Protein synthesis during activation of lymphocytes by mitogens.

When lymphocytes purified from peripheral blood are incubated with mitogenic lectins such as phytohaemagglutinin they initiate DNA synthesis, but only after a lag of about 24 h. There are, however, substantial changes in many aspects of lymphocyte metabolism which begin much more rapidly after lectin addition, and these include large increases in the rates of transcription and translation (Ling & Kay, 1975). The increase in the rate of protein synthesis is first detectable about 2 h after lectin addition, and rises to twice the initial rate after 4 h, and to about 10 times the initial rate per cell after 20h (Jagus & Kay, 1979). There is also an increase in the number of ribosomes per cell by 20h, but if this is selectively suppressed by the addition of low concentrations of actinomycin, the increase in protein synthesis is relatively unimpaired (Kay et al., 1969). The increase in protein synthesis is accompanied by a substantial accumulation of polyribosomes. Most of the ribosomes present in unstimulated lymphocytes are monoribosomes not engaged in protein synthesis, and the majority of those that are active are also monoribosomes, but after activation the proportion of ribosomes actively translating mRNA increases steadily to 7540% by 12-20h (Kay el al., 1971; Ahern & Kay, 1975; Cooper & Braverman, 1977). In contrast, there is no significant change in the elongation rate over this period (Kay et al., 1975), so the increase in protein synthesis must be due entirely to an increase in the rate of the initiation step. Determination of the ability of cell-free protein-synthesizing systems from unstimulated and 20 h-activated lymphocytes to form initiation complexes with [ 35Slmethionyl-tRNA~a has shown that the defective initiation of the unstimulated cells is conserved in cell-free systems derived from them (Ahern et al., 1974). Detailed analysis of this system has shown that, although the capacity of systems from unstimulated lymphocytes to form 40s initiation complexes is decreased, this defect is too small to account for the overall defect in initiation, and that steps between the formation of 40s and 80s initiation complexes are also impaired (Kay et al., 1979). A possible explanation of the defect at the latter step would be a lack of active mRNA in the unstimulated cells. However, although the amount of poly(A)+ mRNA per cell does increase about 4-fold in the 20 h after lectin addition, this increase is too small and too late to account for the increase in protein synthesis (Jagus & Kay, 1979), and at least the earliest increase in protein synthesis is still observed when transcription is suppressed with actinomycin (Wettenhall & London, 1974). In support of these studies with intact cells, addition of an excess of globin mRNA or lymphocyte mRNA to lymphocyte cell-free systems does not overcome the difference in initiation-complex formation between systems from unstimulated and activated cells (Ahern et al., 1974; Wallace et al., 1979). Indeed, the systems from activated cells were able to utilize the added mRNA more efficiently. There is no evidence to suggest that any substantial proportion of the mRNA from unstimulated cells is inactivated, as tests in three different cell-free systems have shown it to be utilized as efficiently as mRNA from activated cells (Wallace et al., 1979). In contrast, the defect in initiation capacity of systems from unstimulated lymphocytes is substantially overcome by addition of unfractionated initiation factors from reticulocytes (Ahern et al., 1974). The defect in 40s initiation-complex formation is largely repaired by addition of eIF-2, although eIF-3 may also have a subsidiary role (Kay et al., 1979). Additional initiation factors are required to repair the defect in formation of 80s initiation complexes, but this has not yet been analysed in detail. Preliminary studies suggest that more than one initiation factor is involved. The decreased capacity for initiation in systems from unstimulated lymphocytes may well be due to the presence of translational inhibitors. Addition of small amounts of lymphocyte cytoplasm or post-ribosomal supernatant to reticulocyte lysates results in a rapid inhibition of protein synthesis (Kay et al., 1978). Steps in the initiation sequence both before and after the formation of 40s initiation complexes are dkcted. This inhibition is not reversible by re-addition of mRNA, and control experiments have established that it is not due to nuclease action on the endogenous mRNA, but it can be temporarily alleviated by unfractionated reticulocyte initiation factors. The inhibition is not reversed by addition of 4mM-adenine or elevated OTP concentrations, procedures that do reverse the superficially similar inhibition caused by haemin deprivation of the reticdocyte lysate. The lymphocyte translational inhibitors are macromolecules, and attempts to purify them and further characterize their mechanism of action are now in progress. These inhibitors are also present in cytoplasm from activated lymphocytes, but in lesser amounts (Kay et al., 1978). Studies on the distribution of lymphocyte poly(A)+ mRNA between free mRNP (messenger ribonucleoprotein) and ribosomes have led to the unexpected conclusion that a high proportion (about 75%) of the mRNA in unstimulated lymphocytes is associated with monoribosomes. During the first 2h after activation, a period in which there is comparatively little change in the overall rate of protein synthesis, almost half this mRNA is released, and becomes free mRNP (Jagus & Kay, 1979). At later times an increasing proportion of the mRNA becomes associated with polyribosomes, in parallel with the increase in protein synthesis. Extensive studies have not revealed any abnormality in the RNP particles in which the mRNA in unstimulated lymphocytes is contained, or in its association with ribosomes. The most probable explanation is that the initial translational controls operated after lymphocyte activation result in preferential utilization of certain mRNA molecules for protein synthesis, and that others are consequently displaced. It thus seems probable that controls acting as the translational level may cause qualitative as well as quantitative changes in lymphocyte protein synthesis after activation by mitogenic lectins.