V o l u m e 2 9 4 , n u m b e r 3, 2 8 5 - 2 8 9 F E B S 10501 ~) 1991 F e d e r a t i o n o f E u r o p e a n B i o c h e m i c a l Societies 0 0 1 4 5 7 9 3 / 9 1 / $ 3 . 5 0
December
f991
The major endogenous bovine brain protein kinase C inhibitor is a h e a t - l a b i l e p r o t e i n Elaine MRC
Group
in S i g n a l
Transduction.
Faculty
D.
Fraser
and
Michael
P. Walsh
of Medicine, University of Calgary. Canada T2N 4N!
Received 26 September
3330 Hospital
1991; r e v i s e d v e r s i o n r e c e i v e d 25 O c t o b e r
D r i v e ,V. W . . C a l g a r y . . 4 1 h e r t a .
1991
A c r u d e c y t o s o l i c f r a c t i o n p r e p a r e d f r o m b o v i n e b r a i n c o n t a i n e d p r o t e i n k i n a s e C . a s s h o w n by i m m u n o b l o t t i n g , b u t its activity w a s tmdetectableo s u g g e s t i n g t h e p r e s e n c e o f i n t e r f e r i n g factors. P h o s p h a t a s e , A T P a s e a n d p r o t e a s e activities did n o t a c c o u n t for t~e a b s e n c e o f detectable protein k i n a s e C activity. T h e m a j o r c o n t r i b u t i n g f a c t o r w a s f o u n d t o be a heat-labile p r o t e i n w h i c h w a s s e p a r a t e d f r o m t h e kinase by i o n - e x c h a n g e c h r o m a t o g r a p h y . T h e cor~tribution to t h e total i n h i b i t o r y activity o f h e a t - s t a b l e p r o t e i n s w a s relatively m i n o r , s u g g e s t i n g t h a t they :nay n o t f u n c t i o n p h y s i o l o g i c a l l y a s p r o t e i n k i n a s e C inhtbitors. P r o t e i n k i n a s e C inhibitor; B o v i n e b r a i n
1. I N T R O D U C T I O N Protein kinase C (PKC) activity is often underestimated or even undetected in crude tissue extracts such as the cytosolic fraction obtained by high-speed centrifugation of homogenates prepared in the presence of divalent cation chelators (e.g. [1,2]). Fractionation of such extracts by ion-exchange or hydrophobic-interaclion chromatography is c o m m o n l y used to separate the kinase from endogenous factors which mask its activity in the crude extract, thereby allowing its activity to be measured (e.g. [1-3]). Such factors may include protein phosphatc.ses, ATPases, proteases and heat-stable and heat-labile protein inhibitors. The purpose of this study was to evaluate the relative importance of these factors in the inability to detect PKC activity in a crude cytosolic fracdon derived from bovine brain. Quantitatively, the most important factor was identified as a heat-labile protein inhibitor. It is proposed that this protein functions to restrict PgC-catalyzed phosphorylations to membrane-, organellean0 cytoskeletal-associatcd protein substrates by inhibiting cytosolic forms of PKC, which is known to phosphorylate a wide range of proteins i n v i t r o [4].
A b b r e v i a t i o n s : E G T A . e t h y l e n e b i s ( o x y e t h y l e n e n i t r i l o ) t e t r a a c e t i c acid; P K C . p r o t e i n kinas¢ C; S D S - P A G E . -sodium d o d e e y l s u l f a t e - p o t y a c r y l a m i d e gel e l e c t r o p h o r e s i s C o r r e s p o n d e n c e a d d r e s s : M . P . W a l s h , M R C G r o u p in Signal "gransd u c t i o n , F a c u l t y o f M e d i c i n e , U n i v e r s i t y o f C a l g a r y , 3330 H o s p i t a l D r i v e N . W . , C a l g a r y , A l b e r t a , C a n a d a T21q 4 N I . F a x : ( I ) (403) 2700737.
PudTlished b y E l s e v i e r S c i e n c e Publisher~ B. IV.
2.
MATERIALS
AND
METHODS
2. I. M a t e r i a l s [¥-~2P]ATP (25 C i / m m o l a n d 4500 CLtmmol) w a s p u r c h a s e d f r o m I C N B i o m e d i c a l s (St. L a u r e n t . Q u e b e c . C a n a d a ) . A m o n o ~ l o n a i antib o d y r e c o g n i s i n g b o t h ,~ a n d fl i s o e n z y m e s o f P K C w a s p u r c h a s e d f r o m A m e r s h a m C o r p . (Oakville, O n t a r i o . C a n a d a ) a n d m o n o c l o n a l a n t i b o d i e s specific for t h e 0t,fl a n d ¥ i s o e n z y m e s o f P K C were p u r c h a s ed f r o m U p s t a t e B i o t o c h n o l o g y , Inc. ( L a k e Placid, NY). C a l f t h y m u s lysine-righ h i s t o n e f r a c t i o n Ill-S, E D T A , E G T A , T r i t o n X-100 a n d p r e s t a i n e d MT m a r k e r s f o r e l e c t r o p h o r e s i s were p u r c h a s e d f r o m S i g m a C h e m i c a l C o . (St. L o u i s . M e ) , D E A E - S c p h a c e l f r o m P h a r m a c i a ( B a i t d ' U r f e , Q u e b e c , C a n a d a ) , L-~.z-phosphatidyi-L-serine ( b e e f b r a i n ) a n d 1,2-diol¢in f r o m S e r d a r y R e s e a r c h L a b o r a t o r i e s ( L o n d o n , O n t a r i o , Canada), and M, marker proteins and electrophoresis reagents from B i o - R a d L a b o r a t o r i e s ( M i s s i s s a u g a , O n t a r i o , C a n a d a ) . G e n e r a l labor a t o r y r e a g e n t s u s e d were a n a l y t i c a l g r a d e o r b e t t e r a n d were p u r c h a sed f r o m F i s h e r Scientific ( C a l g a r y , Alberta, C a n a d a ) . 2.2. P r o t e i n puri)q~ ~tion R a t b r a i n P K C was purified b y a m o d i f i c a t i o n o f the p r o c e d u r e o f W o l f et al. [5], u s i n g D E A E - S e p h a c ¢ ! a n i o n - e x c h a n g e c h r o m a t o g r a phy and phenyl-Scpharose hydrophobic-interaction chromatography. T h e isolated p r o t e i n exhibited a m o l e c u l a r m a s s o f 80 000 D a o n S D S - P A G E a n d w a s t~
December
f991
The major endogenous bovine brain protein kinase C inhibitor is a h e a t - l a b i l e p r o t e i n Elaine MRC
Group
in S i g n a l
Transduction.
Faculty
D.
Fraser
and
Michael
P. Walsh
of Medicine, University of Calgary. Canada T2N 4N!
Received 26 September
3330 Hospital
1991; r e v i s e d v e r s i o n r e c e i v e d 25 O c t o b e r
D r i v e ,V. W . . C a l g a r y . . 4 1 h e r t a .
1991
A c r u d e c y t o s o l i c f r a c t i o n p r e p a r e d f r o m b o v i n e b r a i n c o n t a i n e d p r o t e i n k i n a s e C . a s s h o w n by i m m u n o b l o t t i n g , b u t its activity w a s tmdetectableo s u g g e s t i n g t h e p r e s e n c e o f i n t e r f e r i n g factors. P h o s p h a t a s e , A T P a s e a n d p r o t e a s e activities did n o t a c c o u n t for t~e a b s e n c e o f detectable protein k i n a s e C activity. T h e m a j o r c o n t r i b u t i n g f a c t o r w a s f o u n d t o be a heat-labile p r o t e i n w h i c h w a s s e p a r a t e d f r o m t h e kinase by i o n - e x c h a n g e c h r o m a t o g r a p h y . T h e cor~tribution to t h e total i n h i b i t o r y activity o f h e a t - s t a b l e p r o t e i n s w a s relatively m i n o r , s u g g e s t i n g t h a t they :nay n o t f u n c t i o n p h y s i o l o g i c a l l y a s p r o t e i n k i n a s e C inhtbitors. P r o t e i n k i n a s e C inhibitor; B o v i n e b r a i n
1. I N T R O D U C T I O N Protein kinase C (PKC) activity is often underestimated or even undetected in crude tissue extracts such as the cytosolic fraction obtained by high-speed centrifugation of homogenates prepared in the presence of divalent cation chelators (e.g. [1,2]). Fractionation of such extracts by ion-exchange or hydrophobic-interaclion chromatography is c o m m o n l y used to separate the kinase from endogenous factors which mask its activity in the crude extract, thereby allowing its activity to be measured (e.g. [1-3]). Such factors may include protein phosphatc.ses, ATPases, proteases and heat-stable and heat-labile protein inhibitors. The purpose of this study was to evaluate the relative importance of these factors in the inability to detect PKC activity in a crude cytosolic fracdon derived from bovine brain. Quantitatively, the most important factor was identified as a heat-labile protein inhibitor. It is proposed that this protein functions to restrict PgC-catalyzed phosphorylations to membrane-, organellean0 cytoskeletal-associatcd protein substrates by inhibiting cytosolic forms of PKC, which is known to phosphorylate a wide range of proteins i n v i t r o [4].
A b b r e v i a t i o n s : E G T A . e t h y l e n e b i s ( o x y e t h y l e n e n i t r i l o ) t e t r a a c e t i c acid; P K C . p r o t e i n kinas¢ C; S D S - P A G E . -sodium d o d e e y l s u l f a t e - p o t y a c r y l a m i d e gel e l e c t r o p h o r e s i s C o r r e s p o n d e n c e a d d r e s s : M . P . W a l s h , M R C G r o u p in Signal "gransd u c t i o n , F a c u l t y o f M e d i c i n e , U n i v e r s i t y o f C a l g a r y , 3330 H o s p i t a l D r i v e N . W . , C a l g a r y , A l b e r t a , C a n a d a T21q 4 N I . F a x : ( I ) (403) 2700737.
PudTlished b y E l s e v i e r S c i e n c e Publisher~ B. IV.
2.
MATERIALS
AND
METHODS
2. I. M a t e r i a l s [¥-~2P]ATP (25 C i / m m o l a n d 4500 CLtmmol) w a s p u r c h a s e d f r o m I C N B i o m e d i c a l s (St. L a u r e n t . Q u e b e c . C a n a d a ) . A m o n o ~ l o n a i antib o d y r e c o g n i s i n g b o t h ,~ a n d fl i s o e n z y m e s o f P K C w a s p u r c h a s e d f r o m A m e r s h a m C o r p . (Oakville, O n t a r i o . C a n a d a ) a n d m o n o c l o n a l a n t i b o d i e s specific for t h e 0t,fl a n d ¥ i s o e n z y m e s o f P K C were p u r c h a s ed f r o m U p s t a t e B i o t o c h n o l o g y , Inc. ( L a k e Placid, NY). C a l f t h y m u s lysine-righ h i s t o n e f r a c t i o n Ill-S, E D T A , E G T A , T r i t o n X-100 a n d p r e s t a i n e d MT m a r k e r s f o r e l e c t r o p h o r e s i s were p u r c h a s e d f r o m S i g m a C h e m i c a l C o . (St. L o u i s . M e ) , D E A E - S c p h a c e l f r o m P h a r m a c i a ( B a i t d ' U r f e , Q u e b e c , C a n a d a ) , L-~.z-phosphatidyi-L-serine ( b e e f b r a i n ) a n d 1,2-diol¢in f r o m S e r d a r y R e s e a r c h L a b o r a t o r i e s ( L o n d o n , O n t a r i o , Canada), and M, marker proteins and electrophoresis reagents from B i o - R a d L a b o r a t o r i e s ( M i s s i s s a u g a , O n t a r i o , C a n a d a ) . G e n e r a l labor a t o r y r e a g e n t s u s e d were a n a l y t i c a l g r a d e o r b e t t e r a n d were p u r c h a sed f r o m F i s h e r Scientific ( C a l g a r y , Alberta, C a n a d a ) . 2.2. P r o t e i n puri)q~ ~tion R a t b r a i n P K C was purified b y a m o d i f i c a t i o n o f the p r o c e d u r e o f W o l f et al. [5], u s i n g D E A E - S e p h a c ¢ ! a n i o n - e x c h a n g e c h r o m a t o g r a phy and phenyl-Scpharose hydrophobic-interaction chromatography. T h e isolated p r o t e i n exhibited a m o l e c u l a r m a s s o f 80 000 D a o n S D S - P A G E a n d w a s t~
