s in S e c o n d Messenger and Phosphorylation Research. (Greengard, P. and Robison, G.A. eds.) in press. Pinna, L. A., Meggio, F. and Marchiori, F. (1990) Peptides and Protein Phosphorylation. (Kemp, B.Eo ed.) CRC Press. B o c a Raton, Florida. pp. 145-169. Sommercorn, J., Mulligan, J. A., Lozeman, F. J., and Krebs, E. Go (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838. Klarlund, J. K. and Czech, M. P. (1988) J. Biol. Chem. 263, 15872-15875. Pinna, L. A., Meggio, F., Marchiori, R. and Borin, JG. (1984) FEBS Lett. 171, 211-214. Meggio, F., Marchiori, F., Borin, G., Chessa, G. and Pinna, L. A. (1984) J. Biol. Chem. 259, 14576-14579. Kuenzel, E. A. and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 737-741. Marin, O., Meggio, F., Marchiori, F., Borin, G.r and Pinna, L. A. (1986) Eur. J. Biochem. 160, 239-244. Kuenzel, E. A., Mulligan, J. A., Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem. 262, 9136-9140. Marchiori, F., Meggio, F., Matin, O., Borin, G., Calderan, A., Ruzza, P. and Pinna, L. A. (1988) Biochim. Biophys. Acta 971, 332-338. Meggio, F. and Pinna, L. A. (1988) Biochem. Biophys. Acta 971, 227-231. Meggio, F., Perich, J. W., Johns, R. B., and Pinna, L. A. (1988) FEBS Lett. 237, 225-228. Litchfield, D. W.r Arendt, A., Lozeman, F. J., Krebs, E. G., Hargrave, P. A., a n d P a l c z e w s k i , K. (1990) F E B S Lett. 261, 117-120. DePaoli-Roach, A. A., Ahmad, Z., Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962. Andersson, L. and Porath, J. (1986) Anal. Biochem. 154, 2504. Bradford, M. M. (1976) Anal. Biochem. 72, 248-252. Cheng, H.-C., Kemp, B. E., P e a r s o n , R. B., Smith, A. J., Misconi, L., Patten, S. M. V., a n d Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992.

196