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Protein phosphorylation in intact lymphocytes stimulated by Concanavalin A
Copyright @ 1981 by Academic Press. Inc. All rights of reproduction m any form reserved 0014.4827/81/080409-07$02.00/O
Cell Research 134 (1981) 409-415
Experimental
PROTEIN
PHOSPHORYLATION STIMULATED
TINGCHUNG ‘Department
of Surgery.
IN INTACT
BY CONCANAVALIN
WANG.’ JOHN E. FOKER’ and ALVIN University University
LYMPHOCYTES
of Minnesotu, of Colorado.
Minneapolis, Boulder,
M. MALKINSON”
MN 55455, CO 80309,
A
rind 2School
* of Pharmacy,
USA
SUMMARY The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of azP incorporation into two proteins of 135000 and 150000 mol. wt began to increase, relative to control cells, IO h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of-both proteins. Such treatments included the displacement of conA from its surface receptors by a-methvl-mannoside (aMM), the omission of serum from the culture medium, and the presence-of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135000 and I50000 mol. wt proieins have a common physiological fun&on. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.
The biological activity of many proteins is determined by whether they are in a phosphorylated or dephosphorylated form. Alterations in phosphate content accompany, and are thought to mediate, the rapid adjustment of cell metabolism to many environmental changes [l]. It is not surprising, therefore, that cyclic changes in protein phosphorylation/dephosphorylation occur during cell proliferation. Phosphorylation at multiple sites in specific histones occurs sequentially during the cell cycle, and may regulate chromosomal condensation [2]. Similarly, changes in the phosphorylation of non-histone chromosomal [3], cytosolic [4], and membrane-bound proteins [5] have been observed when the proliferative status 27-811816
of cells was altered. Neither the physiological consequences of these changes nor the functional identity of these phosphoproteins is presently understood. Lymphocytes are normal resting cells which can be stimulated by a variety of mitogens to enter a proliferative cycle. We recently studied the phosphorylation of endogenous proteins in mouse lymphocyte cell lysates [6]. A 135000 mol. wt cytosolic protein was intensely phosphorylated following lymphocyte activation by conA, with maximal phosphorylation occurring concomitantly with the peak of DNA synthesis. Enhanced phosphorylation of a * To whom reprint requests should be
addressed.
Exp Cell Res 134 f/98/)
410
Wang, Foker and Malkinson -Con A
MATERIALS
+Con A
AND METHODS
Lymphocyte culture 150 ooo135 oooe
0
-200
000
.,..%r&r*----
-130 -94
000 000
-24
000
-5
,(
Mouse spleen lymphocytes were isolated from A/Jx C57BL/6F, hybrids and cultured as described TlOl. Spleens w&e-minced in a loose-fitting tissue g&d& to obtain a Ivmahocvte-rich cell susoension. which. after centrifigaiion at 200 g for I min to’ remove tissue debris, was treated with 2 vol of 0.87% NH&I to lyse erythrocytes. Lymphocytes were then washed twice with 0.9% NaCI. once with culture medium (RPM1 1640), and then iesuspended to achieve a cell density of 5x lo6 cells/ml in RPM1 1640 medium containing 2% heat-inactivated human serum, 2 mM Lelutamine. 50 U/ml of oenicillin and 50 me/ml of streptomycin. The cultur& were put in a hu&idified incubator at 37°C under 5% CO,. in the nresence or absence of 2 pglml of conA. Cell number was determined with a hemocytometer. The viability, which was assessed by Trypan blue exclusion, decreased from 100 to 80% during the first 24 h and stabilized at 70% for the rest of the culture period in both control and conA-stimulated cells.
Nucleic acid synthesis
0
The rates of RNA and DNA synthesis were estimated by incorporation of r3H]uridine ([3H]U, 5 &i/ml of cell culture, 6 Cilmmole) and [3H]deoxythymidine VHldT. 2.5 &i/ml of cell culture. 6 Cilmmole), respectively, into &ichloroacetic acid (?CA)-precipitable material during a 1 h labeling period [IO]. Cells were collected on a glass fiber filter with a microculture harvester, washed with 5% TCA, and airdried. The amount of incorporated radioactivity was determined in a Beckman liquid scintillation counter. AL
I. Autoradiogram of a polyacrylamide gel showing 32Pincorporation into protein from intact mouse spleen lymphocytes. Lymphocytes were cultured for 48 h in the absence or presence of conA. They were phosphorylated with’ 32P, and electrophoresis and autoradiography were performed as described in Materials &id Methods. Numbers on the left indicate the estimated mol. wt of two major phosphoprotein bands whose phosphorylation increased following conA stimulation. The numbers on the right indicate the mol. wt of known protein standards.
Fig.
cytosolic protein of identical size has been observed in various proliferating cell types including tumors and fetal tissues [7], serum-fed confluent cultured cells [8], and healing lung tissues [9]. Cell extracts were used in these studies, so it is important to determine whether similar reactions occur in intact cells. We now report that the phosphorylation of a 135000 mol. wt protein, as well as of a 150000 mol. wt protein, increases following conA stimulation in intact mouse lymphocytes. Preliminary reports of some of this work have appeared [29, 301. Exp Cell Res 134 (1981)
~~~~,
~~
,
Protein phosphorylation in intact cell suspensions Cultured cells were washed twice and resuspended to a density of 5x 10’ cells/ml in 30 mM Tris-HCI (pH 7.6) containing 0.15 M NaCl, 4 mM MgCI, 0.3 mM EDTA, and IO mM NaF. Fifty PCi of carrierfree [32P]orthophosphoric acid were added per ml of cell suspension, and incubated at 37°C for 60 min. After incubation the cells were centrifuged at 400 g for 5 min, resuspended in 200 ~1 of lysjs buffer (lo mM Tris-HCI. .oH 7.5. 3 mM MgCI, and 500 units of micrococcal nuclease) and the protein concentration was adiusted to 1 mE/ml with a 1% sodium dodecyl sulfate”(SDS) -40 m& dithiothreitol solution. Protein concentration was determined by the method of Lowry et al. [II].
Gel electrophoresis Phosnhorvlated nroteins from intact cells were fractionated by polyacrylamide slab gel electrophoresis [ 121. Subsequent autoradiography and the quantitative measurement of 32P incorporation into protein bands by microdensitometry were performed as previous1.ydescribed [ 131.
Protein phosphorylation in intact lymphocytes
411
9 12 10 8 6 4 2 0 10 8 6 4 2 0 60
GEL LENGTH (cm)
Fig. 2. Densitometric tracings of autoradiogram from fig. 1 showing phosphoprotein profiles of intact lymphocytes. Intact lymphocytes cultured without (. . .) or with (-) conA for 48 h were phosphorylated, and autoradiographs made. The extent of 32Pincorporation into proteins is indicated by the relative band density of the individual proteins separated by electrophoresis (densitometric units). Peaks A and B represent the 150000 and 135000 mol. wt phosphoprotein bands.
RESULTS ConA-stimulated phosphorylation of specific proteins in intact lymphocytes The effects of conA on protein phosphorylation in intact lymphocytes are shown in an autoradiogram (fig. 1). Similar patterns of distinct phosphorylated bands can be seen in both control and conA-treated cells with the exception that two proteins of 135000 and 150000 mol. wt were consistently more prominent in the conA series. The 135000 mol. wt protein co-migrated with a highly phosphorylated protein obtained when extracts from conA-treated cells were incubated with [y-32P]ATP [6]. The 150000 mol. wt band which was phosphorylated in intact cells was not phosphorylated in the cell-free assay system. Densitometric tracings comparing lymphocytes cultured for 48 h in the presence and absence of conA also showed that the band density in peaks corresponding to 135000 and 150000 mol. wt were markedly increased in the conA-treated cells (fig. 2).
60
50
50
40
40 g
30
30 e
20
20 : F IO"
10 0 0
IO Culture
20
30 time
40
7 0 5c )
(houn)
Fig. 3. Changes in protein phosphorylation, RNA and DNA synthesis in lymphocytes cultured with conA for different lengths of time. 0, 0, Amounts of [32P]phosphate incorporation (densitometric units/l06 viable cells/h) into (A) 135000 and (B) 150000 mol. wt proteins; A, A [3H]U and 0, W [3H]dT incorporation (cpmx 1O-3/1o6viable cells/h) into lymphocytes cultured without (open symbols) or with conA (closed symbols).
The extent of phosphorylation of the 135000 and 150000 mol. wt proteins in intact conA cells increased with culture time (fig. 3). During the first 8-10 h that the cells were cultured in the presence of conA, no difference was observed between the phosphorylation of the two proteins in the control and conA-stimulated intact cells. Phosphorylation in the conA cells began to increase above control levels at approx. 10 h, and had increased lo-fold by 50 h. The time course of phosphate incorporation into both proteins appeared to be identical, and similar to that previously found for the 135000 mol. wt protein using lymphocyte extracts [6]. Compared to the time course of nucleic acid synthesis, increased phosphorylation began several hours after the initial rise in r3H]U incorporation into RNA, but preExp Cell Res 134 (1981)
412
Wang, Foker and Malkinson
Con A serum + ,“dO. -
$ -
1 -
+ -
+ +
:: +
Fig. 5. Effects of serum omission or the addition of
0.2 mM indomethacin on protein phosphorylation and DNA synthesis in intact lymphocytes. Lymphocytes were cultured in the presence or absence of serum for 30 h. Indomethacin was added to some cells beginning 2 h after the cells were placed in culture. All samples were then harvested and assayed for either [32P]phosphate incorporation into proteins (densitometric units/lo6 viable cells/h) or r3H]dT incorporation (cpmx 10-3/106viable cells/h) into DNA. a, 135OOOmol.wt protein; 0, 150000 mol. wt protein; W, r3H]dT incorporation. Fig. 4. Effects of cxMM addition on protein phosphorylation and DNA synthesis in conA-stimulated lymphocytes. [32P]Phosphate incorp. (fold of incorp. compared to cells not treated with conA) into the (A) 135000, and (B) 150000 indicate proteins. (C) r3H]dT incorp. (cpmx 10~“/lo” viable cells/h). Protein phosphorylation and DNA synthesis were measured in cells which were cultured in the presence of conA for 24 h. At various times after the initial addition of conA, unbound conA was removed by washing and resuspending the cells to 5x lo6 cells/ml in RPM1 1640 medium (O---O). Half of the cultures also contained 100 mM aMM in the resuspension medium (O-O), which displaces bound conA [14]. cxMM addition time refers to the length of time that cells had been in the presence of conA prior to the addition of (wMM.
after the initial conA stimulation, endogenous protein phosphorylation in intact cells and [3H]dT incorporation into DNA were measured. CXMMtreatment during the first 5-6 h abolished the conA-stimulated incorporation of 32Pinto the 135000 and 150000 mol. wt proteins, and also prevented [3H]dT incorporation into DNA (fig. 4). As the duration of exposure to conA prior to treatment with (YMM lengthened, progressively more cells synthesized DNA and phosphorylated the two proteins. By 15 h, phosphoceded the onset of [3H]dT incorporation rylation was no longer affected by aMM into DNA (fig. 3). treatment, although [3H]dT incorporation Effects on protein phosphorylation of remained sensitive until 21 h. interrupting conA stimulation The effects of blocking lymphocyte acMitogenic stimulation by conA can be spe- tivation at later times were also investicifically reversed with CXMM, which dis- gated. Lymphocytes ,require serum beginplaces conA from the cell surface and there- ning 10 h after conA stimulation [ 151.When by discontinues the mitogenic signal [14]. cultured continuously in the absence of seAt different times after culture with conA, rum, mitogen-stimulated cells remain in late lymphocytes were washed and resuspended Gl phase but will synthesize DNA if sein a serum-containing medium, with or rum is added. When [3H]dT incorporawithout 100 mM (YMM. Twenty-four hours tion into DNA was inhibited by omitting Exp
Cell
Rcs
134 (19811
Protein phosphorylation in intact lymphocytes
413
ing that the inhibition was not due to a cytotoxic effect of the drug at this concentration. Protein phosphorylation increased 4 h after the lymphocytes were washed free of the drug and incorporation of [3H]dT into DNA began 8 h after removal of indomethacin (fig. 6). This result shows that the temporal relationship between protein phosphorylation and the progression to S phase was maintained in indomethacin-inhibited cells. DISCUSSION Cyclic AMP stimulates the phosphorylation of several lymphocyte proteins in vitro 30 34 38 42 46 [17]. Cyclic nucleotides may also regulate Culture time (hours) the phosphorylation of lymphocyte proFig. 6. Reversible effects of indomethacin on protein teins in intact cells during very early stages phosphorylation and DNA synthesis in intact lymphocytes. ConA-stimulated lymphocytes were cultured of mitogenic signal reception. Increases in without indomethacin (O), with a continuous exposure cellular CAMP [18] and cGMP [ 191 occur to 0.2 mM indomethacin (A), or with 0.2 mM indomethacin for 30 h and then washed to remove indowithin minutes after mitogen treatment, and methacin (A). At the indicated times, cells were ascGMP [20] and monobutyryl CAMP [21] sayed for protein phosphorylation and DNA synthesis. Points with vertical range bars indicate mean + S.E.M. can enhance 32Pincorporation into proteins of duplicate cultures; those without error bars have of a S.E.M.s smaller than dimension of symbols. f3zP]- in intact lymphocytes. Activation phosphate incorp. (densitometric units/<06 viable cells/ CAMP-dependent protein kinase following h); [3H]dT incorp. (cpmx 1O-3/1O6viable cells/h). (A) mitogenic stimulation of lymphocytes oc135000; (B) 150000 mol. wt proteins. curs a few hours after mitogenic stimulation [22]. In addition to these early changes, a surge in intracellular CAMP occurs after serum, phosphorylation of the 135000 and signal transmission and returns to baseline 150000 mol. wt proteins was also inhibited before S phase [16]. The rise in CAMP (fig. 5). concentration starts 10 h after conA treatIndomethacin inhibits pre-S phase rises ment, the time period when increased 32P in lymphocyte cellular cyclic AMP (CAMP) incorporation into the 135000 and 150000 and dT incorporation into DNA, effects mol. wt proteins also begins (fig. 3). This which can be reversed by removing the increase in CAMP content may initiate sevdrug without the re-addition of conA [16]. eral biochemical events which include, or Indomethacin (0.2 mM) blocked phospho- lead to, phosphorylation of these proteins. rylation of the 135000 and 150000 mol. wt Neither the addition of cyclic nucleotides proteins in addition to preventing DNA to lymphocyte cell extracts nor the pressynthesis (fig. 5). The cells can be re- ence of an inhibitor specific for CAMPleased from inhibition by removing indo- dependent protein kinases affected phosmethacin from the culture medium indicat- phorylation of the 135000 mol. wt protein, Exp Cell Res 134 (1981)
414
Wang, Foker and Malkinson
however, suggesting that CAMP does not directly activate the kinase which phosphorylates these proteins [6]. To facilitate discussion, the pre-S phase events following stimulation of lymphocytes by conA can be conveniently divided into two stages. First, an early period of signal reception occurs during which the continued presence of the mitogen on the cell surface is required. The period lasts approx. 8 h, and thereafter the mitogen is no longer necessary, as suggested by experiments in which lectin receptors were blocked [14, 231. A second period is the GUS transition which spans the time from when mitogen is no longer required to the onset of DNA synthesis. When conA activation was blocked during either period, neither enhanced phosphorylation of the 135000 and 150000 mol. wt proteins or DNA synthesis occurred. The phosphorylation of these proteins is thus temporally associated with the initiation of S phase. In this study we found that 0.2 mM indomethacin blocked increased protein phosphorylation and DNA synthesis in conAstimulated lymphocytes. Kantor & Hampton [24] found that low7 M indomethacin partially inhibited CAMP-dependent protein kinase activity in ileal mucosal extracts. Their findings suggest that indomethacin, which can inhibit prostaglandin biosynthesis, may also antagonize the actions of prostaglandins. Goueli & Ahmed [25], however, required 1 mM indomethacin to inhibit protein kinase activities in a variety of tissues. Since many enzymes can be inhibited by indomethacin [26], the mechanism(s) by which this drug inhibits lymphocyte phosphorylation and DNA synthesis is unclear. Intact lymphocytes phosphorylate a 150000 mol. wt protein in response to conA. This protein is not phosphorylatable using lysates from conA-treated cells. In Exp
Cell
Res
134 (1981)
contrast, a 135000 mol. wt protein is phosphorylated both in intact cells and extracts. Perhaps the cell extraction procedures which disrupt the juxtaposition between a protein kinase enzyme and its substrate [27, 281 differentially affect these proteins. Alternatively, the dephosphorylation rates of the 135000 and 150000 mol. wt proteins may differ. Phosphate removal from the 135000 mol. wt protein may be rapid enough that the phosphorylation site becomes vacant and available for phosphorylation upon cell disruption, whereas phosphate removal from the 150000 mol. wt protein may be so slow that once phosphorylated in the intact cell, the phosphate group remains bound in the lysates. The similar time courses of phosphorylation and responses to proliferation inhibitors are consistent with the idea that the 135000 and 150000 mol. wt proteins share a common physiological function. Cytosolic or membrane-bound phosphoproteins may serve as intracellular messengers conveying mitogenic information from the cell membrane to the nucleus [5]. Since increased phosphorylation of the 135000 and 150000 mol. wt proteins begins several hours after addition of conA, it is unlikely that these proteins are involved in signal reception. They may, however, mediate subsequent steps leading to the initiation of DNA synthesis. The size equivalence of the 135000 mol. wt protein with that found in extracts from other proliferating systems [7-91 suggests that these phosphorylation events are an integral feature of cell proliferation. We thank Heman Rengifo and Cathy Marquardt for their excellent technical assistance, and Drs Alan Hoouer and Donald Ross for their hetnful comments on the manuscript. This work was supported by a grant from Medical Education and Research Foundason MERF 8236 (J. E. F.); NIH Grant GM 22492 (A. M. M.); and a Basil O’Connor Starter Grant from the National Foundation (A. M. M.).
Protein phosphorylation in intact lymphocytes REFERENCES 1. Greengard, P, Science 199 (1978) 146. 2. Gurley, L R, Walters, R A & Tobey, R A, J cell biol60 (1974) 356. 3. Johnson, E M, Karn, J & Allfrey, V G, J biol them 249 (1974) 4990. 4. Kletzien, R F, Miller, M R & Pardee, A B, Nature 270 (1977) 57. 5. Carpenter, G, King, Jr, L & Cohen, S, J biol them 254 (1979) 4884. 6. Malkinson, AM, Wang, T & Foker, J E, Exp cell res 113(1978) 442. 7. Malkinson, A M & McSwigan, C E, Biochem j 172 (1978) 423. 8. Wehner, J M. Sheunard, J R & Malkinson, A M, Nature 266 (1977) 842. 9. Malkinson. A M. Life sci 24 (1979) 465. IO. Wang, T, Marquardt, C & Foker,J E, Nature 261 (1976) 702. 11. Lowry, 0 H, Rosebrough, N J, Farr, A C & Randall, R J, J biol them 193 (1951) 265. 12. Fairbanks, G, Steck, T L & Wallach, D F H, Biochemistry 10 (1971) 2606. 13. Ueda, T, Maeno, H & Greengard, P, J biol them 248 (1973) 8295. 14. Powell, A E & Leon, M A, Exp cell res 62 (1970) 315. 15. Wang, T & Foker, J E, J cell biol75 (1977) 19a. 16. Wang, T, Sheppard, J R & Foker, J E, Science 201 (1978) 155. 17. Chaplin, D D, Wedner, H J & Parker, C W, Biochem j 182 (1979) 537.
Printed
in Sweden
415
18. Smith, J W, Steiner, A L, Newberry, W M & Parker. C W. J clin invest 50 (1971) 432. 19. Hadden, J W, Hadden, E M, Haddox, M K & Goldberg, N D, Proc natl acad sci US 69 (1972) 3024. 20. Johnson, E M & Hadden, J W, Science 187 (1975) 1198. 21. Wedner, H J & Parker, C W, Biochem biophys res commun 62 (1975) 808. 22. Byus, C V, Klimpel, G R, Lucas, D 0 & Russell, D H, Nature 268 (1977) 63. 23. Ravid, A & Novogrodsky, A, Exp cell res 97 (1976) 1. 24. Kantor, H S & Hampton, M, Nature 276 (1978) 841. 25. Goueli, S A & Ahmed, K, Nature 287 (1980) 171. 26. Flower, R J & Van, J R, Biochem pharmacol 23 (1974) 1439. 27. Keely, Jr, S L, Corbin, J D & Park, C R, Proc natl acad sci US 72 (1975) 1501. 28. Johnson, E M, Adv cyclic nucleotide res 8 (1977) 267. 29. Foker. J E. Malkinson. A M. Sheopard. J R & Wang, T, Molecular basis of immune’cell function (ed J G Kaplan) p. 57. ElsevierlNorth Holland, Amsterdam (1979). 30. Wang, T, Foker, J E & Malkinson, A M, Adv cyclic nucleotide res 12 (1980) 415. Received May 5, 1980 Revised version received February 26, 1981 Accepted March 5, 1981
Exp Cell RPS 134 (I 981)
Cell Research 134 (1981) 409-415
Experimental
PROTEIN
PHOSPHORYLATION STIMULATED
TINGCHUNG ‘Department
of Surgery.
IN INTACT
BY CONCANAVALIN
WANG.’ JOHN E. FOKER’ and ALVIN University University
LYMPHOCYTES
of Minnesotu, of Colorado.
Minneapolis, Boulder,
M. MALKINSON”
MN 55455, CO 80309,
A
rind 2School
* of Pharmacy,
USA
SUMMARY The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of azP incorporation into two proteins of 135000 and 150000 mol. wt began to increase, relative to control cells, IO h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of-both proteins. Such treatments included the displacement of conA from its surface receptors by a-methvl-mannoside (aMM), the omission of serum from the culture medium, and the presence-of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135000 and I50000 mol. wt proieins have a common physiological fun&on. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.
The biological activity of many proteins is determined by whether they are in a phosphorylated or dephosphorylated form. Alterations in phosphate content accompany, and are thought to mediate, the rapid adjustment of cell metabolism to many environmental changes [l]. It is not surprising, therefore, that cyclic changes in protein phosphorylation/dephosphorylation occur during cell proliferation. Phosphorylation at multiple sites in specific histones occurs sequentially during the cell cycle, and may regulate chromosomal condensation [2]. Similarly, changes in the phosphorylation of non-histone chromosomal [3], cytosolic [4], and membrane-bound proteins [5] have been observed when the proliferative status 27-811816
of cells was altered. Neither the physiological consequences of these changes nor the functional identity of these phosphoproteins is presently understood. Lymphocytes are normal resting cells which can be stimulated by a variety of mitogens to enter a proliferative cycle. We recently studied the phosphorylation of endogenous proteins in mouse lymphocyte cell lysates [6]. A 135000 mol. wt cytosolic protein was intensely phosphorylated following lymphocyte activation by conA, with maximal phosphorylation occurring concomitantly with the peak of DNA synthesis. Enhanced phosphorylation of a * To whom reprint requests should be
addressed.
Exp Cell Res 134 f/98/)
410
Wang, Foker and Malkinson -Con A
MATERIALS
+Con A
AND METHODS
Lymphocyte culture 150 ooo135 oooe
0
-200
000
.,..%r&r*----
-130 -94
000 000
-24
000
-5
,(
Mouse spleen lymphocytes were isolated from A/Jx C57BL/6F, hybrids and cultured as described TlOl. Spleens w&e-minced in a loose-fitting tissue g&d& to obtain a Ivmahocvte-rich cell susoension. which. after centrifigaiion at 200 g for I min to’ remove tissue debris, was treated with 2 vol of 0.87% NH&I to lyse erythrocytes. Lymphocytes were then washed twice with 0.9% NaCI. once with culture medium (RPM1 1640), and then iesuspended to achieve a cell density of 5x lo6 cells/ml in RPM1 1640 medium containing 2% heat-inactivated human serum, 2 mM Lelutamine. 50 U/ml of oenicillin and 50 me/ml of streptomycin. The cultur& were put in a hu&idified incubator at 37°C under 5% CO,. in the nresence or absence of 2 pglml of conA. Cell number was determined with a hemocytometer. The viability, which was assessed by Trypan blue exclusion, decreased from 100 to 80% during the first 24 h and stabilized at 70% for the rest of the culture period in both control and conA-stimulated cells.
Nucleic acid synthesis
0
The rates of RNA and DNA synthesis were estimated by incorporation of r3H]uridine ([3H]U, 5 &i/ml of cell culture, 6 Cilmmole) and [3H]deoxythymidine VHldT. 2.5 &i/ml of cell culture. 6 Cilmmole), respectively, into &ichloroacetic acid (?CA)-precipitable material during a 1 h labeling period [IO]. Cells were collected on a glass fiber filter with a microculture harvester, washed with 5% TCA, and airdried. The amount of incorporated radioactivity was determined in a Beckman liquid scintillation counter. AL
I. Autoradiogram of a polyacrylamide gel showing 32Pincorporation into protein from intact mouse spleen lymphocytes. Lymphocytes were cultured for 48 h in the absence or presence of conA. They were phosphorylated with’ 32P, and electrophoresis and autoradiography were performed as described in Materials &id Methods. Numbers on the left indicate the estimated mol. wt of two major phosphoprotein bands whose phosphorylation increased following conA stimulation. The numbers on the right indicate the mol. wt of known protein standards.
Fig.
cytosolic protein of identical size has been observed in various proliferating cell types including tumors and fetal tissues [7], serum-fed confluent cultured cells [8], and healing lung tissues [9]. Cell extracts were used in these studies, so it is important to determine whether similar reactions occur in intact cells. We now report that the phosphorylation of a 135000 mol. wt protein, as well as of a 150000 mol. wt protein, increases following conA stimulation in intact mouse lymphocytes. Preliminary reports of some of this work have appeared [29, 301. Exp Cell Res 134 (1981)
~~~~,
~~
,
Protein phosphorylation in intact cell suspensions Cultured cells were washed twice and resuspended to a density of 5x 10’ cells/ml in 30 mM Tris-HCI (pH 7.6) containing 0.15 M NaCl, 4 mM MgCI, 0.3 mM EDTA, and IO mM NaF. Fifty PCi of carrierfree [32P]orthophosphoric acid were added per ml of cell suspension, and incubated at 37°C for 60 min. After incubation the cells were centrifuged at 400 g for 5 min, resuspended in 200 ~1 of lysjs buffer (lo mM Tris-HCI. .oH 7.5. 3 mM MgCI, and 500 units of micrococcal nuclease) and the protein concentration was adiusted to 1 mE/ml with a 1% sodium dodecyl sulfate”(SDS) -40 m& dithiothreitol solution. Protein concentration was determined by the method of Lowry et al. [II].
Gel electrophoresis Phosnhorvlated nroteins from intact cells were fractionated by polyacrylamide slab gel electrophoresis [ 121. Subsequent autoradiography and the quantitative measurement of 32P incorporation into protein bands by microdensitometry were performed as previous1.ydescribed [ 131.
Protein phosphorylation in intact lymphocytes
411
9 12 10 8 6 4 2 0 10 8 6 4 2 0 60
GEL LENGTH (cm)
Fig. 2. Densitometric tracings of autoradiogram from fig. 1 showing phosphoprotein profiles of intact lymphocytes. Intact lymphocytes cultured without (. . .) or with (-) conA for 48 h were phosphorylated, and autoradiographs made. The extent of 32Pincorporation into proteins is indicated by the relative band density of the individual proteins separated by electrophoresis (densitometric units). Peaks A and B represent the 150000 and 135000 mol. wt phosphoprotein bands.
RESULTS ConA-stimulated phosphorylation of specific proteins in intact lymphocytes The effects of conA on protein phosphorylation in intact lymphocytes are shown in an autoradiogram (fig. 1). Similar patterns of distinct phosphorylated bands can be seen in both control and conA-treated cells with the exception that two proteins of 135000 and 150000 mol. wt were consistently more prominent in the conA series. The 135000 mol. wt protein co-migrated with a highly phosphorylated protein obtained when extracts from conA-treated cells were incubated with [y-32P]ATP [6]. The 150000 mol. wt band which was phosphorylated in intact cells was not phosphorylated in the cell-free assay system. Densitometric tracings comparing lymphocytes cultured for 48 h in the presence and absence of conA also showed that the band density in peaks corresponding to 135000 and 150000 mol. wt were markedly increased in the conA-treated cells (fig. 2).
60
50
50
40
40 g
30
30 e
20
20 : F IO"
10 0 0
IO Culture
20
30 time
40
7 0 5c )
(houn)
Fig. 3. Changes in protein phosphorylation, RNA and DNA synthesis in lymphocytes cultured with conA for different lengths of time. 0, 0, Amounts of [32P]phosphate incorporation (densitometric units/l06 viable cells/h) into (A) 135000 and (B) 150000 mol. wt proteins; A, A [3H]U and 0, W [3H]dT incorporation (cpmx 1O-3/1o6viable cells/h) into lymphocytes cultured without (open symbols) or with conA (closed symbols).
The extent of phosphorylation of the 135000 and 150000 mol. wt proteins in intact conA cells increased with culture time (fig. 3). During the first 8-10 h that the cells were cultured in the presence of conA, no difference was observed between the phosphorylation of the two proteins in the control and conA-stimulated intact cells. Phosphorylation in the conA cells began to increase above control levels at approx. 10 h, and had increased lo-fold by 50 h. The time course of phosphate incorporation into both proteins appeared to be identical, and similar to that previously found for the 135000 mol. wt protein using lymphocyte extracts [6]. Compared to the time course of nucleic acid synthesis, increased phosphorylation began several hours after the initial rise in r3H]U incorporation into RNA, but preExp Cell Res 134 (1981)
412
Wang, Foker and Malkinson
Con A serum + ,“dO. -
$ -
1 -
+ -
+ +
:: +
Fig. 5. Effects of serum omission or the addition of
0.2 mM indomethacin on protein phosphorylation and DNA synthesis in intact lymphocytes. Lymphocytes were cultured in the presence or absence of serum for 30 h. Indomethacin was added to some cells beginning 2 h after the cells were placed in culture. All samples were then harvested and assayed for either [32P]phosphate incorporation into proteins (densitometric units/lo6 viable cells/h) or r3H]dT incorporation (cpmx 10-3/106viable cells/h) into DNA. a, 135OOOmol.wt protein; 0, 150000 mol. wt protein; W, r3H]dT incorporation. Fig. 4. Effects of cxMM addition on protein phosphorylation and DNA synthesis in conA-stimulated lymphocytes. [32P]Phosphate incorp. (fold of incorp. compared to cells not treated with conA) into the (A) 135000, and (B) 150000 indicate proteins. (C) r3H]dT incorp. (cpmx 10~“/lo” viable cells/h). Protein phosphorylation and DNA synthesis were measured in cells which were cultured in the presence of conA for 24 h. At various times after the initial addition of conA, unbound conA was removed by washing and resuspending the cells to 5x lo6 cells/ml in RPM1 1640 medium (O---O). Half of the cultures also contained 100 mM aMM in the resuspension medium (O-O), which displaces bound conA [14]. cxMM addition time refers to the length of time that cells had been in the presence of conA prior to the addition of (wMM.
after the initial conA stimulation, endogenous protein phosphorylation in intact cells and [3H]dT incorporation into DNA were measured. CXMMtreatment during the first 5-6 h abolished the conA-stimulated incorporation of 32Pinto the 135000 and 150000 mol. wt proteins, and also prevented [3H]dT incorporation into DNA (fig. 4). As the duration of exposure to conA prior to treatment with (YMM lengthened, progressively more cells synthesized DNA and phosphorylated the two proteins. By 15 h, phosphoceded the onset of [3H]dT incorporation rylation was no longer affected by aMM into DNA (fig. 3). treatment, although [3H]dT incorporation Effects on protein phosphorylation of remained sensitive until 21 h. interrupting conA stimulation The effects of blocking lymphocyte acMitogenic stimulation by conA can be spe- tivation at later times were also investicifically reversed with CXMM, which dis- gated. Lymphocytes ,require serum beginplaces conA from the cell surface and there- ning 10 h after conA stimulation [ 151.When by discontinues the mitogenic signal [14]. cultured continuously in the absence of seAt different times after culture with conA, rum, mitogen-stimulated cells remain in late lymphocytes were washed and resuspended Gl phase but will synthesize DNA if sein a serum-containing medium, with or rum is added. When [3H]dT incorporawithout 100 mM (YMM. Twenty-four hours tion into DNA was inhibited by omitting Exp
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ing that the inhibition was not due to a cytotoxic effect of the drug at this concentration. Protein phosphorylation increased 4 h after the lymphocytes were washed free of the drug and incorporation of [3H]dT into DNA began 8 h after removal of indomethacin (fig. 6). This result shows that the temporal relationship between protein phosphorylation and the progression to S phase was maintained in indomethacin-inhibited cells. DISCUSSION Cyclic AMP stimulates the phosphorylation of several lymphocyte proteins in vitro 30 34 38 42 46 [17]. Cyclic nucleotides may also regulate Culture time (hours) the phosphorylation of lymphocyte proFig. 6. Reversible effects of indomethacin on protein teins in intact cells during very early stages phosphorylation and DNA synthesis in intact lymphocytes. ConA-stimulated lymphocytes were cultured of mitogenic signal reception. Increases in without indomethacin (O), with a continuous exposure cellular CAMP [18] and cGMP [ 191 occur to 0.2 mM indomethacin (A), or with 0.2 mM indomethacin for 30 h and then washed to remove indowithin minutes after mitogen treatment, and methacin (A). At the indicated times, cells were ascGMP [20] and monobutyryl CAMP [21] sayed for protein phosphorylation and DNA synthesis. Points with vertical range bars indicate mean + S.E.M. can enhance 32Pincorporation into proteins of duplicate cultures; those without error bars have of a S.E.M.s smaller than dimension of symbols. f3zP]- in intact lymphocytes. Activation phosphate incorp. (densitometric units/<06 viable cells/ CAMP-dependent protein kinase following h); [3H]dT incorp. (cpmx 1O-3/1O6viable cells/h). (A) mitogenic stimulation of lymphocytes oc135000; (B) 150000 mol. wt proteins. curs a few hours after mitogenic stimulation [22]. In addition to these early changes, a surge in intracellular CAMP occurs after serum, phosphorylation of the 135000 and signal transmission and returns to baseline 150000 mol. wt proteins was also inhibited before S phase [16]. The rise in CAMP (fig. 5). concentration starts 10 h after conA treatIndomethacin inhibits pre-S phase rises ment, the time period when increased 32P in lymphocyte cellular cyclic AMP (CAMP) incorporation into the 135000 and 150000 and dT incorporation into DNA, effects mol. wt proteins also begins (fig. 3). This which can be reversed by removing the increase in CAMP content may initiate sevdrug without the re-addition of conA [16]. eral biochemical events which include, or Indomethacin (0.2 mM) blocked phospho- lead to, phosphorylation of these proteins. rylation of the 135000 and 150000 mol. wt Neither the addition of cyclic nucleotides proteins in addition to preventing DNA to lymphocyte cell extracts nor the pressynthesis (fig. 5). The cells can be re- ence of an inhibitor specific for CAMPleased from inhibition by removing indo- dependent protein kinases affected phosmethacin from the culture medium indicat- phorylation of the 135000 mol. wt protein, Exp Cell Res 134 (1981)
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Wang, Foker and Malkinson
however, suggesting that CAMP does not directly activate the kinase which phosphorylates these proteins [6]. To facilitate discussion, the pre-S phase events following stimulation of lymphocytes by conA can be conveniently divided into two stages. First, an early period of signal reception occurs during which the continued presence of the mitogen on the cell surface is required. The period lasts approx. 8 h, and thereafter the mitogen is no longer necessary, as suggested by experiments in which lectin receptors were blocked [14, 231. A second period is the GUS transition which spans the time from when mitogen is no longer required to the onset of DNA synthesis. When conA activation was blocked during either period, neither enhanced phosphorylation of the 135000 and 150000 mol. wt proteins or DNA synthesis occurred. The phosphorylation of these proteins is thus temporally associated with the initiation of S phase. In this study we found that 0.2 mM indomethacin blocked increased protein phosphorylation and DNA synthesis in conAstimulated lymphocytes. Kantor & Hampton [24] found that low7 M indomethacin partially inhibited CAMP-dependent protein kinase activity in ileal mucosal extracts. Their findings suggest that indomethacin, which can inhibit prostaglandin biosynthesis, may also antagonize the actions of prostaglandins. Goueli & Ahmed [25], however, required 1 mM indomethacin to inhibit protein kinase activities in a variety of tissues. Since many enzymes can be inhibited by indomethacin [26], the mechanism(s) by which this drug inhibits lymphocyte phosphorylation and DNA synthesis is unclear. Intact lymphocytes phosphorylate a 150000 mol. wt protein in response to conA. This protein is not phosphorylatable using lysates from conA-treated cells. In Exp
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contrast, a 135000 mol. wt protein is phosphorylated both in intact cells and extracts. Perhaps the cell extraction procedures which disrupt the juxtaposition between a protein kinase enzyme and its substrate [27, 281 differentially affect these proteins. Alternatively, the dephosphorylation rates of the 135000 and 150000 mol. wt proteins may differ. Phosphate removal from the 135000 mol. wt protein may be rapid enough that the phosphorylation site becomes vacant and available for phosphorylation upon cell disruption, whereas phosphate removal from the 150000 mol. wt protein may be so slow that once phosphorylated in the intact cell, the phosphate group remains bound in the lysates. The similar time courses of phosphorylation and responses to proliferation inhibitors are consistent with the idea that the 135000 and 150000 mol. wt proteins share a common physiological function. Cytosolic or membrane-bound phosphoproteins may serve as intracellular messengers conveying mitogenic information from the cell membrane to the nucleus [5]. Since increased phosphorylation of the 135000 and 150000 mol. wt proteins begins several hours after addition of conA, it is unlikely that these proteins are involved in signal reception. They may, however, mediate subsequent steps leading to the initiation of DNA synthesis. The size equivalence of the 135000 mol. wt protein with that found in extracts from other proliferating systems [7-91 suggests that these phosphorylation events are an integral feature of cell proliferation. We thank Heman Rengifo and Cathy Marquardt for their excellent technical assistance, and Drs Alan Hoouer and Donald Ross for their hetnful comments on the manuscript. This work was supported by a grant from Medical Education and Research Foundason MERF 8236 (J. E. F.); NIH Grant GM 22492 (A. M. M.); and a Basil O’Connor Starter Grant from the National Foundation (A. M. M.).
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