Cyclic Adenosine Monophosphate Dependent Protein Kinases in R

0013-7227/91/1295-2345$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 5 Printed in U.S.A.

Identification, Characterization, and Hormonal Regulation of 3',5'-Cyclic Adenosine Monophosphate Dependent Protein Kinases in Rat Sertoli Cells* BRYNJAR F. LANDMARK, BEATHE FAUSKE, WINNIE ESKILD, BJ0RN SKALHEGG, SUZANNE M. LOHMANN, VIDAR HANSSON, TORE JAHNSEN, AND STEPHEN J. BEEBEt Institute of Medical Biochemistry (B.F.L., W.E., V.H., T.J., S.J.B.), University of Oslo, N-0317 Oslo, Norway; Department of Anatomy (B.F.), University of Bergen, N-5009 Bergen, Norway; Institute of Pathology (B.S., T.J., S.J.B.), Rikshospitalet, N-0027 Oslo, Norway; and Medizin Universitdts Klinik, Labor fur Klinische Biochemie, 8700 Wurzburg, Germany

ABSTRACT. Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (~9.2 ± 0.8 pmol/mg protein) and C (~7.3 ± 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mMJ for 24 and 48 h revealed a time-dependent increase in [3H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N3[32P]cAMP photoaffinity labeling, autophosphorylation by y32 [ P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RIa, 25% RIIa, and very

low levels of RII3. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RIO (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII,, (10-fold). Quantification of individual R subunit forms by PH] cAMP binding and immunoimmobilization

dye frontFIG. 5. 8-N3-[32]PcAMP labeling (left) and autophosphorylation (right) of affinity-purified R subunits from control and (Bt)2cAMPstimulated Sertoli cells. Primary Sertoli cell cultures were incubated for 48 h in the absence (C) and presence (S) of 0.1 mM (Bt2)cAMP. Cellular extracts were applied to 8-(6-aminohexyl)-amino-cAMP affinity columns, and R subunits were eluted with 8 M urea. The R subunits were dialyzed and photoaffinity labeled with 8-N3-[32P]cAMP (left) or autophosphorylated with [T- 3 2 P]ATP in the presence of purified PKA C subunit (right). Equal amounts of [3H]cAMP binding activity were subjected to SDS-PAGE in 7.5% acrylamide gels. Purified regulatory subunits (50 ng) from rat brain were used as a standard (Std). Mr

103 52^: 49-

RI i

m

mm

c s

R

RII

* ~ mm C

n

s c s

FlG. 6. Effects of (Bt)2cAMP on R subunit immunoactivity. Primary cultures of Sertoli cells were incubated for 48 h in the absence (A) or presence (B) of 0.1 mM (Bt)2cAMP. Cellular extracts were subjected to SDS-PAGE in 7.5% gels, electroblotted onto nitrocellulose, and incubated with monospecific antibodies for RIO) RIIa) and RIL. Bands were visualized after incubation with [125I]immunoglobulin G (goat antirabbit) and autoradiography. Purified rat brain R subunits were used as standards.

Both RII subunits, but not RI, were autophosphorylated by C subunit (Fig. 5, right two lanes). A smaller mol wt band of approximately 35,000 Mr was autophosphorylated and labeled with 8-N3-[32P]cAMP, indicating that it may be a proteolytic fragment of RII, retaining the hinge region (containing the autophosphorylation site) and the cAMP binding sites of RII. Radioimmunolabeling analysis of the effects of (Bt)2cAMP on Sertoli cell R subunits Extracts from Sertoli cell cultures were separated on 7.5% SDS-PAGE, transferred to nitrocellulose filters, and incubated with antibodies monospecific for each of the R subunits (Fig. 6). As shown, treatment of cultured Sertoli cells for 48 h (S) resulted in a distinct increase in

To perform more quantitative estimates of the absolute levels of the various R subunits in control and (Bt)2cAMP stimulated Sertoli cells, [3H]cAMP-labeled RI and RII subunits were removed from cell extracts using columns containing specific antibodies and protein A Sepharose. Using this procedure the antibodies could distinguish between RI and RII subunits, but poorly between RII a and RII^. Therefore, the results of Western analysis (Fig. 6) were used to estimate the changes in RII a and RILj after (Bt)2cAMP stimulation. From this data we calculated the levels of RIIa and RII^ in control and stimulated cells (Table 1). As shown in the table, (Bt)2cAMP treatment was associated with a 2.1-fold increase in RI subunits (RIa). A similar change in RI a protein levels was observed by Western analysis followed by densitometric scanning. The total amounts of RII were determined by immunoimmobilization to be approximately 25-30% of that of RI a both in control and stimulated cells. The calculated levels of RIIa from cells cultured in the absence and presence of (Bt)2cAMP were 1.9 and 2.3 pmol/mg protein, respectively. In comparison, (Bt)2cAMP stimulated the levels of RIIp from less than 0.1 to 1.1 pmol/mg protein (Table 1). Thus, in nonstimulated Sertoli cells RII a is more than 20 times that of RII/s, whereas in the stimulated Sertoli cells we find comparable amounts of RIIa and RII^. The ratio of RI to RII increased slightly during stimulation with (Bt)2cAMP. TABLE 1. Estimate of specific R subunit levels in Sertoli cells RIa RII Ratio RIIO RII, (pmol/mg) (pmol/mg) (RI/RII) (pmol/mg) (pmol/mg) Control 6.6 ±0.3 (Bt)2cAMP 14.0 ±2.1 (n = 4)

2.0 ± 0.1 3.4 ±0.6 (n = 4)

3.3 4.1

1.9 2.3

0.1 1.1

Sertoli cells were incubated for 48 h in the absence (Control) or presence of 0.1 mM (Bt)2cAMP in the growth medium. R subunits were assayed by [3H]cAMP binding and immunoimmobilization as described in Materials and Methods. Levels of individual RII subspecies were calculated/estimated from the total RII levels and their specific increase from densitometric scanning of the Western blot autoradiograms (shown in Fig. 5). Values for [3H]cAMP binding are means ± SD for four separate Sertoli cell isolations.

PKA IN RAT SERTOLI CELLS

Discussion The role of cAMP in reproductive function is relatively well characterized and involves the covalent modification of protein substrates via phosphorylation by the catalytic subunit of PKA. PKA regulates cell function via posttranslational activation or inactivation of enzymes that regulate metabolism or by activation or inactivation of gene regulatory proteins (2, 33, 34). A number of proteins are induced by cAMP in various cell systems, and some of these are also substrates for PKA (35). In addition, several PKA autoregulatory components have been demonstrated, including autophosphorylation of the regulatory subunits (36) and modification of cAMP-dependent phosphodiesterase activities via PKA phosphorylation (37, 38). cAMP also has been shown to regulate components of its own signal transduction system in that several of the PKA mRNAs are regulated by cAMP via transcriptional and/or posttranscriptional mechanisms (21, 39). In the present study we have characterized the cAMP-stimulated induction of mRNA and protein for all the PKA subunits in rat Sertoli cells. The multiplicity of PKA subunits has recently been more fully defined, primarily through cDNA cloning and sequencing (5, 7-8,12,14-16). In Sertoli cells, the PKAs are represented by RI a, RII a , RUp, and Ca, whereas Cp, Rip, and Cy are absent or present at very low levels (21). Because caveats are inherent in each method for the measurement of PKA protein levels, we have used several methods in the present investigation in order to substantiate the conclusions made. These include Western blotting analysis and [3H]cAMP binding before and after DEAE cellulose chromatography, 8-AHA-cAMP Sepharose, and immunoimmobilization chromatography. Additional data were obtained from 8-N3[32P]cAMP photoaffinity labeling of R subunits and autophosphorylation of RII subunits in conjunction with SDS-PAGE. Functional C subunit levels were obtained by specific activity measurements of heptapeptide phosphorylation. By using 8-N3-[32P]cAMP photoaffinity labeling and DEAE cellulose chromatography, Spruill et al. (32) have demonstrated the presence of two distinct regulatory subunits of PKA in Sertoli cells, corresponding to the RI a and Rlla in our studies. By DEAE cellulose chromatography, Fakunding and Means (40) also demonstrated the presence of two types of protein kinases in Sertoli cell-enriched testes in vivo. However, none of these investigators obtained indications of heterogeneity (RIIn vs. RII/?) among the RII subunits. The present measurements of Sertoli cell PKA subunits proteins indicate that cAMP stimulation is associated with a 2- to 3-fold increase in the ratio of R/C subunits. The C subunit level increased only slightly; therefore, increases in the R subunit account for the ratio

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change observed. In other tissues the levels of PKA subunits have been shown to be subject to regulation during cell growth, after transformation, and during differentiation (for review see Ref. 41). In addition, free RI subunit like that demonstrated in the present study has previously been reported in cells of neural origin (4245), in Chinese hamster ovary cells (46), and in Friend erythroleukemic cells (47). In contrast, a cAMP-dependent increase in the R/C ratio in rat hepatocytes in primary culture has been shown to be due to C downregulation rather than due to an increase in R (48). The most distinctive change in PKA during cAMP stimulation of Sertoli cells was the induction of RII/j mRNA and protein. Interestingly, ovarian granulosa cells, the female counterpart to testicular Sertoli cells, reveal a similar differential regulation of RII^ by cAMP (12, 49). During cAMP stimulation, the increase in RILj mRNA was approximately 30- to 50-fold within the first 24-48 h. Other PKA subunit mRNAs only showed minor increases after cAMP stimulation. After cAMP stimulation, the RI/RII subunit ratio was increased only modestly, as determined by specific immunoimmobilization method. In cAMP-stimulated cells RII0 still represented less than 10% of the total R subunit. This may explain why RII^ has not been identified in Sertoli cells in previous studies (32, 40). Approximately 75% of the total R subunit was RI«, both under basal conditions and after cAMP stimulation. The estimated concentration of RILj in Sertoli cells is low compared to that of RIa but substantial when compared with total levels of regulatory subunits in some other tissues or cells, for example rat hepatocytes (50). In our 8-N3-[32P]cAMP photoaffinity studies of whole cytoplasmic extracts, we always observed labeled RI a and RII a . However, RII^ could not readily be detected under basal conditions. In this case our results are very similar to those obtained by Spruill et al. (32) who detected only two regulatory PKA subunits in cytoplasmic extracts from Sertoli cells. However, after fractionation of the cytoplasmic extracts by DEAE cellulose chromatography or cAMP affinity chromatography, three R subunits could be photoaffinity labeled. In these studies we demonstrated a selective induction of the RII/s protein after cAMP stimulation. Under basal conditions autophosphorylation detected only RIIa; however, after stimulation with (Bt)2cAMP, similar amounts of autophosphorylated RIIa and of RII^ were observed. During the autophosphorylation experiment, a smaller proteolytic fragment of the RII subunits with mol wt of approximately 35,000 M:r was observed. The fact that this protein was observed only in cells treated with cAMP indicates that this may represent a breakdown product of RII^. Another possibility is that cAMP induces a protease degrading both RIIa and RII^. These autophosphoryla-


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tion studies clearly show the selective induction of the RIIp protein, however it does not allow any estimate of the relative RIIa to RILj levels, since the endogenous phosphate content of the RII subunits was not controlled. It has been suggested that R subunit induction may serve to attenuate the cAMP transduction signal by favoring the inactive PKA holoenzyme (44, 48). For such a mechanism, an increase in RII^ would not seem very practical, since this isoform represents only a minor fraction of the total R. Since RI a is the predominant subunit and is increased about 2-fold by cAMP stimulation, such a function would be more appropriate for RI a . Consistent with this hypothesis, much of the RI a subunit appears to be dissociated from the C subunit during cAMP stimulation. cAMP stimulation results in an apparent decrease in type I holoenzyme and an apparent increase in type II holoenzyme. Since quantitative methods are not presently available to adequately separate RII holoenzyme isoforms, it is not possible to determine if the apparent increase in type II holoenzyme is due to RII a or RII^. Overexpression of RII a in transfected NIH3T3 cells results in preferential assembly of PKA type II, even in the presence of excess free RI (51). Whether this preferential association of RII holoenzymes results from in vivo differences in R subunit affinities for C subunit of preferential assembly of type II holoenzyme via compartmentalization is not known. Furthermore, the order of potency of four different analogs is the same as that observed for kinase-mediated mechanisms in other cells (52). The mechanism(s) of this autoregulation has not been specifically determined, but multiple mechanisms are most likely operative, including altered transcription and translation as well as mRNA and protein turnover rates. The stimulatory effects of cAMP on mRNA for RILj in Sertoli cells appears to involve both transcriptional regulation (53) as well as cAMP-mediated mRNA stabilization (53a). The functional significance of the different isomeric forms of R and C in Sertoli cells (or any cell) has still not been established. Their discrete cellular and subcellular localization (41, 54-56), their specific interaction with R binding proteins (57-59), and differential regulation seen in the present study suggest that the isoforms may have a role in redistribution of intracellular PKA activity. Given the various PKA gene products, a great number of PKA holoenzymes could result. It remains to be determined whether isoforms of PKA holoenzymes contribute to differential regulation of the great diversity of cAMP-mediated responses.

Acknowledgments We gratefully acknowledge the technical assistance of Ms. Guri Opsahl and Ms. Gladys Josefsen. We thank Ms. Liv St0ttum for assisting in typing the manuscript.

Endo•1991 Voll29«No5

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Ninth International Congress of Endocrinology The Ninth International Congress of Endocrinology will be held in Nice (Acropolis, Convention Center, Nice, France) from August 30-September 5, 1992. The program will cover the entire field of basic and clinical endocrinology and will include plenary lectures, symposia, meet-the-professors sessions, and poster presentations. For information, contact: Ninth International Congress of Endocrinology SOCFI 14, Rue Mandar 75002 Paris, France Phone: 33 (1) 42 33 89 94 Fax: 33(1) 40 26 04 44.