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N-0317 Oslo 3, Norway, and the **Labor fur Klinische Biochemie, Medizinische Universitats Klinik, Josef-Schneider Str. 2,. 8700 Wurzburg, Germany. We have ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267,No. 8, Issue of March 15,pp. 5374-5379, 1992 Printed in U.S.A.

Identification, Purification, and Characterizationof Subunits of CAMP-dependent Protein Kinase in Human Testis REVERSEMOBILITIES OF HUMAN RII,AND RIIB ON SODIUM DODECYL SULFATE-POLYACRYLAMIDE GELELECTROPHORESIS COMPARED WITHRAT AND BOVINE RIIs* (Received for publication, April 30,1991, and in revised form, December 2, 1991)

Bjern S.SkilheggSQ1,Brynjar LandmarkII, Kari B. FossSJI,Suzanne M. Lohmann**, Vidar Hansson 11, Tor Lea#, and Tore JahnsenS 11 From the $Institute of Pathology, Rikshospitalet, N-0027 Oslo 1, Norway, the $Institute of Immunology and Rheumatology, Rikshospitalet, N-0027 Oslo 1, Norway, the lllnstitute of Medical Bwchemistry, University of Oslo, P.O. Box 1112 Blindern, N-0317 Oslo 3, Norway, and the **Labor fur Klinische Biochemie, MedizinischeUniversitatsKlinik, Josef-Schneider Str. 2, 8700 Wurzburg, Germany

We have previously identifiedand characterized reg-the purifiedhuman testis subunits, and only the smallulatory (R) subunitsof cyclic AMP-dependent protein est RII subunit(RIIJ revealed a distinct mobility shift kinase, particularly the RII subunits in rat tissues on SDS-PAGE after phosphorylation/dephosphoryla(Jahnsen, T., Lohmann, S. M., Walter, U.,Hedin, L., tion. This study supports the conclusion that the moand Richards,J. S. (1986)J. Biol. Chem. 260,16980- bilities of human RII subunits (RII,,RII,)on SDS15987;Jahnsen, T., Hedin, L., Lohmann, S. M., Wal- PAGE are reversed in contrast with those of other ter, U.,and Richards, J. S. (1986)J. Biol. Chem. 261, species such as rat and bovine. This has to be taken 6637-6639;Jahnsen, T., Hedin, L., Kidd, V. J., Beat- into consideration when examining R subunits in other tie, W. G., Lohmann, s. M., Walter, U., Durica, J., human tissues bymethods such as photoaffinity labelSchulz, T. Z., Schiltz, E., Browner, M., Lawrence, C. B., Goldman, D,, Ratoosh, S. L., and Richards, J. S. ing which cannot distinguish subtype identities. (1986)J.Biol. Chem. 261,12352-12361).These studies showed that rat RII, and RII, had apparent molecular masses of 64 and 62 kDa, respectively. The aim Regulatory effects of cAMP in mammalian cells are meof the present study was to purify and characterize diated via activation of CAMP-dependent protein kinase (1). CAMP-dependent protein kinase R subunits in human In the testis, cAMP/cAMP-dependent protein kinase mediate testis and to examine which of the subunits (mRNAs the effects of the gonadotropins on testicular somatic cells and proteins)are present in thistissue. Our results show that human testis contains mRNAs and also influence sperm motility (2-6). The CAMP-dependfor five out of the seven known subunits of CAMP- ent protein kinase holoenzyme consists of a regulatory (R) dependent protein kinase.We observed strong expres- subunit dimer and two catalytic (C) subunits which are dission of mRNAs for RI, (1.6 and 3.2 kilobases (kb)), sociated by the binding of four molecules of CAMP, two to RII, (2.2, 2. and 4, 7.0 kb), and RII, (3.3kb). We also each R subunit. Binding of cAMP to R releases the active C demonstrated mRNAs for two of the three catalytic subunit responsible for subsequent phosphorylation of key subunits, C, (2.7kb) andC, (1.7kb). substrates (7,8). Type I and typeI1 CAMP-dependentprotein Purification of R subunits by DEAE-cellulose and kinase have been described and are distinguished by the salt cAMP affinity chromatography revealed three distinct 61, and concentration at which they elute from DEAE-cellulose colforms with apparent molecular masses of 49, 63 kDa, respectively. Characterization of these R sub- umns (9, 10). Molecular cloning has demonstrated isoforms of both R units by their 8-azido-CAMPphotoaffinity labelingand immunoreactivity, as well as by a phosphorylation- and C subunits. At present four different forms of R subunits dependent mobility shift on sodium dodecyl sulfate- (RI,, RIB,RII,, RIIB)l(11-21) and three different C subunits polyacrylamide gel electrophoresis (SDS-PAGE), in- (C,, CB,C,) (22-24) have been identified at the mRNA/gene dicated subunit sizes of RII, (63 kDa) > RII, dephos- level. RI,, RII,, and C, represent the ubiquitous mRNA forms phoform (51kDa) > RI, (49kDa). This conclusion was found in most tissues, whereas the mRNA for RIIBseems to verified by the analysis of RII subunitsproduced by in be cell- and tissue-specific and is hormonally regulated in vitro transcription/translation of full-length cDNAs ovarian granulosa cells and testicular Sertoli and Leydig cells for both human RII, and RII, in wheat germ lysates. The in vitrotranslated products were the same size as (25, 26). RIBmRNA has so far been detected in mouse brain and testis(13), rat brain and testis(27), and in several human tissues, with the highest expression in the brain (28). The * This work was supported by the Norwegian Cancer Society, the highest levels ofCB mRNA have been observed in rat and Norwegian Research Council for Science and the Humanities, a Torsteds grant, the Anders Jahres Foundation for the Promotion of mouse brain and inthe human prostate, intestine, brain, and ~

~~

~~

Science, the Nordic Insulin Foundation, andthe Deutsche Forschungsgemeinschaft (Grant KO 210/11-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. II To whom correspondence should be addressed Institute of Pathology, Rikshospitalet, N-0027 ,Oslol, Norway.

The abbreviations used are: RI,, RIB,and RIL, RIIp, isoforms of the regulatory subunits of CAMP-dependent protein kinase; C,,CB and C,, isoforms of the catalytic subunit of CAMP-dependentprotein kinase; cAK, CAMP-dependent protein kinase; cAKI and cAKII, CAMP-dependentprotein kinase type I and II; SDS, sodium dodecyl sulfate, PAGE, polyacrylamide gel electrophoresis.

5374

CAMP-dependent Protein Kinase Testis Human in

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cDNA for RIBhas been cloned and its mRNA localized to the brain and testis in mouse. Expression of the RIBcDNA in NIH-3T3 cells produced a 55-kDa protein which was similar in size to the native RIB detected in mouse brain, using a specific anti-RIB antibody (13). In our studies we have not been able to detect any 55-kDa proteins either by photoaffinity labeling or Western analysis before or afterR subunit purification. These resultsindicate that theRI, protein is not present in the testisof elderly men. High level expression of mRNAs for both human RII, and RII, indicated that the two larger proteins purified from human testis were RII, and RIIB,respectively. Both proteins werespecifically photoaffinity-labeled with 8-azido-CAMP and autophosphorylated by T-[~’P]ATP.Our immediate assumption was that the larger subunit (53 kDa) represented RII, and thesmaller (51 kDa) subunitRIIP However, further analysis based on Western blots and the phosphorylationdependent mobility shift on SDS-PAGE indicated that this might not be the case. Antibodies specific for RII, in rats exclusively reacted with the smaller 51-kDa protein on Western blots. In contrast, the antibodies against RIIB detected primarily the larger protein band (53 kDa) on Western blots. A characteristic featureof the RIIsubunits isthat they are autophosphorylated in the presence of y-[32P]ATP and catalytic subunits (30). In several species a clear cut phosphorylation-dependent mobility shift has been observed for RII,, but not for RII,. Whereas rat and bovine RII, has a greater molecular weight than RIIs, the purified human testis RII subunit which showed a mobility shift in response to autophosphorylation was the smaller (51 kDa) subunit. Thus, both MATERIALS AND METHODS AND RESULTS3 immunoreactivity as well as phosphorylation-dependent change in apparent size by SDS-PAGE clearly indicated that DISCUSSION the smaller (51 kDa) human RII subunit was RII,, whereas In thepresent studywe have purified and characterized the the larger (53 kDa) represented the RII, form. These data regulatory subunits of CAMP-dependent protein kinase from suggest that the phosphorylation-dependent mobility shift is human testis. We have further shown that mRNAs for five a more universal characteristic of the RII, subunit than out of seven known R and C subunits are expressed in the apparent size on polyacrylamide gels. testis of elderly men. Strong signals for RI,, RII,, RIIB,C, Cho-Chung and collaborators (39) have recently examined and for the newly cloned C, were seen. We observed small CAMP-dependent protein kinase R subunits in human leumolecular weight messages for RI, and RII, which have been kemic cells (HL60 cells) and in humancolon carcinoma cells. shown to be germ cell-specific in rats (25). In the rat, RIB In these studies they claim that the smaller RII subunit in mRNA has also been primarily found in germ cells (25). these human cell lines represents RII,. Considering the presHowever, the fact that this subunit was nearly undetectable ent studies and in light of the fact that the skeletal muscle in our human RNA preparations may indicate that the sper- RII antibody used (40) in their studiesspecifically recognizes matogenesis in elderly men is reduced to such an extent that the RII, subunit, it is possible that the changes in protein the germ cell specifically containing this mRNA is lacking. levels attributed to RII, were instead changes in RII,.An Despite the fact that C, mRNA has been demonstrated in alternative possibility is that the isomeric RII subunits of human testis of a 14-year-old boy (25), we could not find CAMP-dependentprotein kinase in human display tissue- as significant levels of this message in our RNA preparations. well as cell-specific differences in size. However, results from Purification of R subunits by DEAE-cellulose and CAMP our laboratory indicate that normal human peripheral blood affinity chromatography revealed three distinct bands with T lymphocytes express an RII, subunit of 51 kDa as well.’ In apparent molecular masses of 49,51, and 53 kDa, respectively. addition to these findings, previous results from @yen et al. The three protein bands were shown to be CAMP-binding (16) demonstrate that human RII, diverge markedly in the proteins by photoaffinity labeling using 8-a~ido-[~*P]cAMP.N-terminal end (amino acids 45-87) compared with species The smallestprotein (49 kDa) was identicalin size and such as rat, bovine, mouse, and porcine, whereas the RIIB immunoreactivity to rat RI, and was considered to represent sequence is more conserved. Taken together, these data the human testis RI, subunit. Further support for this was strongly suggest that thehuman RII, may display a different the fact that thissubunit could not be autophosphorylated in apparent molecular weight compared with RII, in other spethe presence of -y-[32P]ATPand catalytic subunit (results not cies and may be smaller than RII,. shown). In a final attempt to prove this conclusion, we performed in uitro transcription/translation of full-length cDNAs for * K. Task&, S. M. Lohmann, B. Landmark, 0. @yen,B. Skilhegg, RII, and RIIP We also determined on SDS-PAGE the extent and T. Jahnsen, manuscript in preparation. a Portions of this paper (including “Materials and Methods,” “Re- to which the in uitro translatedproteinsdemonstrated 8ult8,” Figs. 1-9, and Table 1) are presented in miniprint at the end mobility shift upon phosphorylation/dephosphorylation.In

testis (23, 25); whereas the message for C, has only been detected in human testis(24). In thespecies so far examined R subunit sizes follow the order RII,, 54-56 kDa (14) > RII,, 51-52 kDa (17-19,29) > RI,, 49 kDa (11).Both RII, and RIIB are autophosphorylated (30), but only RII, shows an autophosphorylation-dependent mobility shift as on SDS-PAGE (30). The RIBprotein has been detected in mouse brain with an apparent molecular mass of 55 kDa (13), and C, has an apparent molecular mass of 40 kDa (31), whereas CBand C, have so far not been demonstrated at theprotein level. In rat testis, CAMP-dependent protein kinase subunits for RI,, RIIa, RII,, and C, have been identified both at themRNA and protein level, whereas the RIB andCBsubunits have only been described at the mRNA level. In our laboratory, human cDNAs for RI,, RIB,RII,, RII,, C,, CB,and C, have been cloned (12,16,21,24). In the present study we have used Northern blotanalysis to examine mRNAs for the various CAMP-dependent protein kinase subunits in human testis. We have further purified and characterized the R subunits of CAMP-dependent protein kinase from human testis anddemonstrate that thelargest RII subunit isRIIB(53 kDa) followed by the dephosphorylated form of RII, (51 kDa) and RI, (49 kDa). We have verified this by showing that RII, and RII, produced by in uitro transcription/translation behave identically to the nativeRIIsubunits purified from human testis. We also demonstrated that the mobility shift produced by phosphorylation/dephosphorylation is a more reliable characteristic for identificationof RII, than isapparent size determination by SDS-PAGE.

of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

‘B. S. Skilhegg, B. Landmark, S. 0. D!askeland, V. Hansson, T. Lea, and T. Jahnsen, manuscript in preparation.

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CAMP-dependent Testis Human inKinase Protein

these studies we clearly found that the in vitro transcribed/ translated subunits possess properties identical to the purified human testis RII subunits, both with respect to size and mobility shift upon phosphorylation/dephosphorylation. RII, (dephosphorylated) had an apparent molecular mass of 51 kDa, whereasRIIBrepeatedly was determined to 53 kDa. After in vitro phosphorylation, the RII, subunits increased its molecular mass to about 54 kDa, whereas the in vitro translated RIIBshowed no apparent change in size. In support of previous data from several species, our results demonstrate that the autophosphorylation-dependentmobility shift is a characteristic feature of the RII, subunit and a useful criterion for distinguishing RII,from RIIP It further suggests that the mobility shift due to autophosphorylation is a more characteristic property of the RII, subunit than is its apparent size on SDS gels. Our results indicate that some of the previous studies assuming that the51-kDa RII subunit found in human cells represents the RII, subunit need to be reevaluated. Acknowledgment-The antiserum against rat liver RI, was a gift from Dr. Stein 0. Dbskeland, Institute of Anatomy, University of Bergen, N-5009Bergen, Norway. REFERENCES 1. Kuo, J. F., and Greengard, P. (1969)Proc. NatL Acad. Sci. U.S. A. 64,1349-1355 2. Dorrington, J. H., Roller, N. F., and Fritz, I. B. (1975)Mol. Cell. Endocrinol. 3,57-70 3. Means, A. R., Fakunding, J. L., and Tindall, D. J. (1976)BioZ. Reprod. 14,54-58 4. Steinberger, A., Hintz, M., and Heind, J. J. (1978)Bwl. Reprod. 19,566-571 5. Spurill, W. A., Steiner, A. L., Tres, L. L., and Kierszenbaum, A. L. (1983)Proc. NatZ. Acad. Sci. U. S. A. 80,993-996 6. Tash, J. S., Hidaka, H., and Means, A. R. (1986)J. CellBwl. 103,649-655 7. Glass, D.B., and Krebs, E.G. (1980)Annu. Rev. Pharmucol. Toxicol. 20,363-388 8. Bramson, H. N., Kaiser, E. T., and Mildvan, A. S. (1984)CRC Crit. Rev. Biochem. 15,93-124 9. Corbin, J. D., Keely, S. L., and Park, S. R. (1975)J. BioZ. Chem. 250,218-225 10. Corbin, J. D., Sugden, P. H., West, L., Flochart, D.A., Lincoln, T. M., and McCarthy, D. (1978)J. Bwl. Chem. 253,3997-4003 11. Titani, K., Sasagawa, T., Ericsson, L. H., Kumar, S., Smith, S. B., Krebs, E. G., and Walsh, K. A. (1984)Biochemistry 23, 4193-4199 12. Sandberg, M., Taskin, K., @yen, O., Hansson, V., and Jahnsen, T. (1987a)Biochem. Bwphys. Res. Commun. 149,939-945 13. Clegg, C. H.,Cadd, G. G., and McKnight, G. S. (1988)Proc. Natl. Acad. Sci. U. S. A. 86,3703-3707 14. Takio, K., Smith, S. B., Krebs, E. G., Walsh, K. A., and Titani, K. (1984)Biochemistry 23,4200-4206 15. Hemmings, B. A., Schwartz, M., Adavani, S. R., and Jans, D. A. (1986)FEBS Lett. 209,219-222

16. @yen,O., Myklebust, F., Scott, J. D., Hansson, V., and Jahnsen, T. (1989)FEBS Lett. 246.57-64 17. Jahnsen, T., Lohmann, S. M., Walter, U., Hedin, L., and Richards, J. S. (1985)J. Biol. Chem. 260,15980-15987 18. Jahnsen, T., Hedin, L., Lohmann, S. M., Walter, U., and Richards, J. S. (1986)J. BwZ. Chem. 261,6637-6639 19. Jahnsen, T., Hedin, L., Kidd, V. J., Beattie, W. G., Lohmann, S. M., Walter, U., Durica, J., Schulz, T. Z., Schiltz, E., Browner, M., Lawrence, C. B., Goldman, D., Ratoosh, S. L., and Richards, J. S. (1986)J. Biol. Chem. 261, 12352-12361 20. Sandberg, M., Levy, F. O., Oyen, O., Hansson, V., and Jahnsen, T. (1988)Bwchem. Biophys. Res. Commun. 154,705-711 21. Levy, F. O., @yen, O., Sandberg, M., Taskin, K., Eskild, W., Hansson, V., and Jahnsen, T. (1988)Mol. Endocrinol. 2,13641373 22. Showers, M. O., and Maurer, R. A. (1986)J. Biol.Chem. 261, 16288-16291 23. Uhler, M. D., Chrivia, J. C., and McKnight, G. S. (1986)J. BioZ. Chem. 261,15360-15363 24. Beebe, S. J., @yen, O., Sandberg, M., Freysa, A., Hansson, V., and Jahnsen,T.(1989)Mol. Endocrinol. 4,465-475 25. @yen,O., Myklebust, F., Scott, J. D., Cadd, G. G., McKnight, S. G., Hansson, V., and Jahnsen, T. (1990)BioL Reprod. 43.4654 26. Frbysa, A., Ascoli, M., Segaloff,D., Beebe, S. J., Jahnsen, T., and Hansson, V. (1988)in The Molecular and Cellular Endocrinology of the Testis (Cooke, B. A., and Sharpe, R. M., eds) pp. 6572,Raven Press, New York 27. Massa, J. S., Fellows, R. E., and Maurer, A. (1990)Mol. Reprod. Deu. 26,129-133 28. Solberg, R., Taskin, K., Keiserud, A., and Jahnsen, T. (1991) Bwchem. Bwphys. Res. Commun. 176,166-171 29. Stein, J. C., and Rubin, C. S. (1985)J. Bwl. Chem. 260, 1099110995 30. Robinson-Steiner, A. M., Beebe, S. J., Rannels, S. R., and Corbin, J. D. (1984)J. Biol. Chem. 259,10596-10605 31. Carlson, G. M., Bechtel, P. J., and Graves, D. J. (1979)Adu. Enzyml. Relat. Mol. Bwl. 50, 41-48 32. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutters, W. J. (1979)Biochemistry 18,5294-5299 33. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982)Molecular Cloning: A Laboratory Manual, p. 447, Cold SpringHarbor Laboratory, Cold Spring Harbor, NY 34. Laemmli, U. K. (1970)Nature 227,680-685 35. Walter, U., and Greengard, P. (1983)Methods Enzyml. 99,154162 36. Bradford, M. (1976)Anal. Bwchem. 72,248-254 37. Nilsen, D.A., and Shatiro, D. J. (1986)Nuchic Acids Res. 14, 5963 38. Beebe, S. J., and Corbin, J. D. (1986)Enzymes (Basel) 17, 43111 39, Tortora, G., Clair, T., and Cho-Chung, Y. S. (1990)Proc. Natl. Acad. Sci. U. S. A. 87, 705-708 40. Ekanger, R., Sand, T. E., @greid, D., Christoffersen, T., and Dbskeland, S. 0.(1985)J. Bwl. Chem. 260,3393-3401 41. Nimmo, H. G., and Cohen, P. (1977)Adu. Cyclic Nucleotide Res. 48,923-927 42. Hofmann, F., Beavo, J. A., Bechtel, P. J., and Krebs, E. G. (1975) J. BwL Chem. 250,7795-7801

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CAMP-dependent Protein Kinase in Human Testis

milk (blacking medial a t room temperature for 30 minutes and incubated with antisera against either rat

RI,, ret RII, or rat ~ (diluted l l ~ 11100, ili000, or ii200, In blocking media. respecriveiyl Overnight. Afterwards fiicers were washed 10 min inTBS. 10 mi" in TBS containing 0.1 % Nonider P-40 and twlce for

I O mi"

I"

TBS. Filters were again blocked for 30 minutes and then incubated with goat anti-rabbit li2511-

IgG (NEX 155, New England Nuclear) (1 u C I l l 0 ml of 5 %skim milk-TBSI for two and a half hour. Filters were agam washed

as described above, air dried and erpmed for autoradiography (Kodak XAR-5 or

Amenham hyperfilm HPI.

Furif?EatlonOf Rlld I l l 1 Suhrmlts. A method described byCorbin

RI and RII. After

et ai. ( i o ) was modified and used lorpurifying

centrifugation (see preparation of cyfoplamic extracts), the supernatant was mixed with a suspensionof DEAE-cellulose (Whatman DE-52) suspended i n PEMT (four YolUmes of DEAE su~pemionper. gram of

imsue), gently mixed for ac l e s s 3 hours at 4'C and

then applied to a column IPharmacia K 50i60).

Gel msreriai was l e f t to settle and thereafter washed with 2wO m i of PEMT. The RI Subunit was eluted

from rhe column wlth one column volume O f 100 mM NsCl snd the RII subunits wlfh one column volume

of 400 mM NsCi. and both fractions subsequently added benramidlne to a final conCenfratlOn of 25 mM. The two f m ~ f i o n were s thereafter siowiy appiied to separate affinity Columns, each containing Iml oi 816-amin0hexyiamln0l-cAMP Sepharose (Pharmacial and washed wlrh 200 ml 2 M NaCi-PEMT over night.

Thereafrerthe

IO mi PEMT, and washed with

resins were equilibratedwith

10 ml 10 mM 5'AMP

imehringer Mannheiml and 10 m i PEMT. After the last PEMT IOlutlon had entered the resin completely. I m i of cGMP (1.25 mMI (Emehringer Mannhelml war added. The cGMP ~oiutionswere allowed t o enter the resins completely, and t o equilibrate for 10 minutes before being collected. The G M P equiiibratlonr were repeated three times. After the lest =GMP fractions were collected, i mi 01 cAMP (30 mM) (Sigma) was added and collected in thesame way as the cGMP frsctions except for allowing the cAMPt o equilibrate

for I2 hours, before rhey were collected. Finally, the affinity CoIUmns were equilibrated for 15 minutes

m

room temperature with I m i PEMT containing

8 M urea, and fracrlons coliected.

A i l fractions were analyzed by SDS-PAGE and chase containing R subunlts were dialyzed five times against PEMT 1500 ml per. I ml fraction) for 12 hours. Finaiiy fracrlons were cmcenrrared by diaiym against PEMT containing 50% glycerol and stored a t -20.C. The yield from 50 g of human testis Llssuc were 170 pg RI and 550 pg RIB, whereas 50 g Of rat brain gave 75 r g RI and 750 pg of RII respeccwely. All protein

measurements were done u s n g the 010 Rad protein assay ( X 500-00061, BI described by Bradford (361.

AutoOhosDhorvlatlonof RII subunits.

Phosphoryiafmn of RII subunits (either

1W

ng of purified RII subunits. 3 pl of

a

translared RII

products or 100 pg of cell extract1 was carried out In the presence of 10 mM porsss~um phosphate (pH 7.4). I mM EDTA, 10 mM magnesium acefare. I pg purifiedbovineheartC

subunir and i pl y-132PlATP

(Amershaml 15000 mCiimmoi) or 0.2 mM caidATP in B total volume of 10 pi. The mixture was incubated for 30 mmutes at 30'C, and

one hour a t 4'C. The reaction was terminated by adding SDS sample buffer

and bollmg for 5 minutes. Samples were analysed by SOS-PAGE gel electrophoresis and dried gels were exposed for autoradiography.

p Dephosphorylatux of R i i subunits (either 50 ng of Purified RII Proreina. 3 1 L i of

in rllrp

translated RII

products or 100 vg of cell extracts] were performed i n the presence of 50 mM Tris HCi (pH 8.0) 0.1 mM EDTA and12-20

U of calf aikaline phosphatase (Emehringer Mannheim) i n a

total volume of 10 pl. The

mlxtlues were incubated for 30-40 min at 37 'C and the reaction terminatedby adding SDS sample buffer and borling for 5 mi". Samples were analysed by SDS-PAGE electrophoresis, the gels dried and erpased for autoradiography.

In v i m f ~ ~ ~ ? p y l lud i o ntrawlation sf u n a M p . The cDNAI

encoding fuii length human RII,

and RIIo subunits of cAK (16,211 weye subcloned into a

Bluescript (pSK*)vector (Srraragene).Cesium chloride purified plasmid DNAwas passed over a Sepharose C L ~ 2 BIPharmacial column, RNA-free fracrlons were ethanol precipitated and linearized with BsmHl or Ndel reStriCtiOnenzymes, respectively. The DNA fempiafes were Pmteinase K-treated for 30 minuter a t

30 -C before phenol chloroform eitraCfiOn, ethanol plecipltationand resuspension m Rnase free TE buffer (pH 8.01. One pg of each linearized plasmid was transcribed

in rltrn using

10-20 U T7 RNA polymerase

(Stratagene) in a final reaction volume of 25 p i as previoUJly described (371. Afrer 1 hour lncubsrlon at 37

T the RNA was precipitated (2.5 voi of ethanol. 3M Na-ecefafe pH 5.01 and resuspended i n diethyl PYr(r8rbonate veered water 125 pi1containing

75 mMDTT

synthesized mRNA was examined quanrltatlvely

and 10 U RNmn (Promegal.

In

by spectrophotometry and qualitatively by gel

In ykm trmslation using 1-2 pg of RII, or RliB mRNA was acccamplahed in rltrn fransi*fion system according IO the manufactures descrlprion IPromegsl,

eleCfrOpholese ( 1 % agarose).

by using e wheat germ

in the Presenceof 135Slmethionine (Amerrhaml.Labeled translation products were analysed by SDS-PACE electrophoresis, the gels dried and exposed for autoradiography.

CAMP-dependent Protein Kinasein HumanTestis

5378

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7.5.

RII,

c

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4.4m

28s

2.418s 1.4-

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49

37

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t67

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CAMP-dependent Protein Kinasein Human Testis RII

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Dephospho.

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Human Rat

5379 Human Rat

Human RII

Bovine RII,

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