Lymphocyte blastogenesis. Post-transcriptional controls of protein synthesis

Biochimica et Biophysica Acta 824 (1985) 365-368 Elsevier

365

BBA Report

BBA 90039

Lymphocyte blastogenesis. Post-transcriptional controls of protein synthesis Eric P. Lester

a

and Herbert L. Cooper h

a Department of Medicine, Unioersity of Chicago, 5841 South Maryland Aoe., Chicago, IL 60637, and b Laboratory of Pathophysiology, National Cancer Institute, Bethesda, MD 20205 (U. S.A.) (Received December 6th, 1984)

Key words: Protein synthesis; Lymphocyte blastogenesis; Phytohemagglutinin; (Human)

To better understand the regulation of macromolecular synthesis in the response of lymphocytes to a mitogen, we have used two-dimensional electrophoresis to search for specificity in the early increase seen in protein synthesis in human iymphocytes treated with phytohemagglutinin and examined the role of new RNA synthesis in this response. Our results confirm a major increase in overall protein synthesis after 4 h of phytohemagglutinin treatment. A further disproportionate increase in the synthetic rates of certain polypeptides was observed using two-dimensional polyacrylamide gel electrophoresis. While actinomycin-D reduced protein synthesis to a level below that of untreated cells, phytohemagglutinin nonetheless enhanced total protein synthesis even in the presence of actinomycin. Some, but not all, of the disproportionate increases in synthesis seen for certain polypeptides are blocked by actinomycin. These results imply the existence of multiple mechanisms controlling protein synthesis early in the course of lymphocyte stimulation. At least some of these do not require new RNA synthesis and thus operate at a post-transcriptional level.

Human peripheral blood lymphocytes were isolated as previously described [1] and cultured as four aliquots at 2.107 cells/ml in 1.5 ml of methionine-free RPMI-1640 (Gibco, Selectamine) + 2% autologous serum at 37°C, 5% CO2, for 1.8 h. [35S]methionine (Amersham), was added for a subsequent 2.5 h at a final concentration of 166 /xCi/ml. Culture A (control) had no other additives. Cultures B and D contained phytohemagglutinin, 5 /~g/ml for the final 4 h of culture. Cultures C and D contained actinomycin-D, 5 ~tg/ml for the entire 4.3 h culture period. At the end of culture, the cells were washed with cold, complete RPMI-1640 and taken up in 200 #1 of lysis buffer, consisting of 9.5 M urea, 2% ampholytes (pH 3.5-10, LKB Instruments), 2% Nonidet P-40 and 5% mercaptoethanol. Trichloroacetic acid-precipitable radioactivity was measured on 10-/~1 aliquots in triplicate. Data from three separate experiments are shown. In Expt. 1, cells were

precultured for 47 h in RPMI-1640 + 10% autologous plasma before use and labelled with 89 /~Ci/ml, thus accounting for lower total protein synthesis than in Expts. 2 and 3. For each experiment 25-/~1 aliquots from each 200 /~1 suspension of cells in lysis buffer were subjected to two-dimensional electrophoresis in polyacrylamide gel according to the method of O'Farrell [2] followed by fluorography [3]. All gels were exposed to X-ray film (XR-5, Kodak) at - 8 0 ° C for 24 h, except for sample C, which required 72 h to provide a comparable image. Analysis of gel patterns was accomplished by visual inspection of relative spot intensity and focused on only a few polypeptides showing highly reproducible changes. No attempt at exhaustive analysis was made. Rather, we sought changes in a few polypeptides indicative of various mechanisms of regulation. Thus, four aliquots of lymphocytes were ex-

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Fig. 1. Experimental protocol and total protein synthesis data for 4 h phytohemagglutinin (PHA) stimulation. Human peripheral blood lymphocytes were isolated [1] and cultured as four aliquots as shown. Culture A (control) had no other additives. Cultures B and D contained phytohemagglutinin, 5 ,ug/ml for the final 4 h of culture. Cultures C and D contained actinomycin-D (Act D), 5/.tg/ml for the entire 4.3 h culture period.

amined: control (resting), actinomycin-treated, phytohemagglutinin-treated, and cells treated with actinomycin and phytohemagglutinin (Fig. 1, experimental protocol). The dose of actinomycin-D (5 #g/ml) was sufficient to abolish RNA synthesis entirely within 20 min. [4,5]. Cells treated with phytohemagglutinin alone for 4 h showed an increase in total protein synthesis to 160% of control levels, as measured by the incorporation of [35S]methionine into trichloroacetic acid-precipitable material during the final 2.5 h of culture (Fig. 1). Fig. 2 shows representative two-dimensional gel fluorographs from Expt. 2. Similar results were observed in the electrophoreses from Expts. 1 and 3. Fig. 2B suggests that the increase in protein synthesis seen with phytohemagglutinin was a general one, since the relative intensity of each spot in the fluorograph of these pulse-labeled proteins was largely unchanged after 4 h of phytohemagglutinin when compared with the control fluorograph (Fig. 2A). The overall intensity was increased, reflecting the increased synthesis of each polypeptide. Nonetheless, a few polypeptides showed an additional, disproportionate increase in intensity (Fig. 2B, arrows) in all three experiments. Most prominent was a polypeptide with an estimated molecular weight of about 25000 (Fig. 2B, lower heavy arrow). These results are similar to our previously published studies [6].

.7"

Fig. 2. Two-dimensional fluorographs of samples from one of the experiments shown in Fig. 1, Panel B points out four polypeptides (arrows) whose relative synthetic rates increase with phytohemagglutinin, in addition to the overall increase in density of most spots. Panel C shows that actinomycin-D alone produced little, if any, change in relative synthetic rates. Panel D shows that actinomycin prevented the relative increase in synthesis of some polypeptides (thin arrows) but not others (heavy arrows) produced by phytohemagglutinin. Thus, multiple post-transcriptional control mechanisms appear to regulate the synthesis of different polypeptides during early lymphocyte stimulation by phytohemagglutinin.

Cells treated only with actinomycin-D showed a fall in overall protein synthesis to 42% of control, as has been reported previously for lymphocytes [4,5,7] and other cell systems [8]. The qualitative fluorographic pattern of proteins synthesized during the last 2.5 h of a 4.3 h incubation with actinomycin-D alone revealed very little change when compared with the pattern of those from resting cells, except that one of the proteins (Fig. 2C, lower heavy arrow), whose synthesis is also disproportionately enhanced by phytohemagglutinin alone, also appeared to be relatively enhanced by actinomycin-D, However, the absolute synthetic rate of this protein was still probably little changed in comparison with its rate in the control cells, since it has nearly identical apparent densities in films exposed for equal times and representing proteins from equal numbers of resting and actinomycin-treated cells. Since the enhancement of synthesis of this protein by phytohemagglutinin is not blocked by actinomycin (see

367 below), it clearly seems subject to an unusual control mechanism. Addition of phytohemagglutinin after complete inhibition of RNA synthesis by actinomycin-D did not enhance overall protein synthesis above resting cell levels, but it did produce a relative stimulation of protein synthesis when compared with actinomycin-treated cells not receiving phytohemagglutinin (Fig. 1) - 67 versus 42% of untreated controls. Again, the bulk of this stimulation appears to be a general one in view of the minimal changes present in comparison with the control fluorograph. However, we still saw a disproportionate increase in the synthesis of a few proteins (Fig. 2D, heavy arrows), although the differential enhancement by phytohemagglutinin of others (Fig. 2D, thin arrows) had been abolished by the addition of actinomycin. Similar changes were seen in all three experiments. Pulse-chase experiments have shown no enhancement of any of these polypeptides by addition of phytohemagglutinin during the chase, indicating that the previously seen disproportionate enhancement of certain polypeptides was a result of a true increase in their synthetic rates, rather than a result of prolonged half-lives due to a decrease in their catabolic rates (data not shown). Our results are similar to those of Wettenhall and London [9], who found that actinomycin-D did not block the early stimulation of protein synthesis in porcine lymphocytes treated with concanavalin A (ConA). Their results, however, dealt only with overall rates of protein synthesis, rather than changes in the synthesis of specific polypeptides. Schmidt-Ullrich et al. [10]. utilizing onedimensional electrophoresis of continuously labeled proteins from rabbit thymocytes, were able to detect increased specific activities in certain protein bands after 24 h of ConA stimulation. Two-dimensional electrophoresis has permitted us to obtain a much more detailed view of protein synthesis early in the course of mitogenesis. Braude et al. [11] have also provided evidence for a posttranscriptional mechanism controlling the synthesis of certain polypeptides in other eukaryotic cells. They used fluorographs of two-dimensional electrophoreses of pulse-labeled proteins to demonstrate that the fertilization of a mouse ovum triggers the enhanced synthesis of a family of

M r = 35000 polypeptides independent of new RNA synthesis. We have demonstrated here both a similar specificity in the stimulation of protein synthesis triggered by the action of phytohemagglutinin on lymphocytes, and the independence of a portion of that stimulation from new RNA synthesis. Our data suggest that the mechanisms producing an early increase in protein synthesis after phytohemagglutinin addition may be divided into two basic classes. The first produces an increase in the synthesis of all or nearly all polypeptides and the second produces an additional, disproportionate enhancement of the synthesis of a few specific proteins. Furthermore, each of these classes can be subdivided into actinomycin-D-sensitive and-insensitive sub-classes. Previous studies have shown that the availability of functional mRNA in lymphocytes in the resting state and during the early hours of activation is not limiting to the general rate of protein synthesis [12]. Evidence also has been presented to show that the general level of protein synthesis is limited, in resting cells, by the availability of a short-lived, non-messenger RNA and that during the early rise in overall protein synthesis following phytohemagglutinin this material ceases to be limiting [4,5]. Thus, the general actinomycin-sensitive increase in protein synthesis with phytohemagglutinin, described here, would appear to be mediated by the mRNA-independent, RNA-requiring mechanism, qualifying it as a post-transcriptional control. The general, but actinomycin-resistant increase in total protein synthesis which occurred in cells treated with actinomycin and phytohemagglutinin may indicate a second independent translational control mechanism affected by phytohemagglutinin treatment. It also may represent stabilization by phytohemagglutinin treatment of the ratelimiting RNA mentioned above. In either case, the effect is deafly a post-transcriptional one. The disproportionate actinomycin-inhibited stimulation of synthesis of several specific proteins (Fig. 2B, small arrows) by phytohemagglutinin has the highest likelihood of being a transcriptionally controlled event while the actinomycin-resistant specific stimulation of at least two proteins deafly is not (Fig. 2B, heavy arrows). The latter can only be conceived of as a phytohemagglutinin-induced

368 p r e f e r e n t i a l i n i t i a t i o n on specific p r e e x i s t i n g m R N A s , unless these m R N A s are of m i t o c h o n d r i a l origin, a n d their increased synthesis is thereby resistant to the effects of a c t i n o m y c i n - D [13]. O u r e x p e r i m e n t s have shown that we are not dealing here with a r e d u c e d c a t a b o l i c rate for these p o l y peptides. O u r d a t a thus suggest the existence of at least two, a n d p o s s i b l y three, s e p a r a t e p o s t - t r a n s c r l p tional m e c h a n i s m s which are m o d i f i e d b y p h y t o h e m a g g l u t i n i n t r e a t m e n t to increase the p r o d u c tion of proteins. M i l n e r [14] has p r e s e n t e d evidence for a r e q u i r e m e n t for a m a n i t i n - s e n s i t i v e R N A synthesis for c o m m i t m e n t to growth a n d earlier showed that p r o t e i n synthesis is necessary for that c o m m i t m e n t [15]. A t r a n s c r i p t i o n a l control m e c h a n i s m also m a y be i n d i c a t e d b y o u r d a t a ( a c t i n o m y c i n - s e n s i t i v e specific stimulation). Thus, m e c h a n i s m s controlling the expression of genetic i n f o r m a t i o n d u r i n g l y m p h o c y t e blastogenesis m a y o p e r a t e at b o t h t r a n s c r i p t i o n a l a n d p o s t - t r a n s c r i p tional levels.

References 1 Cooper, H.L. (1974) Methods Enzymol. 32, 633-639 2 O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007-4021 3 Bonnet, W.M. and Laskey, R.A. (1974) Eur. J. Biochem. 46, 83-88 4 Cooper, H.L. (1968) J. Biol. Chem. 243, 34-43 5 Cooper, H.L. and Braverman, R. (1980) Exp. Cell Res. 127, 351-359 6 Lester, E.P., Lemkin, P., Lipkin, L. and Cooper, H.L. (1981) J. Immunol. 126, 1428-1434 7 Cooper, H.L. and Braverman, R. (1977) Nature 269, 527-529 8 Penman, S., Scherrer, K.K., Becker, Y. and Darnell, J.E. (1963) Proc. Natl. Acad. Sci. USA 49, 654-662 9 Wettenhall, R.E.H. and London, D.R. (1974) Biochim. Biophys. Acta 349, 214-225 10 Schmidt-Ulrich, R., Wallach, D.F.H. and Farber, E. (1974) Biochim. Biophys. Acta 356, 288-299 11 Braude, P., Pelham, H., Flach, G. and Lobatto, R. (1979) Nature 282, 102-105 12 Cooper, H.L. and Braverman, R. (1979) J. Cellular Physiol. 93, 213-225 13 Godman, G.C., Keneklis, T.P. and King, M.E. (1973) Exp. Cell Res. 77, 159-166 14 Milner, J. (1978) Nature 275, 660-661 15 Milner, J. (1978) Nature 272, 628-629