A possible involvement of type II cAMP-dependent protein kinase in the initiation of DNA synthesis by rat liver cells

Preliminary 3. Kato, H, Int rev cytol49 (1977) 55. 4. Fogel, M & Sachs, L, Virology 40 (1970) 174. 5. Rothschild, H & Black, P H, Virology 42 (1970) 251. 6. Kaplan, J C, Wilbert, S M, Collins, J J, Rakusanova, T, Zamansky, G B & Black, P H, Virology 68 (1975) 200. 7. Aaronson, S A & Stephenson, J R, Biochim biophys acta 458 (1976) 323. 8. I2b;sch, M S &Black, P H, Adv virus res 19 (1974) 9. Kaplan, J C, Zamansky, G B, Black, P H & Latt, S A, Nature 271 (1978) 662. 10. Zamansky, G B, Little, J B, Black, P H & Kaplan, J C, Mutat res 51 (1978) 109. 11. Zamanskv, G B, Kleinman. L F. Little. J B. Black. P H & Kaplan, J C, Virology 73 (1976) 468. 12. Rakusanova, T, Kaplan. J C. Smales. W P & Black, P H, J virol I9 (1976) 279. 13. Teich, N, Lowy, D R, Hartley, J W & Rowe, W P, Virology 51 (1973) 163. 14. Lieber, M M, Livingston, D M & Todaro, G J, Science 181 (1973) 443. 15. Stephenson, J R & Aaronson, S A, Proc natl acad xi US 71 (1974) 4925. 16. Fischinger, P .I, Tuttle-Fuller, N, Hiiper, G & Bolognesi, D P, J virol 16 (1975) 267. 17. Paskind, M P, Weinberg, R A & Baltimore, D, Virology 67 (1975) 242. 18. Perry, P&Wolff, S, Nature 251 (1974) 156. 19. Latt, S A, Allen, J W, Rogers, N E & Juergens, L A, Handbook of mutagenicity test procedures (ed B Kilbey, M Legator & C Ramel) p. 275. Elsevier-North Holland, Amsterdam (1977). 20. Lieber, M M, Sherr, C J & Todaro, G J, Int j cancer 13 (1974) 587. 21. Rowe, W P, Pugh, W E & Hartley, J W, Virology 42 (1970) 1136. 22. Brown, R L & Crossen, P E, Exp cell res 103 (1976) 418. 23. Nichols, W W. Bradt, C I. Toii, L H. Godlev. M & . Segawa, M, Cancer res 38 (1978) 960. 24. Kate, H. EXP cell res 82 (1973) 383. 25. - Ibid 85 (i974) 239. i 26. Weerd-Kastelein, E A, Keijer, W, Rinaldi, G & Bootsma, G, Mutat res 45 (1977) 253. 27. Palitti, F & Becchetti, A, Mutat res 45 (1977) 157. 28. Waksvik, H, Brogger, A & Stene, J, Hum genet 38 (1977) 195. 29. F.ihlman. B A & Sturelid, D, Hereditas 88 (1978) 30. Ishii, Y &Bender, M A, Mutat res 51 (1978) 419. 31. Vogel, W & Bauknecht, T, Hum genet 40 (1978) 193. 32. Faed, M J W & Mourelatos, D, Mutat res 49 (1978) 437. 33. Tenant, R W, Rascati, R J & Lavelle, G C, International symposium on radiation-induced leukemogenesis and related viruses (ed J F Duulan) D. 179. Elsevier-North Holland. Amsterdam (19?7). 34. Carrano, A V, Thompson, L H, Lindl, P A & Minkler, J L, Nature 271 (1978) 551. Received October 8, 1979 Accepted December 10, 1979

notes

471

Copyright (0 19X0 by Academic Pres\, Inc. All I-jghf> of reproduction many form reserved 0014-4x?7/x0/040477-05$0?.lxf/0

A possible involvement of type II CAMP-dependent protein kinase in the initiation of DNA synthesis by rat liver cells A. L. BOYNTON and J. F. WHITFIELD, Animal und Cell Physio/ogy Group. Division of Biological Sciences, Nationul Reseclrch Council of Ctrnudrr. Ottcrwu, Ont., Crinclda K/A OR6 A brief, protein kinase-activating CAMP surge and initiation of DNA synthesis occurred within 1 h after the addition of calcium to calcium-deprived cultures of T5lB epithelioid rat liver cells. DNA synthesis was also stimulated by a preparation of type II CAMP-dependent protein kinase from rabbit muscle, but not by an equally active preparation of type I CAMP-dependent protein kinase or by a boiled type II protein kinase preparation. The stimulatory actions of calcium and the active type II protein kinase preparation were both blocked by a specific inhibitor (from beef heart) of the catalytic subunits of CAMP-dependent protein kinases. These observations suggest that type II protein kinase might function specifically in the initiation of DNA synthesis.

Summcq.

Early observations on cultivated cells such as BALBlc 3T3 mouse cells suggested that CAMP (adenosine 3’,5’-monophosphate) regulates cell proliferation by blocking an early stage of prereplicative development [4, 171. Therefore, the cellular CAMP content must drop before cells arrested in early Gl phase can resume cycling. However, substantial CAMP surges at the beginning of the prereplicative development of hepatocytes, kidney tubule cells and parotid gland acinar cells [5, 11, 16, 19, 221 invalidate this idea. Moreover, even quiescent BALB/ c 3T3 cells under certain circumstances resume cycling without lowering their CAMP content [4]. Although CAMP may not regulate early prereplicative development, a brief accumulation of it later in the Gl phase of cells such as hepatocytes in vivo, parotid gland acinar cells in vivo, spleen lymphocytes in vitro, and TSlB epithelioid rat

478

Preliminary notes Table 1. An example of the effect of boiling on the actions of types Z and ZZCAMPdependent protein kinases on the DNA-synthesis activity of T51B epithelioid rat liver cells in low (0.02 mM) calcium medium

01 0 I2 34 Hours of incubation

d

IO loo pglrn cone

Fig. I. An example of (A) the CAMP surge [O] and

prompt initiation of DNA synthesis [0] caused by adding calcium to cultures of TSlB epithelioid rat liver cells which had been incubated for 24 h in low (0.02 mM) calcium medium. Note: The CAMP content and DNA-synthesis activity did not change in untreated cultures. The points are the means f S.E.M. of the values in 4-10 cultures. (B) Abscissa: CAMP-dependent protein kinase inhibitor cont. &g/ml); ordinate: (A, right; B, left) % of cells with [3H]thymidine-labeled nuclei (B) at 2 h after calcium and inhibitor additions. An example of the prevention of calcium’s stimulatory action by an inhibitor of the catalytic subunits of CAMP-dependent protein kinases. Cultures of T5lB epithelioid rat liver cells were incubated in low (0.02) calcium medium for 24 h. Calcium was then added to a final cont. of 1.25 mM and inhibitor was added at the same time. The points are the means? S.E.M. of the values in 4 cultures.

Addition

% of DNAsynthesizing (i.e., [“HIthymidinelabeled) cells at 2 h after protein kinase addition

None Type I protein kinase Boiled type I protein kinase Type II protein kinase Boiled type II protein kinase

20.3+0.5 21.621.7 20.710.9 45.5k2.2 20.350.9

Type I or II protein kinase preparation was added to a final concentration of 25 pg/ml which had an enzyme activity of 7.5 phosphorylation U/ml. The stock kinase solutions (containing 1 mg (300 phosphorylation U)/ml) in phosphate-buffered saline were inactivated by boiling for 10 min. The values are the means f S.E.M. of the values in 4 cultures.

sibility of type II CAMP-dependent protein kinase being specifically involved in the initiation of DNA synthesis. Materials and Methods

liver cells in vitro is needed for the initiation of DNA synthesis [3, 16, 19, 21, 221. Therefore protein kinase activity is probably involved in the initiation of DNA synthesis, because the many other actions of CAMP seem to be due to the activation of such enzymes [13]. Indeed, synthesis and activation of one of these enzymes, the type II CAMP-dependent protein kinase [13, 151, at a specific point in the Gl phase seems to be linked to the initiation of DNA synthesis by Chinese hamster cells [6-10, 121. In this communication we will present evidence obtained with T5 IB epithelioid rat liver cells [18] which supports the posExp Cell Res 126 (1980)

T5lB epithelioid rat liver cells were isolated by Swierenga et al. [18]. Proliferation of these cells can be inhibited by lowering the extracellular calcium concentration [3, 18, 221. To do this, cells were first planted at a density of 0.7~ 104/cmZin a high (1.8 mM) calcium medium consisting of 10% (v/v) FBS (fetal bovine serum from Flow Lab, Rockville, MD), 90% (v/v) BME (Eagle’s basal medium, also from Flow) and the antibiotic gentamicin (from Microbiological Ass., Bethesda, MD). They were then incubated for 24 h at 3PC (in an atmosphere consisting of 95% air and 5% CO,) to ensure maximum attachment and spreading, after which the high-calcium medium was replaced by low (0.02 mM)-calcium 10% (v/v) FBS, 90% (v/v) BME medium. Methods for adjusting the free calcium concentration in serum and BME have been described elsewhere [2, 4, 181. Experiments began 24 h later when the percentage of cells making DNA in the low-calcium medium had fallen from the normal level of 60% [3] to less than 20%. A protein inhibitor (from beef heart) of the catalytic subunits of types I and II CAMP-dependent protein kinases [20] was obtained from Sigma Chemical Co.

Preliminary notes

Depending on its protein content, each dried cell extract was dissolved in either 0.5 or 1.Oml of sodium acetate buffer (pH 6.2), and its CAMP acetylated at the 2’0 position by adding 10 ~1 of triethylamine and 5 ~1 of acetic anhydride [l4]. The CAMP content of each sample was determined by radioimmunoassay with kits from Becton Dickinson lmmunodiagnostics (Orangeburg, NY). The DNA-synthetic activity was determined autoradionraohicallv (i.e.. from the orooortion of cells with [SH]thymidine-iabellkd nuclei) according to Boynton & Whitfield f2l. a orocedure in which f3Hlthvmidine is specifically incorporated into DNA idu;ing a 1 h exposure to the nucleoside) and yields a minimum of 500 grains/nucleus in developed autoradiographs.

Type1 Pruteii kirmse

lOI

0’b.l

IO

10.0 loo.0

479

0

I2

34

Results and Discussion Boynton & Whitfield [3] have shown that Fig. 2. (A) Abscissa: cont. of CAMP-dependent prolowering the calcium concentration in the tein kinase preparation [phosphorylation U/ml]: ordinate: % of cells with [“Hlthymidine-labeled nuclei at 10% PBS-90% BME medium from 1.8 2 h after protein kinase addition. to 0.02 mM stops the proliferation of TSlB An example of the effects of various concentrations of partially purified preparations of rabbit muscle types epithelioid liver cells in late Gl phase, and I and II CAMP-dependent protein kinases on the DNAsynthesis activity of T51B epithelioid rat liver cells in that subsequent calcium addition causes a low (0.02 mM) calcium medium. 0, Type I protein CAMP surge which peaks at 15 min and is kinase; 0, type II protein kinase. The points are the responsible for the initiation of DNA synmeans I!ZS.E.M. of the values in 4 cultures. (B) Abscissa: hours of incubation; ordinare: % of thesis which follows soon afterwards. A cells with [3H]thymidine-labeled nuclei. An example of typical example of these calcium-induced the prompt initiation of DNA svnthesis bv T51B enithehoid rat liver cells (in low (0.02 mk) calcium events is provided in fig. 1A. medium) after addition of type II protein kinase to a CAMP-dependent protein kinases (which final concentration of 25 pg (7.5 phosphorylation U)/ ml. l , No addition; 0, type II protein kinase added are strongly activated in T51B cells by calat time “0”. The points -are the means f S.E.M. of cium addition [22]) appeared to be involved the values in 4 cultures. in the calcium-induced initiation of DNA synthesis, because calcium-deprived T5 1B cells did not initiate DNA synthesis when [St Louis, Mol. Partiallv purified prenarations of tvoes ] and II (or peaks I and il) CAMP-dependent protein the CAMP-dependent protein kinase inkinases ]13, 151,isolated from rabbit muscle according to Beavo et ai. [l], were also obtained from Sigma hibitor was added (at concentrations beChemical Co. In both preparations, the phosphorylattween 20 and 100 pg/ml) at the same time ing activity was 0.3 U/pg of protein (biuret), 1 U as calcium (fig. 1B). Protein kinase involvebeing the amount of protein kinase activity which transfers 1.0 pmole of phosphate from [-,J*P]ATP ment was further indicated by the observato hydrolysed and partially dephosphorylated casein tion that addition of type II CAMP-dependin 1 min at pH 6.5 and 30°C. To determine cellular CAMP levels, the cultures ent protein kinase preparation (at a maxiwere very rapidly (10 set) rinsed three times in icemally effective concentration such as 25 cold phosphate-buffered saline and immediately floated on an acetone-dry ice mixture. The frozen pg (7.5 phosphorylation units)/ml [fig. 2A]) cells were then scraoed from the Petri dishes in an stimulated calcium-deprived T5 1B cells as ice-cold solution of -90 % ethanol containing 0.1 N HCl and disruoted bv_ sonication. Part of each soni- effectively as calcium (fig. 2B), although . cated homogenate was set aside for the determination the DNA-synthetic response to this enzyme of protein content usine Biorad orotein assav kits (B&ad Laboratories, Richmond, CA). The remainder preparation, unlike the response to calcium, was centrifuged at 700 a for 15 min at 4°C. and the supematant dried unde; reduced pressure ‘at 6S”C. was transient (compare figs IA and 2B). Cont.

(U/ml)

Hours of

incubation

E.vp Cdl Re.\ 126 (IYXU)

480

Preliminary

notes

I-1

J

OL -60

-30

0

t30

Min

Fig. 3. Abscissa:

time of addition of CAMP-dependent protein kinase inhibitor (min before (-) or after (+) addition of type II protein kinase preparation); ordinate: % of cells with [3H]thymidine-labeled nuclei at 2 h after protein kinase addition. An example of the ability of CAMP-dependent protein kinase inhibitor to prevent type II protein kinase from stimulating the initiation of DNA synthesis by TSlB epithelioid rat liver cells in low (0.02 mM) calcium medium. The protein kinase was added to a final cont. of 25 pg (7.5 phosphorylation U)/ml and the inhibitor was added to a final cont. of 100 pg/ ml various times before or afterwards. The shaded area represents the mean (5S.E.M.) DNA-synthetic activity in 4 cultures exposed to type II protein kinase only. The points are the means k S.E.M. of the values in 4 cultures variously treated with the type II protein kinase and its inhibitor.

Indeed it appeared that the cells responded specifically to the active type II protein kinase in this preparation because neither a heat-inactivated (boiled) type II protein kinase preparation nor an active type I CAMP-dependent protein kinase preparation stimulated T51B cells to initiate DNA synthesis in low (0.02 mM) calcium medium (fig. 2A; table 1). As expected, the DNA-synthetic response to the type II protein kinase preparation was preventable by the CAMP-dependent protein kinase inhibitor. However, because the exogenous protein kinase acted swiftly, the inhibitor was completely effective only when added between 60 min before and 1 min after the enzyme preparation

(fig. 3). It did not stop the initiation of DNA synthesis when added more than 5 min after the enzyme (fig. 3). Incidentally, this failure of a later addition of the inhibitor to affect the stimulation of DNA synthesis indicated that its inhibitions of the DNA-synthetic responses to calcium and the type II protein kinase were not due to inhibition of DNA synthesis itself or to a general toxicity. These observations support those on cultivated hamster cells [6-10, 121 which indicate that an activation of type II protein kinase by endogenous CAMP is specifically needed for the initiation of DNA synthesis. Moreover, the large size of the type II protein kinase molecule and the rapidity of the DNA-synthetic response both to it and to low concentrations of exogenous CAMP, which stimulate protein kinase activity without readily entering cells [3, 221, suggest that the initiation of DNA synthesis might start with the phosphorylation of some protein in the cell membrane. Thus, in normally cycling cells, the Gl CAMP surge [3, 6-10, 12, 16, 19, 21, 221 would provide the active catalytic subunits from endogenous type II protein kinase needed to phosphorylate this membrane protein and thereby start the DNA-synthetic processes. References 1. Beavo, J A, Bechtel, P J & Krebs, E G, Methods in enzymology (ed J C Hardman & B W O’Malley) vol. 38, p. 299. Academic Press, New York, San Francisco & London (1974). 2. Boynton, A L & Whitfield, J F, Proc natl acad sci US 73 (1976) 1651. 3. - J cell physiol 101 (1979) 139. 4. Boynton, A L, Whitfield, J F, Isaacs, R J & Tremblay, R, Life sci 22 (1978) 703. 5. Comber, H J & Taylor, D M, Biochem sot trans 2 (1974) 74. 6. Costa, M, Molecular biology and pharmacology of cyclic nucleotides (ed G Folco & R Paoletti) p. 109. Elsevier-North Holland Biomedical Press, Amsterdam, Oxford & New York (1978). 7. Costa, M, Gemer, E W & Russell, D H, Biochim biophys acta 425 (1976) 246.

Preliminary 8. - J biol them 251 (1976) 3313. 9. - Biochim biophys acta 538 (1978) I. 10. Cress, A E & Gemer, E W, J cell biol 75 (1977) 14a. 11. Durham, J P. Baserga, R & Butcher, F R, Biochim biophys acta 372 (1974) 196. 12. Gerner, E W, Newsome, V L & Holmes, D K, J cell biol75 (1977) l4a. 13. Greengard, P, Cyclic nucleotides, phosphorylated proteins and neuronal function. Raven Press, New York (1978). 14. Harper, J F & Brooker, G, J cyclic nucleotide res 1 (1975) 207. 15. Lincoln, T M & Corbin, J D, J cyclic nucleotide res 4 (1978) 3. 16. MacManus, J P, Braceland, B M, Youdale, T & Whitfield, J F, J cell physiol 82 (1973) 157. 17. Pastan. I. Johnson. G S & Anderson. W B. Ann rev b&hem 44 (19;s) 491. 18. Swierenga. S H H. Whitfield. J F & Karasaki. S. Proc nag acad sci US 75 (1978) 6069. 19. Tsang, B K, Rixon, R H & Whitfield, J F, J cell uhysiol. In press. 20. Walsh, D A-& Ashby, C D, Recent progr horm res

notes

481

sional gel electrophoresis (NEPHGE). Visual inspection of the fluorograms revealed that the injection of globin mRNA (up to 14000 molecules/cell) does not alter significantly the relative intensity of the major acidic (IEF) and basic (NEPHGE) polypeptides synthesized by the cells.

The use of the technique of direct microinjection with micropipets to study biological processes in vivo has been hampered in many cases by the lack of suitable assays to detect the effect of the microinjected macromolecule(s). Usually due to the small number of cells that can be injected detection assays rely mainly on immunofluorescent, autoradiographic or virus plaque assays [l-6]. We report here experiments in which microinjection of as few as 100 mouse 3T3 cells with rabbit globin mRNA 29(1973)329. 21. Wang, T, Sheppard, J R & Foker, J E, Science 201 followed by incubation with [“5S]methionine (1978) 155. 22. Whitfield, J F, Boynton, A L, MacManus, J P, results in the synthesis of a basic polySikorska, M & Tsana. B K. Mol cell biochem 27 peptide (mol. wt 15 K) that co-migrates (1979)155. with [3H]leucine-labelled rabbit globin in Received October 8, 1979 high resolution two-dimensional gels Accepted December 10, 1979 (NEPHGE [7]). This basic polypeptide is the only new protein that can be detected among 500 polypeptides (acidic+basic) that can be reproducibly separated by high resolution two-dimensional gel electroCopyright @ 1980 by AcademK Press, Inc. phoresis (IEF [8], NEPHGE [7]). These reAll rights of reproduction in any form reserved 0014.4827/80/040481-05$n2.00/0 sults confirm and extend observations of Graessmann (personal communication) and Direct microinjection of rabbit globin mRNA Stacey & Allfrey [3] who have shown by into mouse 3T3 cells. Analysis of the means of immunofluorescent techniques polypeptides synthesized in vivo synthesis of a globin-like protein in somatic RODRIGO BRAVO and JULIO E. CELIS,’ Division cells microinjected with rabbit globin of Biostructural Chemistry, Department of Chemistry, mRNA. Aarhus University, DK-8000 Aarhus C, Denmark 3T3 cells grown attached to 9 mm2 coverslins have been microiniected in the cvtonlasm with tob rabbit globin mRGA and the polipeptides synthesized after injection have been labelled with [35S]methionine under conditions in which the product of as few as 100 cells could be analvsed bv high resolution two-dimensional gel eIectropGoresis- followed by 10 days’ fluorography. Microinjection of rabbit globin mRNA results in the synthesis of a basic polypeptide of mol. wt 15 K that is not present in control cells, and that co-migrates with purified [3H]leucine-labelled globin as determined by high resolution two-dimen-

Summary.

Printed

in Sweden

Material

and Methods

Mouse 3T3 cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and containing 50 units of penicillin and 50 pg of streptomycin per ml. For microinjection and or labelling the cells were seeded in 9 mm2 sterile coverslips (Micro coverglass 3 x3 mm, Bellco Glass, Inc.) placed in NUNC Microtest plates (NUNC, catalog 1480, Den-

Cell cultures.

’ To whom correspondence should be addressed.