Protein kinase in the hair follicles of the human scalp and beard

Biochimica et Biophysica Acta, 315 (1973) 333-342 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA

66944

P R O T E I N K I N A S E IN T H E H A I R F O L L I C L E S OF T H E HUMAN SCALP AND B E A R D

K U N I H 1 K O Y O S H I K A W A * AND K E N J I A D A C H I *

Oregon Regional Primate Research Center, Beaverton, Oreg. 97005 (U.S.A.) (Received D e c e m b e r 27th, 1972)

SUMMARY

Protein kin ase (ATP: protein phosphotransferase, EC 2.7- I. 37) which phosphor ylates histone but not significant amounts of protamine or casein, was demonstrated in follicular tissues from the human scalp and beard. I t was activated up to 6 times by 5" lO-7 M cyclic AMP and up to 5 times b y the same concentration of cyclic IMP. Cyclic AMP activated the enzyme by dissociating the cyclic AMP receptor subunit from the catalytic subunit. In hair follicles from both scalp and beard the Km values for cyclic AMP and ATP were about 2.3" IO-8 M, and 1. 4. IO-5 M, respectively, and the optimal p H was 6. 7. The biochemical characteristics for the enzymes of the scalp and beard hair follicles did not differ appreciably.

INTRODUCTION

Increasing numbers of studies show that cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) phosphorylates divergent kinds of proteins including histone, protamine, casein and some membrane1, ~ or ribosomal protein ~ and concomitantly activates or inactivates phosphorylase b kinasO, glycogen synthetase 14 and lipaseS, ~ in various mammalian tissues. The biological importance of protein kinase in cell function appears to depend upon its activation b y the cyclic AMP system 5-12. In our focal studies on the pathogenesis of male pattern baldness, we have shown that androgen inhibits adenyl cyclase activity in the human scalp hair follicle la. This finding led us to examine the possibility that androgenic action proceeds via the adenyl cyclase-protein kinase system, even though steroid hormones generally exert their action via a specific receptor protein system. Since androgens clinically cause biphasic results on hair follicles, i.e., a dystrophic effect on scalp hair follicles and a stimulatory effect on beard follicles, we have tried to characterize and compare the protein kinases of these different follicles. * P r e s e n t a d d r e s s : T h e D e r m a t o l o g y Service of t h e M i a m i V e t e r a n s A d m i n i s t r a t i o n Hospit a l a n d U n i v e r s i t y of M i a m i School of Medicine, Miami, Fla. 33125, U.S.A.

334

K. Y O S H I K A W A , K. A D A C H I

MATERIALS AND METHODS

Cyclic nucleotides, calf thymus histone, salmon sperm protamine and a-casein were obtained from Sigma; cyclic Jail]AMP was purchased from Schwarz; and [~,-32P]ATP is the product of New England Nuclear. Hair follicles were obtained from the scalps and beards of three Japanese adult males. Plucked hair follicles were immediately homogenized in a glass homogenizer for 4 Inin in 20 mM potassium phosphate buffer (pH 7.0) containing 2 mM EDTA ((ethylenedinitrilo)-tetraacetic acid) with a ratio of 9 ° scalp follicles/5oo/*1 and 20 beard follicles/25o/*I. After tile homogenate had been centrifuged at IO ooo × g for 3 ° min, the clear supernatant solution was preincubated with or without 5"Io 7 M cyclic AMP at 3o °C for 5 rain. An aliquot (2oo/*1) was layered on the top of 4.8 ml of 5-20% sucrose density gradient solution containing 2o0 /*g/ml of histone or albumin, then centrifuged at 47 ooo rev./min for 17 h in a Beckman SW 5o rotor. Without these proteins in the sucrose density gradient solution, the enzyme lost most of its activity during centrifugation. All procedures were carried out at o-3 °C. Centrifuged solutions were divided into 2o fractions, and protein kinase and the cyclic AMP binding activity were assayed in each fraction. Protein kinase was assayed by the method of Maeno et al. 14 with a modification in the washing step. The standard reaction system contained 5 /,moles of sodium acetate buffer (pH 6.o), o.I mg of calf thymus histone, i/*mole of magnesium acetate, i/*mole of NaF, 0.2/,mole of theophyline, 0.03/*mole of ethyleneglycol bis-(fi-aminoethyl ether)-N,N'-tetraacetic acid, 0.05 nmole of cyclic AMP, 0.5 nmole of [V-32P]ATP (product that had a specific activity of 13-16 Ci/mmole was diluted about 30 times with nonradioactive ATP before use), and enzymes for a total volume of IOO/*1. Reaction was started by adding ATP after 5 rain preincubation at 30 °C, and the entire reaction mixture was incubated for 5 min at tile same temperature. Then 1.5 nil of lO% trichloroacetic acid was added to stop the reaction, tile aliquot was passed through a millipore filter (HA 0.45 /*m), the trapped precipitate was washed with 30 ml of 5% trichloroacetic acid, and radioactivity on the millipore filter was counted in IO ml of toluene-Omnifluor (New England Nuclear)-Bio-solv (Beckman) counting medium (800/*1 of Bio-solv-BBS3 were dissolved in IO ml of toluene containing 4 g/1 of Omnifluor) in the Packard Tricarb liquid scintillation spectrometer, i unit of enzyme activity was expressed as pmoles P incorporated into precipitated protein/rain. Cyclic AMP binding activity was assayed by the method of Gilman 15 in a total volume of 50/*1. Namely, reaction mixture containing 2.5/,moles of sodium acetate buffer (pH 4.o), 0.5 pmole of cyclic [all]AMP (spec. act. 2o.8 Ci/mmole), protein kinase inhibitor and enzyme fraction was incubated at o °C for 60 rain. At the end of incubation period, mixture was diluted with 0. 5 ml of chilled 20 mM potassium phosphate buffer (pH 6.o), and passed through Millipore filter with a o.45-/*m pore size. The filter was washed with 20 ml of 20 mM potassium phosphate buffer (pH 6.0) and its radioactivity counted in the same way as for the protein kinase assay. Protein kinase inhibitor prepared from guinea pig skeletal muscle gave, at the optimal concentration, a more than 3-fold increase of binding as compared to assay in its absence. Under the condition described above, the radioactivity (the binding) of cyclic [all]AMP is linear to the concentration of the

PROTEIN KINASE OF HAIR FOLLICLES

335

binding protein in the sample analyzed. Protein was measured by the method of Lowry et al. 16 with bovine serum albumin as a standard. RESULTS

Sucrose density gradient centrifugation pattern of the enzyme When samples (homogenates of scalp hair follicles) were Centrifuged after preincubation without cyclic AMP, the curve of protein kinase activity showed a single peak (at 7 S) that was greatly activated by 5" lO-7 M cyclic AMP in the assay system. The cyclic AMP binding activity was accompanied to this peak. Centrifuging the samples preincubated with 5" lO-7 M cyclic AMP showed a different sedimentation .'.~

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Fig. I. Sucrose density gradient centrifugation p a t t e r n s of p r o t e i n kinase a n d cyclic AMP binding activity in follicles f r o m scalp hair. Samples were i n c u b a t e d at 3 ° °C for 5 min w i t h (B) or w i t h o u t (A) 5" io ~ M cyclic AMP before centrifugation. 40/,1 of each fraction were used for p r o t e i n kinase and cyclic AMP binding assay. P r o t e i n kinase a c t i v i t y was assayed w i t h 5' IO-~ M cyclic AMP ( 0 - - 0 ) or w i t h o u t it ( O - - - - - - © ) at 3 ° °C for io min, o m i t t i n g preincubation, x • • • × , cyclic AMP binding activity. Sucrose did n o t significantly affect either a s s a y at the concentrations used. P r e i n c u b a t i o n w i t h o u t cyclic AMP before centrifugation is necessary to diminish endogenous cyclic AMP as well as to standardize the conditions. W h e n t h e crude h o m o g e n a t e was centrifuged w i t h o u t preincubation, we o b t a i n e d t w o a l m o s t e q u a l p e a k s of cyclic AMP binding activity, which were indicative of partial dissociation of the subunits.

336

K. Y O S H I K A W A , K. A D A C H I

profile of protein kinase activity (at 4 S) and cyclic AMP binding activity (5-6 S) with the latter activity peak dissociated from the protein kinase activity peak. This protein kinase shifted to 4 S was not further activated by the presence of 5" lO-7 M cyclic AMP (Figs IA and IB). Since significant concentrations of free cyclic AMP were not present in this fraction we concluded that the enzyme had been converted into a cyclic AMP-independent form by preincubation with cyclic AMP. These data are consistent with the proposed mechanism for protein kinase activation; that is, cyclic AMP binds to a receptor subunit to dissociate it from a catalytic subunit which is active in its free subunit form. Such an activation mechanism has been proposed for protein kinase from various tissues such as rabbit reticulocyteT,17, bovine adrenal cortexS, ~s, rabbit skeletal muscle 9, bovine heart muscle~% bovine brain ~°, rat liver 2°, bovine pineal gland 2~, bovine anterior pituitary gland 2", and rabbit red blood celP 2. Fig. 2 shows the results of an ultracentrifugation study of beard hair follicles. Enzymes from scalp hair follicles and beard follicles did not differ qualitatively in either the sucrose density gradient centrifugation profile or the cyclic AMP activation mechanisms of the protein kinase, ttowever, the follicles I0'

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337

PROTEIN KINASE OF HAIR FOLLICLES TABLE I PROTEIN

KINASE

ACTIVITY

OF CRUDE

EXTRACTS

AND

PARTIALLY

PURIFIED

ENZYMES

Gross specific activities of partially purified e n z y m e were calculated on the basis of d a t a on e n z y m e activity and protein c o n c e n t r a t i o n o b t a i n e d in s e p a r a t e samples, i.e., to m e a s u r e protein c o n t e n t in each fraction, the centrifugation was p e r f o r m e d w i t h the sucrose m e d i u m w i t h o u t added protein. A p p a r e n t l y the low purification factor is due to the partial loss of the e n z y m e activity (luring centrifugation (the recovery of total enzyme activity is a b o u t 3 ° %). Complete inactivation is noted w h e n the h o m o g e n a t e is centrifuged in the sucrose m e d i u m w i t h o u t histone or bovine s e r u m albumin,

Activity/ follicle (pmoles/ follicle per rain)

Specific activity (pmoles/ mg per min)

Purification

Scalp hair follicle

Crude e x t r a c t Purified

1.32 --

233 392

I 1.7

Beard hair follicle

Crude e x t r a c t Purified

3.37 --

225 455

i 2.o

may differ quantitatively in the relative amounts of cyclic AMP receptor and catalytic subunit of protein kinase. Comparing Figs IB and 2B, we see that the cyclic AMPbinding activity peak of the beard follicle is relatively higher than that of the scalp. The cyclic AMP-dependent protein kinase fraction (Figs IA and 2A) was collected, kept frozen at --20 °C, and used as a partially purified enzyme in the following kinetic studies. Table I summarizes the enzyme activities of crude extracts and partially purified samples. Further purification was not possible because of limited amounts of tissues.

Activation of protein kinase by cyclic A M P The activity of the partially purified enzyme was assayed with various concentrations of cyclic AMP. It was activated with increasing concentrations of cyclic AMP reaching its maximum activation (3-4 times the activity without cyclic AMP) at a concentration of about 5" lO-7 M. Further increase in cyclic AMP concentration 4

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10-6 5x10-6 10-5 10-4 IO-I 10-8 10-7 cAMP Concentration (M) Fig. 3. Cyclic AMP activiation of p r o t e i n kinase in hair follicles. 3° ~1 of partially purified enzyme was used for each assay. Relative enzymic activities were calculated on the basis of activity w i t h o u t cyclic AMP. E a c h p o i n t represents the average value o b t a i n e d b y 2 - 4 different experiments. The scalp ( © - - - - - - O ) and beard ( 0 - - - 0 ) .

338

K. Y O S H I K A W A , K. A D A C H I

P incorporated (p mole)

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F i g . 4. Km f o r c y c l i c A M P . D a t a w e r e c o r r e c t e d b y t h e v a l u e f o u n d w h e n n o c y c l i c A M P w a s added to the reaction mixture. Each point represents the mean of duplicate assays. Protein kinase of the hair follicles from scalp (O---C)) and beard (O---O).

i n h i b i t e d t h e e n z y m e a c t i v i t y (Fig. 3). P r o t e i n kinase from b e a r d hair follicles a p p e a r s to be a c t i v a t e d more t h a n t h a t from scalp hair follicles. However, the difference is p r o b a b l y n o t due to an essential q u a l i t a t i v e difference between t h e enzymes from the two tissues as discussed below. Km value for cyclic A M P is a b o u t 2.3" lO -8 M for t h e e n z y m e s from b o t h tissues (Fig. 4).

Effects of other cyclic nucleotides The results are s u m m a r i z e d in Table II. E a c h cyclic nucleotide was t e s t e d at c o n c e n t r a t i o n s of 5" lO-7 M a n d lO -4 M in the r e a c t i o n m i x t u r e . The e n z y m e s from scalp a n d b e a r d hair follicles r e s p o n d e d in the same m a n n e r a g a i n s t these cyclic nucleotide compounds. Cyclic A M P is most effective at the c o n c e n t r a t i o n of 5" i o 7 M a n d less effective a t lO -4 M. Cyclic I M P is n e a r l y 9 0 % as effective as cyclic A M P at 5" Io-V M a n d is more active at lO -4 M t h a n at lO -7 M. Cyclic GMP, cyclic U M P

TABLE

II

A C T I V A T I O N O1~" P R O T E I N

KINASE BY VARIOUS CYCLIC NUCLEOTIDES

Partially purified enzyme was prepared by centrifugation in sucrose density gradient solution containing bovine serum albumin instead of histone. Activity was expressed as pmoles P incorp o r a t e d i n t o p r o t e i n / 5 r a i n . All c y c l i c n u c l e o t i d e s o l u t i o n w a s p r e p a r e d f r e s h .

Cyclic nucleotides

Scalp hair follicle

Beard hair follicle

Activity

A ctivation

Activity

.4 ctivation

o Cyclic AMP 5'I° 7M I • lO -4 M C y c l i c I M P 5" IO-~ M I • lO -4 M C y c l i c G M P 5" IO 7 M I • lO -4 M C y c l i c C M P 5" I ° - T M

0.27 1.72 1.46 1.5t 1.81 0-75 1.72 o.61

I 6.27 5.32 5.52 6.61 2.74 6.27 2.21

0.44 2.28 I.,53 2.04 2.12 0.83 2.15 0.60

I 5.21 3-49 4.65 4.85 1.89 4.91 1.37

I • 10 -4 M

1.91 0.58

6.95 2.11

2.21

5.04

1.53

5.56

0.60 2.22

1.37 5.07

C y c l i c U M P 5" l O - 7 M I • 10 -4 M

339

PROTEIN KINASE OF HAIR FOLLICLES

and cyclic CMP are much less effective at the concentration of 5" lO-7 M, although they fully activate the enzyme at lO -4 M. The rates of activation in this experiment were much higher than those in the previous experiment (Fig. 3) because albumin was used instead of histone as a stabilizer in the sucrose density gradient solution. As reported earlier 1°, histone added to the sucrose density gradient solution causes partial dissociation of the receptor subunit from the catalytic subunit at the preincubation step of the enzyme assay and possibly during other procedures. This partial dissociation activates the enzyme without cyclic AMP; thus the activation rates by cyclic AMP are lower with the protein kinase prepared in the histone medium. Activation by protamine has also been reported recently 2~. Effect of A T P concentration on the enzyme activity The change of ATP concentration in the assay system caused the same response in the protein kinase from both scalp and beard hair follicles. The enzyme activity was increased in proportion to the increase in ATP concentration. The Km value for ATP is about 1. 4- lO -5 M for the enzymes from both tissues (Fig. 5).

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ATP concentration (M) Fig. 5. Effect o f A T P c o n c e n t r a t i o n on t h e p r o t e i n kinase activity. 20/,1 of partially purified e n z y m e from scalp hair follicle ( O - - - - - O ) and io/~1 of t h a t from b e a r d hair follicle ( Q - - O ) were assayed.

Proteins as a phosphate accepter Calf thymus histone, salmon sperm protamine and a-casein were tested as phosphate accepters in the protein kinase reaction (Table Histone was the best substrate for the enzymes from both tissues. Except for a slight phosphorylation of casein b y the crude extract of hair follicles, little or no phosphorylation was noted when protamine or casein was used as a substrate.

III).

34 °

K. YOSHIKAWA, K. ADACHI

TABLE lI[ PHOSPHORYLATION OF VARIOUS PROTEINS BY PROTEIN KINASE The e n z y m e p r e p a r a t i o n was the same as t h a t in Table lI. Assays were carried o u t for every s u b s t r a t e p r o t e i n (o.i m g / i o o / , l of reaction system) in duplicate with duplicate blanks. The b l a n k t u b e s were i n c u b a t e d w i t h o u t s u b s t r a t e p r o t e i n which was added after the incubation. E n d o g e n o u s soluble p r o t e i n p h o s p h o r y l a t i o n , in the case of crude extract, was not noted in a n y significant a m o u n t at this p r o t e i n c o n c e n t r a t i o n (scalp hair follicle (o.8/~g), beard hair follicle (I.I pg) in i o o # 1 reaction system). Protein p h o s p h o r y l a t i o n was expressed as pmoles P incorp o r a t e d / m g e n z y m e protein per min . Protein

Protamine a-Casein Histone

S c a l p hair ]bllicle

B e a r d hair follicle

Crl*de exh, acl P u r i f i e d

Crude extract P u r i f i e d

o o 491

o

21 231

o 36 274

28 o lO5O

4

0)

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I

///

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g

o'-'°-.o,

o_. N

g

I-Acetate--I

~

~ Tr is

~pH I

Fig. 6. Effect of p H on p r o t e i n kinase activity. 3°/~l of aliquots of the cyclic A M P - d e p e n d e n t p r o t e i n kinase fraction of scalp hair follicle ( 0 - - - - - - @ ) and beard hair follicle ( e - - O ) were assayed. E a c h p o i n t represents the m e a n value of duplicate assay.

The effect of pH The m a x i m u m e n z y m e a c t i v i t y was o b t a i n e d at a p H between 6.5 a n d 7.0, a n d the e n z y m e was r e l a t i v e l y sensitive to pH. I t was more active in a c e t a t e buffer t h a n in Tris buffer (Fig. 6). O p t i m a l p H a p p e a r s to be the same for the enzymes from b o t h tissues. DISCUSSION

A c t i v e p r o t e i n kinase was n o t e d in hair follicles from the h u m a n scalp a n d beard. Crude e x t r a c t from a single hair follicle is sufficient for this a s s a y s y s t e m , a n d

P R O T E I N K I N A S E OF H A I R F O L L I C L E S

341

easily plucked hair follicles provide fresh and convenient tissue samples when needed. Although m a n y studies on this enzyme have shown histone rather than protamine or casein to be the preferred nonspecific phosphate acceptor, the opposite results have been found with enzymes from the bovine heart 19 and pineal gland21; thus, the substrate specificity varies with different so-called protein kinases. Histone is a good substrate for enzyme from human hair follicle while protamine is not significantly phosphorylated b y either crude extract or partially purified enzyme. The slight phosphorylation of casein by crude extract seems to have been due to another enzyme such as phosvitin kinase. This kinase which appears to be in human epidermis phosphorylates casein as well as phosvitin but little histone 24. The activation mechanism by cyclic AMP for follicular protein kinase is considered to l~e tl:e same as that reported for kinases from various mammalian tissues. Although only 2 distinct peaks of protein kinase activity were noted in our sucrose density gradient centrifugation experiments, the small shoulder peak between cyclic AMP-dependent and independent protein kinase activity suggests either another form of this enzymeT,18,2°,2a,26, or a dimerous component as shown in bovine brain protein kinase 1° where two main activity peaks are interpreted as tetramers (composed of 2 catalytic and 2 receptor subunits) and monomers of catalytic subunits, respectively. The preliminary values for the cyclic AMP content of scalp and beard hair follicles obtained with the method of Gillman ~5 were o.o 4 and 0.16 pmole per follicle, respectively. I f we assume that the volumes of these hair follicles were o.5 #1 and 2 k,l, the gross concentration of cyclic AMP in these tissues was o.8. I0 -7 M, which is enough to regulate the activity of protein kinase in hair follicles in vivo (the Km and maximal activation concentrations are 2.3" I0 -s M and 5" 10-7 M, respectively). Thus all the data, including the Km value for ATP, p H dependency, and nucleotide specificity show essentially the same characteristics for protein kinase from the scalp and beard hair follicles. The protein kinase of human hair follicles closely resembles that of the bovine anterior pituitary gland 22 and rabbit skeletal muscle 25 and also, with a few differences, is similar to the protein kinases of rat liver, rat fat pad, and bovine brain 27-29. The data suggest the presence of similar protein kinases in a wide variety of mammalian tissue. Protein kinase of human skin, recently reported by K u m a r et a/. a°,3~ also has essentially the same characteristics as hair follicle protein kinase except that the optimal p H is in the alkaline range. Adenyl cyclase has previously been shown in human hair follicle13; in this report we have shown the presence of cyclic AMP and cyclic AMP-dependent protein kinase. Together these data appear to substantiate the presence of the adenyl cyclase-cyclic AMP-protein kinase system in tissue from human hair follicles. The different effects of androgenic hormone on the hair follicles of the scalp and beard must be explained by some other differences than those in the characters of the protein kinases themselves.

ACKNOWLEDGMENTS Publication No. 659 of the Oregon Regional Primate Research Center supported in part by Public Health Service, National Institutes of Health Grants

342

g. YOSHIKAWA, K. ADACHI

RRooI63 of the Animal Resources Branch, Division of Research Resources, and AM 08445 of the National Institute of Arthritis and Metabolic Diseases.

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