The stimulation by adrenocorticotropin of the phosphorylation of adrenal inhibitor-1: a possible role in steroidogenesis

Molecular and Ceil&r Endocrinology, Elsevier Scientific Publishers Ireland.

60 (1988) 61-69 Ltd.

61

MCE 01940

The stimulation by adrenocorticotropin of the phosphorylation inhibitor-l: a possible role in steroidogenesis Radha Depumnent of Biochemistry,

B. Iyer,

Mount Sinai

Abha Chauhan School of Medicineand The

Adrenal

cortex;

Phosphoprotein

B. Koritz

Graduate School, The City Uniuersrty of New York, New York, NY 10029, U.S.A.

(Received

Key words:

and Seymour

of adrenal

25 April 1988; accepted

phosphatase;

Inhibitor-l;

1 July 1988)

Steroidogenesis

Summary

Adrenocorticotropin (ACTH) acts via protein kinase A and the putative phosphorylation of a regulatory protein(s). We have examined a role in this process for inhibitor-l which, following phospho~lation by protein kinase A, inhibits a phosphoprotein phosphatase activity. In the tissues we have examined inhibitor-l was found primarily in the cytosol (90%) with the rest in the mitochondrial pellet. The highest concentration was in the adrenal cortex. Using adrenal cortex slices, the stimulation of steroidogenesis by ACTH and dibutyryl CAMP is paralleled by a corresponding increase in the phosphorylation of inhibitor-l and this is not affected by inhibitors of protein synthesis which inhibit the steroidogenic response. The increase in the phospho~lation of in~bitor-1 occurs in the cytosol, while that in the mit~hond~al pellet is not affected. Exogenous phospho~lated inhibitor-l, however, was found to inhibit phosphoprotein phosphatase activity in the mitochondrial pellet. The results suggest that the ACTH-induced increase in phosphorylated inhibitor-l in the cytosol can affect susceptible phosphoprotein phosphatase activity both in the cytosol and the mitochondrial pellet and, hence, the level of phosphorylation of regulatory protein(s) involved in steroidogenesis.

Introduction The stimulation by adrenocorticotropin (ACTH) of steroidogenesis in the adrenal cortex is mediated by cAMP (Haynes et al., 1959: Grahame-Smith et al., 1967; Hayashi et al., 1979; Sala et al., 1979) and affects the formation of pregnenolone from cholesterol (Stone and Hechter, 1954; Karaboyas and Koritz, 1965) in the ~tochond~al (Halkerston et al., 1961). It has been shown that ACTH and CAMP increase

Address for correspondence: Seymour B. Koritz, Department of Biochemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, U.S.A. 0303-7207/88/$03.50

0 1988 Elsevier Scientific

Publishers

Ireland.

adrenal protein kinase activity (Gill and Garren, 1970; Richardson and Schulster, 1973) and that mutant adrenal cells with reduced CAMP-dependent protein kinase activity had reduced steroidogenie responses to ACTH or CAMP (Rae et al., 1979). Both ACTH and CAMP bring about changes in the phosphorylation of a number of ribosomal, cytosolic and mitochondrial proteins (Murakama and I&ii, 1973; Roos, 1973; Beckett and Boyd, 1977; Podesta et al., 1979; Korascil and Gallant, 1980; Pon et al., 1986) and mitochondrial from ACTH-treated adrenals show increased in vitro phosphorylation of a protein (Bhargava et al., 1978). The level of phospho~lation of a protein will depend on the rates of phospho~lation and deLtd

62

phosphorylation and, in a number of cases, dephosphorylation is critical (Ingebritsen and Cohen, 1983). Phosphoprotein phosphatase activity is present in all subcellular fractions in the rat adrenal (Ullman and Perlman, 1975) and partial purification and characterization of some of these activities from beef adrenal cortex have been out (Merlevede and Riley, 1966; Ullman and Perlman, 1975; Li, 1979). Little, however, has been done on the possible role of the phosphoprotein phosphatase in the action of ACTH on steroidogenesis. Podesta et al. (1979) have reported that ACTH causes a rapid stimulation of the phosphorylation of an adrenal cytoplasmic protein which corresponded to the increase in the synthesis of corticosterone. The turnover of phosphate in this protein, as well as in other proteins not affected by ACTH, was rapid and the data are consistent with a regulatory role of a phosphatase. Korosil and Gallant (1980) have found that ACTH enhanced both phosphorylation and dephosphorylation of adrenal proteins. These changes preceded corticosterone stimulation, depended on ACTH concentration and are brought about also by CAMP. The phosphoprotein phosphatase activity of tissue extracts is largely due to four enzymes with broad substrate specificities, a phosphoprotein phosphatase 1 and three types of phosphoprotein phosphatase 2 (Ingebritsen and Cohen, 1983). In order that a phosphatase have a regulatory role in the determination of the level of phosphorylation of the putative regulatory protein(s) involved in the action of ACTH on steroidogenesis, the activity of the phosphatase must respond to ACTH, i.e., CAMP. Such a nexus can take place via a heat-stable protein, inhibitor-l, which will inhibit phosphoprotein phosphatase only after phosphorylation by CAMP-dependent protein kinase (Huang and Glinsmann, 1975, 1976) so that an effect of this phosphatase on a regulatory protein phosphorylated also by a CAMP-dependent protein kinase would be diminished and the extent of phosphorylation of the regulatory protein increased. Inhibitor-l is present in a variety of tissues including the adrenal cortex (Huang et al., 1977). A possible involvement of inhibitor-l in CAMP-dependent hormone action has been indicated by an increase, in skeletal muscle, of its level

of phosphorylation following the administration of adrenaline (Tao et al., 1978; Foulkas and Cohen, 1979) and in cardiac tissue by isoproterenol (Iyer et al., 1988). Using beef adrenal cortex slices, we have found that both ACTH and N6,2-O-dibutyryl CAMP (dibutyryl CAMP) increase the level of phosphorylation of inhibitor-l and that this increase correlated with the stimulation of steroidogenesis. This, and additional data, suggest that inhibitor-l, by affecting the level of phosphorylation of regulatory phosphoprotein(s), may be involved in the stimulation of adrenal steroidogenesis by ACTH. Materials and methods Materials

ACTH, dibutyryl CAMP, the catalytic subunit of CAMP-dependent protein kinase and phosphorylase h kinase were obtained from Sigma Chemical Company and [ y-‘*P]ATP from Amersham. Phosphoprotein phosphatase 1 from rabbit skeletal muscle was prepared and stored according to the procedures described by Antoniw and Cohen (1976). Phosphorylase b was prepared from rabbit skeletal muscle by the method of Fisher and Krebs (1958) and [ 32P]phosphorylase a was prepared by Dr. Li of this Department by the method of Tores and Chelala (1970). Assays

The samples for assay for inhibitor-l were prepared by the procedure of Foulkes et al. (1980) except that the first lyophilization step was omitted. The analysis for inhibitor-l was carried out by the procedures of Nimmo and Cohen (1978) and Foulkes and Cohen (1979) in which the sample was assayed directly to give the level of inhibitor-1-P present, after exhaustive phosphorylation to give the total amount of inhibitor-l present and after exhaustive dephosphorylation by phosphoprotein phosphatase 1 to assure that the inhibitory activity is due to inhibitor-l. In all the inhibitory effects were eliminated cases, (955100%) following the dephosphorylation step. The determinations were carried out in duplicate and the values obtained were i 5% of the average. A unit of inhibitor-l, the amount which inhibits 0.02 U of phosphoprotein phosphatase 1 by 50%,

63

is that given by Nimmo and Cohen (1978). The assay and units for phosphoprotein phosphatase activity with [ 32P]phosphorylase a as the substrate (Table 6) were also those of Nimmo and Cohen (1978). The 2 min preincubation was carried out with a 8 ~1 of the mitochondrial preparation and, when present, 5 ~1 of inhibitor-l or phosphorylated inhibitor-l. The assays were carried out in duplicate and the values obtained were &5% of the average. Steroids were determined in methylene chloride extracts of the incubation medium by the procedure of Finn et al. (1976) and are expressed in terms of cortisol. Protein was determined by the method of Lowry et al. (1951). Preparation of subcellular fractions The subcellular fractions used for the determination of inhibitor-l levels (Tables 1 and 5) were obtained by the following procedure. The sliced or minced tissues in a volume of 250 mM sucrose-50 mM NaF-4 mM EDTA, pH 7.0, adjusted with NaOH, 4-5 times the weight of the tissue, were homogenized by five or six strokes of a motordriven Teflon pestle. The homogenate was centrifuged at 4 o C at 750 x g for 10 min and the pellet, largely nuclei and cell debris, discarded. The supernatant was centrifuged at 6600 X g for 10 min, the mitochondrial pellet removed and the supernatant centrifuged at 100000 x g for 60 min to separate the microsomal pellet from the cytosol. The mitochondrial and microsomal pellets were taken up in a volume of 50 mM NaF-4 mM EDTA, pH 7.0, equal to the original weight of the tissue. To isolate the mitochondrial pellet used in the phosphoprotein phosphatase (Table 6) beef adrenal cortex slices were homogenized in 10 ~01s. of 0.25 M sucrose-12 mM Tris . HCl, pH 7.4. The mitochondrial pellet was isolated as given above and suspended in one volume of sucrose-Tris to give the isotonic mitochondrial preparation. This mitochondrial pellet was washed once with sucrose-Tris, twice with 12 mM Tris-1 mM EDTA (pH 7.4), once with 12 mM Tris (pH 7.4) and suspended in 1 vol. of 12 mM Tris to give the hypotonic mitochondrial preparation. Incubation Slices of beef adrenal

cortex

were prepared

as

described previously (Bhargava et al., 1978). The slices (1.0 g) were preincubated for 15 min in 12 ml of Krebs-Ringer bicarbonate solution containing 200 mg glucose per 100 ml (KRB-glucose) under 95% 0, : 5% CO, at 37°C. After the preincubation, the slices were removed, blotted on filter paper, and placed in 12 ml of fresh KRB-glucose containing the additions indicated in Tables 2, 3 and 4. The final incubation was for 20 min at 37 o C under 95% 0, : 5% CO,. The slices were immediately removed, blotted on filter paper, transferred to cold sucrose-NaF-EDTA homogenization medium, homogenized and inhibitor-l determined in the fractions indicated in the Tables. It requires 3-4 g of adrenal cortex for the determination of inhibitor-l in the 750 x g supernatant of the homogenate and 8-10 g for the determination of inhibitor-l in the subcellular fractions obtained from the 750 x g supernatant. Results The distribution of inhibitor-l in the major subcellular components of the 750 x g supernatant in the beef adrenal cortex and some additional tissues is given in Table 1. It was found in all tissues examined except rat liver, an observation in accord with that of Huang et al. (1977) and, on the average, about 90% was present in the cytosol and 10% in the mitochondrial pellet. None was found in the microsomal pellet. The beef adrenal cortex had a larger amount (U/g tissue) and a greater specific activity (U/mg protein), by a factor of two, in both fractions, than any other tissue examined. About 85% of the total inhibitor in the adrenal cortex was present in the cytosol and the rest in the mitochondrial pellet. The adrenal medulla and the corpus luteum had a similar intracellular distribution while the other tissues had about 95% in the cytosol. In terms of specific activity, however, the level in the mitochondrial pellet is greater than 60% of that in the cytosol in all tissues except kidney and testis. Since inhibitor-l has been found in dog and rabbit liver (Goris et al., 1978; Knight and Teal, 1980) its absence in rat liver could be a species difference or could be due to a substance in rat liver which interferes with the assay for inhibitor-l. To test this latter possibility, the inhibitor-l from

64 TABLE

1

THE SUBCELLULAR

DISTRIBUTION

OF INHIBITOR-l

IN VARIOUS

TISSUES

The adrenal cortex and medulla and the corpus luteum were obtained from beef. The other tissues were from Sprague-Dawley rats. The results, i SEM, represent four experiments for the adrenal cortex and medulla and three experiments for the rest of the tissues. Abbreviations: Hm, the 750 x g supernatant of the initial homogenate; Mt, the mitochondrial pellet; MC, the microsomal pellet; Ct. the post-microsomal supernatant, the cytosol. The weight of the tissue in this and other tables refers to wet weight.

U/g tissue U/mg protein U/g tissue U/mg protein U/g tissue U/mg protein U/g tissue U/mg protein U/g tissue U/mg protein U/g tissue U/mg protein U/g tissue

Adrenal cortex Adrenal medulla corpus luteum Skeletal muscle Kidney Testis Liver

Hm

Mt

MC

Ct

3020 &70 49.5+ 1.2 2070 570 23.Ozb 1.0 535 * 9 10.7* 0.2 761 k20 21.2+ 0.5 1010 +30 13.1 f 0.4 926 k50 21.0* 1.2 0

448 *30 37.3? 2.5 453 *21 17.1 f 0.8 66.0* 3.0 7.1 * 0.3 35.0* 1.8 17.5* 0.8 32.0* 6.0 2.1 f 0.4 34.1 f 3.0 5.75 0.5 0

0

2270 k50 55.1* 1.1 1480 170 30.3+ 1.4 418 i39 13.5f 1.3 727 +18 24.2* 0.6 907 *40 19.6i 0.9 810 133 27.1 + 1.1 0

the adrenal cortex was assayed in the absence and presence of a corresponding preparation from rat liver. No effect on the assay for inhibitor-l in the adrenal cortex was found (data not given). A role for inhibitor-l in the determination of the level of phosphorylation of the putative regulatory protein(s) involved in the action of ACTH on steroidogenesis would be supported by an increase in the phosphorylation of inhibitor-l which corresponds to the increase in steroidogenesis. The effects of ACTH and dibutyryl CAMP on steroidogenesis and on the phosphorylation of inhibitor-l are given in Tables 2 and 3. With ACTH, a

TABLE

0 0 0 0 0 0

Recovery 90% 93% 90% 100% 93% 91%

maximum of a 2-fold increase in steroidogenesis was obtained with adrenal slices while with dibutyryl CAMP, a 3-fold increase was obtained. It is seen that with ACTH and with all concentrations of dibutyryl CAMP, the effects on steroidogenesis and on the phosphorylation of inhibitor-l were identical. This is seen also in the data in Tables 4 and 5. There is a considerable decrease in the level of total inhibitor-l in the tissue following the incubations of the adrenal cortex slices and this is due largely to a general loss of protein. Thus, the total units of inhibitor-l in the untreated adrenal cortex

2

THE EFFECT OF ACTH IN THE ABSENCE PHOSPHORYLATION OF INHIBITOR-l AND

AND PRESENCE OF INHIBITORS ON STEROIDOGENESIS

OF

PROTEIN

SYNTHESIS

ON THE

ACTH was present at 1 U/ml and cycloheximide and puromycin at 1.0 mM. The inhibitors of protein synthesis were present in both the preincubation and final incubation of the beef adrenal cortex slices. Phosphorylated inhibitor-l was determined in the 750~ g supernatant of homogenates of the adrenal slices. The results, + SEM, represent three experiments. Additions

Phosphorylated U/g

ACTH ACTH + cycloheximide ACTH + puromycin il The difference

with respect

187k 6 372+16 a 358528 a 363? 4” to the control

Cortisol

inhibitor-l

tissue

are significant

% Change

pg/g

+103*13 +98+22 +looFlo

4.4kO.l 9.810.1 i) 4.5 * 0.9 5.1 +0.6

at P < 0.01.

tissue

% Change

+123i-9 0 +16

65 TABLE

3

THE EFFECT PHORYLATION SYNTHESIS

no effect on the stimulation of the phosphorylation of inhibitor-l by these substances (Tables 2 and 4). These results suggest that any involvement of inhibitor-l in the action of ACTH would be anterior to the site of action of the rapidly turning-over protein(s) sensitive to cycloheximide and puromycin. Since ACTH acts at the formation of pregnenolone from cholesterol (Stone and Hechter, 1954; Karaboyas and Koritz, 1965) in adrenal mitochondria (Halkerston et al., 1961; Koritz and Kumar, 1970) the intracellular location of the increase in phosphorylated inhibitor-l is pertinent. The data in Table 5 show that the dibutyryl CAMP-induced increase occurs in the mitochondrial supernatant but not in the mitochondrial pellet. The same results were obtained with ACTH in a single experiment (data not shown). Since no inhibitor-l was found in the microsomal pellet (Table l), the increase in phosphorylated inhibitor-l due to ACTH and dibutyryl CAMP (Tables 2-5) occurs in the cytosol. The possibility of an undetected increase in phosphorylated inhibitor-l in a fraction of the mitochondrial pellet was investigated in one experiment. Using ACTH which resulted in a 2.2-fold increase in steroidogenesis, the mitochondrial pellet obtained was sonicated with 20 s bursts for 2 n-tin in ice with a Heat Systems Ultrasonic Processor at the microtip limit, centri-

OF DIBUTYRYL CAMP ON THE PHOSOF INHIBITOR-l AND ON CORTISOL

The levels of phosphorylated inhibitor-l were determined in the 750X g supematant of homogenates of the slices of beef adrenal cortex. Theophylhne (5 mM) was present in the final incubation. The results, + SEM, represent four experiments and the differences with respect to the control are significant at P c 0.001. Dibutyry1 CAMP

Phosphorylated inhibitor-l U/g

tissue

Cortisol

% Change

pg/g

tissue

+58* 7 + 137 f 14 +1s3+17 +199& 6

5.5 * 0.2 8.8 + 0.4 12.1 k 0.6 15.4kO.9 17.5+1.3

% Change

(mM) 0

2.5 5 10 30

239&13 398 f 22 573 f 33 685 + 35 728 * 24

+61_+ 8 +133+11 +185f 9 +219f14

(Table 1) and in the slices (Table 2) are, per g tissues, 3020 and 850, a 72% decrease, while per mg protein they are 49.5 and 40.5, an 18% decrease. These results would indicate a relative retention of inhibitor-l. The inhibitors of protein synthesis, cycloheximide and puromycin, which sharply reduced the stimulation of steroidogenesis by ACTH and CAMP (Ferguson, 1963; Garren et al., 1965) had

TABLE

4

THE EFFECT OF CYCLOHEXIMIDE ON THE STIMULATION OF INHIBITOR-l AND ON CORTISOL SYNTHESIS

BY DIBUTYRYL

CAMP ON THE

The levels of phosphorylated inhibitor-l were determined in the 750 x g supematant of homogenates Cycloheximide (1 mM) was present in both the preincubation and the final incubation. Theophylline incubation. Dibutyryl CAMP

Cycloheximide

(mM)

0 2.5 10 30

+ _ + _ + _ +

Experiment

Experiment

1

PHOSPHORYLATION

of slices of beef adrenal cortex. (5 mM) was present in the final

2

Phosphorylated inhibitor-l

Cortisol

Phosphorylated inhibitor-l

Cortisol

U/g

pg/g

U/g

rig/g

235 226 362 350 600 530

tissue

4.9 5.0 7.3 5.4 12.1 6.8

tissue

tissue

255 250

5.7 5.4

658 675 720 720

12.5 7.2 14.5 6.0

tissue

66 TABLE

5

THE INTRACELLULAR LOCATION IN BEEF ADRENAL INCREASED BY DIBUTYRYL CAMP Theophylline

(5 mM) was present

Experiment

Dibutyryl CAMP

Mitochondrial pellet

(mM)

Phosphorylated

2 3 4

INHIBITOR-l

Cortisol pg/g

tissue

% Change

inhibitor-l

tissue

U/g

tissue

% Change

243 490 225 502 250 639 264 700

6.1 12.2 5.6 12.2 5.6 16.0 6.0 17.3

+102 + 123 +156 +165

fuged at 100000 x g for 60 min and the supernatant and pellet (membranes) analyzed. Neither fraction showed an increase in phosphorylated inhibitor-l. The supematant had twice the amount of inhibitor-l than the membranes. This ratio of 2 : 1 in the sonicated mitochondrial pellets was found also in beef adrenal medulla and rat testis, kidney and skeletal muscle. These data suggest that some of the inhibitor-l in the mitochondrial pellet is associated with membranes. Since neither ACTH nor dibutyryl CAMP increased the phosphorylation of inhibitor-l in this fraction the precise location of the inhibitor-l was not further investigated.

TABLE

OF THE PHOSPHORYLATED

Mitochondrial supernatant

38 38 38 39 40 43 42 45

0 5 0 5 0 30 0 30

SLICES

in the final incubation.

U/g 1

CORTEX

+ 100 +11s +186 +188

The absence of an effect of dibutyryl CAMP on the phosphorylation of inhibitor-l in the mitochondrial pellet suggests that a role for inhibitor-l on the level of phosphorylation of a regulatory protein(s) in this fraction would involve an interaction of a responsive phosphoprotein phosphatase with cytosolic phosphorylated inhibitor-l. The data in Table 6 indicate that such an interaction can take place. It is seen that phosphorylated inhibitor-l inhibits the dephosphorylation of phosphorylase a added to isotonic or hypotonic mitochondrial pellets while nonphosphorylated inhibitor-l has no effect. The phosphorylated inhibitor-l present in these experi-

6

THE INHIBITION BY EXOGENOUS

OF PHOSPHOPROTEIN PHOSPHATASE PHOSPHORYLATED INHIBITOR-l

IN THE

ADRENAL

CORTEX

MITOCHONDRIAL

PELLET

Non-phosphorylated inhibitor-l (inhibitor-l) and phosphorylated inhibitor-l (inhibitor-l-P) were present at 125 U per incubation. For each experiment, the isotonic and hypotonic mitochondrial pellets were prepared from the same adrenals. The results, + SEM, are based on four experiments and the differences with respect to the control are significant at P -c0.04 for the isotonic pellets and P c 0.01for the hypotonic pellets. Mitochondrial pellet

Additions

Phosphoprotein

Isotonic

_ Inhibitor-l Inhibitor-l-P _

900 * 140 940 * 100 48Ok 70 220* 20 240+ 20 62* 5

Hypotonic

mU/g

Inhibitor-l Inhibitor-l-P

tissue

phosphatase % Change

-48*2

-71*4

mU/mg 60+7 62k7 32k4 25+3 26k3 6kO.6

protein

% Change

-52*2

-73*6

67

ments was at saturation levels since the same inhibition was found at one-half the concentration (data not given). The data in Table 6 also indicate that a greater percentage of the total phosphoprotein phosphatase activity than of the total protein is retained during the preparation of the hypotonic mitochondrial pellet. Thus, the amount of phosphoprotein phosphatase activity (mu/g tissue) in the hypotonic pellet is 25% of that in the isotonic pellet while the specific activity (mU/mg protein) is 42% of that in the isotonic pellet. This would suggest an association of part of the phosphoprotein phosphatase activity with membranes of the mitochondrial pellet. The preparation of the hypotonic pellet involves a number of washes which would serve to minimize phosphatase adventitiously bound to the membranes. In addition, there is an increase in the inhibition of the phosphatase activity by phosporylated inhibitor-l from 50% to 72% in the hypotonic mitochondrial pellet. This may reflect an increased concentration of a phosphoprotein phosphatase sensitive to inhibitorI-P, in the membranes of the mitochondrial pellet. It is also possible, however, that modifications of the membranes by hypotonic conditions may result in an increased interaction between phosphorylated inhibitor-l and the phosphatase. Both possibilities emphasize that a responsive phosphoprotein phosphatase in the preparation can react which external phosphorylated inhibitor-l. Discussion The stimulation of CAMP-dependent protein kinase activity in the adrenal cortex by ACTH will increase not only the phosphorylation of regulatory proteins involved in steroidogenesis but also, by increasing the phosphorylation of inhibitor-l, may decrease this dephosphorylation and, hence, augment the effect of ACTH. The data presented are consistent with such a role for inhibitor-l. The inhibitor is present in beef adrenal cortex at a concentration greater than that found in any other tissue examined. Of greater consequence, steroidogenesis and the phosphorylation of inhibitor-l are stimulated to the same extent by ACTH and by various concentrations of dibutyryl CAMP, suggesting an effect at the regulated step in steroidogenesis. As would be expected, the phosphoryla-

tion of inhibitor-l is cycloheximide and puromytin insensitive since it would be a function primarily of the activation of protein kinase A and not the concentration of a labile regulatory protein. The inhibitor-l whose phosphorylation is increased is present only in the cytosol, where about 85% of the inhibitor-l is located. The extent of phosphorylation of the remaining 15%, found in the mitochondrial pellet is not affected. The data, however, also show the phosphoprotein phosphatase activity in the mitochondrial pellet to be inhibited by external phosphorylated inhibitor-l. Phosphoprotein phosphatase activity in cardiac microsomes has been found also to be inhibited by external phosphorylated inhibitor-l (Iyer et al., 1988). Phosphorylated proteins involved in the effect of ACTH on steroidogenesis include the putative phosphorylated, puromycinand cycloheximidesensitive, rapidly turning-over protein involved in the stimulation of the rate-limiting step in steroidogenesis (Ferguson, 1963; Garren et al., 1965; Davis and Garren, 1968) the transformation of cholesterol to pregnenolone in the mitochondria (Stone and Hechter, 1954; Halkerston et al., 1961) and cholesterol ester hydrolase. Cholesterol ester hydrolase is activated by protein kinase A and acts upon cytosolic lipid droplets to release cholesterol for steroidogenesis (Trzeciak and Boyd, 1973, 1974; Beckett and Boyd, 1977). It is also activated by y-melanotropins by a mechanism which does not involve CAMP. These potentiate the ACTH stimulation of steroidogenesis but in the presence of ACTH, they have no effect on steroidogenesis (Pedersen and Brownie, 1980; Pedersen et al., 1980). In addition, cycloheximide, which inhibits stimulation of steroidogenesis by ACTH and CAMP, does not affect their stimulation of cholesterol ester hydrolase (Davis and Garren, 1966; Trzeciak and Boyd, 1973). Thus, the effect of ACTH and CAMP on the hydrolase is not involved in the limiting step of steroidogenesis but serves to assure an ample supply of cholesterol. An inhibition of a phosphoprotein phosphatase acting on the phosphorylated form of cytosolic cholesterol ester hydrolase would contribute to this process. Recently, evidence concerning the nature of the putative phosphorylated rapidly turning-over pro-

68

tein has been presented (Pon and Orme-Johnson, 1966; Krueger and Orme-Johnson, 1983; Pon et al., 1986). It was found that, in rat adrenal cortex cells, an M, 28000 protein, which is cotranslationally phosphorylated, increases concomitant with steroidogenesis in response to ACTH and CAMP and that this response is sensitive to cycloheximide. This phosphoprotein is not synthesized in the absence of ACTH or CAMP and is formed also in the corpus luteum but not in a non-steroidogenic tissue, such as adipocytes, which also responds to hormones whose actions are mediated by CAMP. Since the phosphorylations are cotranslational, a possible role for a cytosolic phosphoprotein phosphatase and its inhibition by cytosolic phosphorylated inhibitor-l is likely. If this phosphoprotein acts directly on the conversion of cholesterol to pre~enolone its extent of dephosphorylation in the mitochondria may be diminished since we have found that external phosphorylated inhibitor-l can inhibit phosphoprotein phosphatase activity in the mitochondrial pellet. Although the location of the phosphoprotein phosphatase in this pellet is not known at present, the inhibition of the phosphatase activity in the hypotonic mitochondrial pellet would support an interaction with membrane-bound phosphoprotein phosphatase. The precise role of cytosolic phospho~lated inhibitor-l, resulting from the action of ACTH or dibutyryf CAMP, on the activity of a phosphoprotein phosphatase which can affect steroidogenesis, must await the demonstration of a phosphatase in the adrenal cortex which acts upon this steroidogenic phosphoprotein and which is inhibited by phospho~lated inhibitor-l. Since phosporylated inhibitor-l acts on phosphoprotein phosphatase 1, which has a broad substrate specificity (Ingebritsen and Cohen, 1983), this is a likely candidate.

This work was supported by Grant AM-31757 from the United States Public Health Service. References Antoniw, 45-54.

J.F.

and

Cohen,

P. (1976)

Eur.

J. Biochem.

68,

Beckett, G.J. and Boyd, G.S. (1977) Eur. J. B&hem. 72, 223-233. Bhargava, G., Schwartz, E. and Koritz, S.B. (1978) Proc. Sot. Expt. Biol. Med. 158, 183-186. Davis, W.W. and Garren, L.D. (1966) Biochem. Biophys. Res. Commun. 24, 805-810. Davis, W.W. and Garren, L.D. (1968) J. Biol. Chem. 243, 5153-5157. Ferguson, Jr., J.J. (1963) J. Biol. Chem. 238. 2754-2759. Finn, F.M., Johns, P.A., Nishi, N. and Hofmann, K. (1976) J. Biol. Chem. 251, 3576-3585. Fisher, E.H. and Krebs, E.G. (1958) J. Biol. Chem. 231.65-73. Foulkes, J.G. and Cohen, P. (1979) Eur. J. B&hem. 97, 251-256. Foufkes, J.G., Jefferson, L.S. and Cohen, P. (1980) FEBS Lett. 112, 21-24. Garren, L.D., Ney, R.L. and Davis, W.W. (1965) Proc. Natl. Acad. Sci. U.S.A. 53, 1443-1450. Gill, G.N. and Garren, L.D. (1970) Biochem. Biophys. Res. Commun. 39, 335-343. Goris, J., Defreyn, G., Vandenheede, J.R. and Merlevede, W. (1978) Eur. J. Biochem. 91, 457-464. Grahame-Smith, D.G., Butcher, R.W., Ney, R.L. and Sutherland, R.W. (1967) J. Biol. Chem. 242, 5535-5541. Halkerston, I.D.K., Eichorn, J. and Hechter. 0. (1961) J. Biol. Chem. 236. 374-380. Hayashi, K., Sala, G., Catt, K.J. and Dufau, M.L. (1979) J. Biol. Chem. 254. 6678-6683. Haynes, Jr.. R.C., Peron, F.G. and Koritz, S.B. (1959) J. Biol. Chem. 234, 1421-1423. Huang, F.L. and Glinsmann, W.H. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3004-3008. Huang, F.L. and Glinsmann, W.H. (1976) Eur. J. Biochem. 70, 419-426. Huang, F.L., Tao, S. and Glinsmann, W.H. (1977) B&hem. Biophys. Res. Commun. 78, 615-623. Ingebritsen, T.S. and Cohen, P. (1983) Science 221, 331-338. Iyer, R.B. Koritz, S.B. and Kirchberger, M.A. (1988) Mol. Cell. Endocrinol. 55, 1-6. Karaboyas, G.C. and Koritz, S.B. (1965) Biochemistry 4, 462-468. Knight. B.L. and Teal, T.K. (1980) Eur. J. B&hem. 104. 521-528. Koritz, S.B. and Kumar, A.M. (1970) J. Biol. Chem. 245, 152-159. Koroscil, T.M. and Gallant, S (1980) J. Biol. Chem. 255, 6276-6283. Krueger, R.J. and Orme-Johnson, N.R. (1983) J. Biol. Chem. 258,10159-10167. Li, H.-C. (1979) Eur. J. Biochem. 102, 363-374. Lowry, O.H., Rosebrough, N.J., Farr, H.L. and Randall, A.L. (1951) J. Biol. Chem. 193, 265-275. Merlevede, W. and Riley, G.A. (1966) J. Biol. Chem. 241, 3517-3524. Murakami, N. and I&ii, S. (1973) Endocrinol. Jpn. 40,421-424. Nimmo, G.A. and Cohen. P. (1978) Eur. J. Biochem. 87, 341-351. Pedersen. R.C. and Brownie, A.C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2239-2243.

69 Pedersen. R.C., Brownie, A.C. and Ling, N. (1980) Science 208, 10441046. Podesta, E.J., Milani, A., Steffen, H. and Neher, R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5187-5191. Pon, L.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6594-6599. Pon, L.A., Hart&an, J.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 13309-13316. Rae, P.A., Gutman, N.S., Tsao, J. and Schimmer, B.P. (1979) Proc. Nat]. Acad. Sci. U.S.A. 76, 1896-1900. Richardson, M.C. and Schulster, D. (1973) B&hem. J. 136, 993-998. Roos, B.A. (1973) Endocrinology 93, 1287-1293. Sala, G.B., Hayashi, K., Catt, J.J. and Dufau, M.L. (1979) J. Biol. Chem. 254. 3861-3865.

Stone, D. and Hechter, 0. (1954) Arch. B&hem. Biophys. 51, 457-469. Tao, S.H., Huang, F.L.. Lynch, A. and Glinsmann, W.H. (1978) Biochem. J. 176, 347-350. Torres, H.N. and CheIala, C.A. (1970) Biochem. Biophys. Acta 198, 495-503. Trzeciak, W.H. and Boyd. G.S. (1973) Eur. J. Biochem. 37, 327-333. Trzeciak, W.H. and Boyd, G.S. (1974) Eur. J. B&hem. 46, 201-207. Ullman, B. and Perlman, R.L. (1975) B&him. Biophys. Acta 403, 393-411.