Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters.

Exposure of quiescent, serum-starved 3T3-L1 cells to insulin promotes phosphorylation of initiation factors eIF-4F, eIF-4B, and eIF-3 p120, as well as ribosomal protein S6. Phosphorylation of both the p25 and p220 subunits of eIF-4F is stimulated typically by 2.5-5-fold, with a 2-4-fold increase in phosphorylation of eIF-4B and eIF-3 p120. Optimal stimulation is observed by 10(-9) M insulin. A similar pattern of stimulation is seen upon treatment of 3T3-L1 cells with 1 x 10(-6) M phorbol 12-myristate 13-acetate (PMA). Two-dimensional phosphopeptide mapping of p25, isolated from quiescent, insulin- or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the p220 subunit. Following PMA-stimulation of 3T3-L1 cells, phosphopeptide mapping of p220 results in a pattern similar to that observed in vitro with Ca2+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of p220 results in the appearance of novel peptides. Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4b, eIF-3 p120, or S6 via a protein kinase C-dependent mechanism is observed. Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F p220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 p120 is observed. Thus, eIF-4F p220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 p120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F p220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.

A similar pattern of stimulation is seen upon treatment of 3T3-Ll cells with 1 x lo-' M phorbol 12-myristate 13-acetate (PMA). Twodimensional phosphopeptide mapping of ~25, isolated from quiescent, insulin-or PMA-stimulated cells, results in a single tryptic phosphopeptide, indicating a single phosphorylation site identical to that obtained with protein kinase C. A more complex phosphopeptide map is observed with the ~220 subunit. Following PMA-stimulation of 3T3-Ll cells, phosphopeptide mapping of ~220 results in a pattern similar to that observed in vitro with Ca'+/phospholipid-dependent protein kinase (protein kinase C). Following insulin stimulation, mapping of ~220 results in the appearance of novel peptides.
Upon prolonged exposure to PMA, the cells are no longer responsive to this mitogen and no stimulation of phosphorylation of eIF-4F, eIF-4B, eIF-3 ~120, or S6 via a protein kinase C-dependent mechanism is observed.
Addition of insulin to these down-regulated cells leads to stimulation of phosphorylation of eIF-4F ~220, ribosomal protein S6, and to a lesser extent, eIF-4B; little or no stimulation of phosphorylation of eIF-4F p25 and eIF-3 ~120 is observed.
Thus, eIF-4F ~220, eIF-4B and ribosomal protein S6 are phosphorylated via PMA-dependent and insulin-dependent pathways, whereas phosphorylation of eIF-4F p25 and eIF-3 ~120 is stimulated only upon activation of protein kinase C. Phosphopeptide maps of eIF-4F ~220 and ribosomal protein S6 suggest that protease-activated kinase II is one of the protein kinases involved in the insulin-stimulated response in protein kinase C-depleted cells.
Protein phosphorylation is a fundamental regulatory mechanism involved in the control of cellular processes by hormones. Treatment of cells with insulin causes specific changes in the phosphorylation of a number of cellular proteins as a result of insulin-induced alterations in the activity of specific  tion (4, 5). A number of different protein kinases appear to be involved in the insulin response, including the tyrosine protein kinase of the insulin receptor, serine/threonine protein kinases modulating ribosomal protein S6 (4, 6), and microtubule-associated protein-2 kinase (1,4,7,8). Other hormones involved in growth and differentiation mediate changes in cellular metabolism by activation of the Ca*+/ phospholipid-dependent protein kinase (9,10). Protein kinase C is regulated by modulation of diolein levels through activation of phospholipase C. The enzyme also serves as a receptor for PMA,' which substitutes for diolein in the activation process.
One of the initial events occurring in response to stimulation with insulin or PMA is the multiple phosphorylation of ribosomal protein S6 (4, 11). The same sites are modified in uiuo in response to a number of mitogenic compounds, including PMA and insulin. To date, four translational initiation factors, 2,3,4B, and 4F have been shown to be phosphorylated in uivo (12)(13)(14)(15)(16)19). Phosphorylation of eIF-3 ~170, eIF-4B, and eIF-4F p25 and ~220 is stimulated in reticulocytes in response to PMA (15). eIF-4B and eIF-4E/eIF-4F p25 have also been shown to be dephosphorylated upon heat shock, and during mitosis or serum-starvation of exponentially growing cells (13,14,19).
All four of the phosphorylated initiation factors play a crucial role in the translation process (20-22). eIF-2, which has been studied extensively, is involved in the binding of initiator tRNA to the 40 S preinitiation complex (20). eIF-4F is required for the efficient translation of capped mRNA (22-24) and is a major discriminatory factor in protein synthesis initiation (25). The p25 subunit is known to specifically recognize the mRNA cap structure (16,26), while the function of ~220, a critical component of eIF-4F (23, 27-29), remains unclear. It has been suggested that ~220 is involved in the alignment of eIF-4A with the mRNA cap and p25 (21). eIF-3 and eIF-4B often co-purify with eIF-4F (15, l&30). Although they are not stable components of the cap-binding protein complex, they associate with eIF-4F during the initiation process (30). eIF-3 is required for mRNA binding to the 43 S initiation complex (31, 32) and has ribosomal subunit dissociation activity (33). eIF-4B is involved in the recycling of eIF-4F between successive mRNAs (21,30,34,35 in response to PMA (15). Protease-activated kinase II, implicated in the insulin response (37), modifies the ~220 subunit in uitro (36). In these studies, we utilized eIF-4F to examine differences in the metabolic activation of protein kinases in response to insulin and PMA. At the same time, phosphorylation of eIF-3 and eIF-4B were examined, due to association of these factors with eIF-4F, and the data correlated with phosphorylation of S6. We found that phosphorylation of initiation factors in response to insulin and PMA is mediated by PMA-dependent and insulin-dependent pathways, the latter of which is mediated, at least in part, through proteaseactivated kinase II. Labeling of 3T3-Ll Cells with ["P/Orthophosphate-3T3-L1 cells were grown to confluency as described previously (37) and washed twice with phosphate-free Dulbecco's modified Eagle's medium. Cells were then incubated in the same medium with ["'Plorthophosphate (2 mCi/plate) in the absence of serum for 1.5 h. At that time, either insulin in 1 mM HCl or 1 mM HCl alone was added: the cells were incubated further at 37 "C as indicated in the figure legends. For PMA treatment, cells were incubated with 1 x lo-" M PMA in dimethyl sulfoxide or dimethyl sulfoxide alone (15), at 37 "C, for the times indicated. Cell lysates were prepared as described (37) in the presence of phosphatase and protease inhibitors (50 mM NaF, 1 mM GTP, 80 mM fl-glycerophosphate, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 40 fig/ml each of leupeptin, pepstatin, and antipain), and "P-labeled eIF-4F was isolated immediately by affinity chromatography on m'GTP-Sepharose, as described by Morley and Traugh (15). Phosphorylated proteins were analyzed by gel electrophoresis (15). Protein was stained with Coomassie Blue and the radioactivity was quantified by scanning the autoradiograms at 660 nm with a transmission densitometer.
Two-dimensional Phosphopeptide Mapping of eIF-QF-Following polyacrylamide gel electrophoresis, individual subunits of eIF-4F were excised from the gel and subjected to trypsin digestion and twodimensional phosphopeptide mapping as described (15,36). The phosphopeptides were detected by autoradiography.

Stimulation
of Phosphorylation of eIF-4F in Quiescent 3T3-Ll Cells in Response to Insulin-To examine whether the phosphorylation state of eIF-4F in quiescent, serum-starved 3T3-Ll cells was changed in response to insulin, cells were incubated with ["'Plorthophosphate in the absence of serum for 1.5 h, and then incubated in the absence or presence of 10m7 M insulin for 1.5 h. eIF-4F was isolated by m7GTP-Sepharose chromatography in the presence of protease and phosphatase inhibitors and equal amounts of protein analyzed by polyacrylamide gel electrophoresis. Little incorporation of "'Pi into eIF-4F was obtained in the absence of insulin (Fig.  1A). However, upon insulin stimulation a large increase in phosphorylation of both p25 and ~220 was observed. As shown in the autoradiograms in Fig. lA, all of the eIF-4F adhered to the column. In agreement with data from studies on this initiation factor from reticulocytes (15, 38), changes in phosphorylation of p25 and ~220 had no effect on the binding and recovery of eIF-4E and eIF-4F from m7GTP-Sepharose. Phosphoamino acid analysis of both p25 and ~220 showed only the presence of phosphoserine (data not shown). Typically, the increase in phosphorylation in response to insulin was 2.5-5fold for ~220 and ~25, although stimulation as high as lo-fold was seen with the ~220 subunit. This coincided with a 2.5-3fold increase in phosphorylation of ribosomal protein S6 (data Equal amounts of protein were analyzed-b; gel electrophoresis as described under "Experimental Procedures"; the resultant autoradiogram is shown. Lanes 1 and 4, cell lysate; lanes 2 and 5. protein which did not adhere to m7GTP-Sepharose; lanes 3 and 6, protein eluted from m'GTP-Sepharose with 0.075 mM m7GTP. Arrows indicate the nosition of eIF-4F ~25 and ~220, eIF-4B, and _ eIF-3 ~120. These data are representative of results obtained in six separate experiments. B, 3T3-Ll cells were preincubated with ["'PI orthophosphate, and then incubated with the indicated final concentrations of insulin for 1.5 h. 32 P-Labeled eIF-4F was isolated and analyzed as described in A. Radioactivity was quantified by scanning the autoradiogram at 660 nm with a transmission densitometer. not shown). Identical results were obtained when cells were washed prior to the addition of insulin to remove the radiolabel. Identical results were also obtained following a 24-h preincubation with 32Pi with or without cell washing prior to the addition of insulin. These controls rule out any effect of '"Pi transport on the insulin-or PMA-induced phosphorylation observed.
To determine whether stimulation of phosphorylation was physiologically relevant, cells were incubated with insulin at concentrations from 10-l' to 10m7 M and "P-labeled eIF-4F Insulin and PIVIA Stimulate Phosphorylation of Initiation Factors 10613 was isolated and analyzed. As shown in Fig. lB, optimal stimulation of phosphorylation of both the ~220 and ~25 subunits of eIF-4F was observed at lo-" M insulin. This indicates that the stimulation was dependent on physiological concentrations of insulin. In some experiments, an apparent increased level of phosphorylation of eIF-4F ~25, as shown in these data, reflects a higher level of phosphorylation during the preincubation period. A similar effect has been reported in studies utilizing reticulocytes (15). Phosphorylation of eIF-3 and eIF-4B in Response to Insulin-By virtue of association with eIF-4F during the initiation process (30), additional "'P-labeled proteins (5-10% of the total protein) were recovered with eIF-4F during chromatography on m'GTP-Sepharose.
As judged by co-migration with purified eIF-3 and eIF-4B upon gel electrophoresis, these phosphoproteins were identified as eIF-3 ~120, and eIF-4B. Upon insulin stimulation of cells, a 2-4-fold increase in the phosphorylation of both eIF-4B and eIF-3 ~120 was observed at physiologically relevant insulin concentrations (Fig. 1). Phosphoamino acid analysis of eIF-4B and eIF-3 ~120 showed only the presence of phosphoserine (data not shown). Stimulation of Phosphorylation of eIF-4F, eIF-4B, and eIF-3 in Response to PMA-To determine whether PMA could influence the phosphorylation state of eIF-4F in quiescent 3T3-Ll cells, the cells were preincubated with ["*P]orthophosphate in the absence of serum and then incubated further for 45 min in the absence or presence of 1 X lo-" M PMA. In the absence of PMA, low levels of phosphorylation of eIF-4F were seen (Fig. 2). Upon PMA stimulation, an increase in phosphorylation of ~25 and ~220 of 4-5-fold was observed. In addition, a 2-3-fold increase in phosphorylation of eIF-4B and eIF-3 ~120 (Fig. 2) was detected in the PMA-stimulated cells. The amounts of eIF-3 and eIF-4B associated with eIF- 3T3-Ll cells were preincubated with [3LP]orthophosphate and then incubated in the absence or presence of 1 X lo-" M PMA for 45 min. "P-Labeled eIF-4F was isolated, and equal amounts of protein were analyzed by polyacrylamide gel electrophoresis followed by autoradiography. Arrows indicate the position of eIF-4F p25 and ~220, eIF-3 ~120, and eIF-4B on the autoradiogram. 4F during chromatography on m7GTP-Sepharose were similar, if not identical, from cells incubated in the presence or absence of insulin or PMA. Thus, under all conditions examined, complexation of the factors was not altered by the phosphorylation state. Two-dimensional Phosphopeptide Maps of p25 and p220-Radioactive subunits of eIF-4F were isolated from control and insulin-stimulated or PMA-treated 3T3-Ll cells, subjected to extensive trypsin digestion and analyzed by two-dimensional phosphopeptide mapping followed by autoradiography. Analysis of ~25 from quiescent and insulin-stimulated cells showed a single phosphopeptide (Fig. 3, A and B), the intensity of which was increased with insulin (Fig. 3B). Similarly, a single highly phosphorylated peptide was observed with PMA-treatment (Fig. 3C). This phosphopeptide was identical to that observed upon PMA-induced phosphorylation of ~25 in reticulocytes and by phosphorylation of purified eIP-4P in vitro with protein kinase C (15). eIF-4F ~220 from quiescent, insulin-or PMA-stimulated cells, showed a more complex phosphopeptide pattern upon trypsin digestion. Analysis of eIF-4F ~220 isolated from insulin-stimulated cells showed that, relative to quiescent cells, the intensity of existing phosphopeptides was increased in addition to the presence of new labeled species (Fig. 3, D and E). Upon PMA treatment, similar analyses of ~220 showed a phosphopeptide pattern distinct from that obtained with insulin-stimulated cells (Fig.  3F). However, this pattern was very similar to that obtained from ~220 isolated from PMA-stimulated reticulocytes (15) and from ~220 phosphorylated by protein kinase C in vitro W-3.

Stimulation of Phosphotylation of Initiation Factors in Cells Down-regulated
with Phorbol Esters-Total cellular protein kinase C activity can be down-regulated by prolonged exposure of cells to phorbol esters (9). These protein kinase Cdepleted cells were used to determine whether protein kinase C was the sole mediator of phosphorylation of eIF-4F in Co. Quiescent cells were treated with 1 X lo-' M PMA at 37 "C for 24 h and then incubated with ["*P]orthosphosphate for 1.5 h in the absence of serum. Cells were then challenged with 1 X 10m6 M PMA or 10m7 M insulin for 45 min; following lysis, '"P-labeled eIF-4F was isolated immediately by m7GTP-Sepharose affinity chromatography.
As shown in Fig. 4A, addition of fresh PMA led to little or no change in the level of phosphorylation of ~25 or ~220. This indicates that the cells were deficient in protein kinase C. Similarly, little stimulation  A, quiescent 3T3-Ll cells were rendered protein kinase C-deficient by pretreatment for 24 h with 1 X 10mG M PMA. Subsequently, cells were incubated with ["'Plorthophosphate for 1.5 h in the absence of serum, followed by incubation for 45 min with no additions as a control (C), with 1 x lo-" M PMA (P) or with lo-' M insulin (I). Cell lysates were prepared and ""P-labeled eIF-4F was isolated and equal amounts of protein analyzed by polyacrylamide gel electrophoresis as described under "Experimental Procedures. " "P was quantified by scanning the resulting autoradiograms at 660 nm with a transmission densitometer. Ribosomes were isolated by ultracentrifugation from the material which did not adhere to the m'GTP-Sepharose affinity column. The ribosomes were resuspended (39) and equal amounts of protein were analyzed by gel electrophoresis and the autoradiogram was quantified as described. B, ""P-labeled eIF-4F ~220 was isolated from downregulated control (i) or insulin-stimulated cells (ii), as described in A, and subjected to two-dimensional tryptic phosphopeptide mapping. The autoradiograms are presented and arrows indicate the origin in each case.
of phosphorylation of eIF-4B or eIF-3 ~120 was detected. This was in contrast to the results presented in Fig. 2, where a 4fold stimulation of phosphorylation of p25 and ~220, and a 2-2.5-fold stimulation of phosphorylation of eIF-4B and eIF-3 ~120 was observed in direct response to PMA.
When the down-regulated cells were stimulated with insulin, there was little or no change in the level of phosphorylation of eIF-4F ~25. However, "*Pi incorporation into ~220 was stimulated 5-fold, a degree similar to that obtained after insulin addition to quiescent cells (Fig. 1). As shown in Fig.  4A, insulin treatment, but not PMA treatment, of the downregulated cells, led to stimulation of S6 phosphorylation. Phosphorylation of eIF-4B was also increased, but to a lesser extent, with little change in the phosphorylation state of eIF-3 p120.
Two-dimensional phosphopeptide mapping of eIF-4F ~220 isolated from the down-regulated cells was carried out as described. As shown in Fig. 4B, tryptic phosphopeptide maps of eIF-4F ~220 isolated from insulin-stimulated down-regulated cells showed a pattern similar to that obtained with insulin-stimulated quiescent cells (Fig. 3E), and distinct from cells exposed to PMA (Fig. 3F). Phosphoamino acid analysis of eIF-4F ~25, ~220, and eIF-4B from these cells showed only phosphoserine (data not shown). Two-dimensional phosphopeptide mapping of S6, isolated from such cells, showed the presence of five major phosphopeptides; these were the same species observed with S6 phosphorylated in vitro by proteaseactivated kinase II (Ref. 37 and data not shown).
These data show PMA-stimulated phosphorylation of eIF-4F ~25 and eIF-3 ~120 by a protein kinase C-dependent pathway, whereas stimulation of phosphorylation of eIF-4F ~220, eIF-4B, and S6 was in response to insulin and PMA. This suggests that ~220, eIF-4B, and S6 were phosphorylated by a PMA-dependent pathway, via protein kinase C, and an insulin-dependent pathway, which appears to include several protein kinases including protease-activated kinase II. DISCUSSION We have demonstrated that two subunits of eIF-4F (~25 and ~220) are phosphorylated in 3T3-Ll cells and the phosphorylation is stimulated in response to insulin. Phosphorylation of both the ~25 and ~220 subunits of eIF-4F is stimulated typically 2.5-5-fold, respectively, although stimulation as high as lo-fold has been seen for the ~220 subunit. Optimal stimulation of phosphorylation of both subunits is observed at lo-' M insulin, indicating the response is dependent upon physiologically relevant insulin concentrations. Cell extracts prepared from insulin-stimulated cells retain the ability to phosphorylate eIF-4F in uitro (data not shown). Phosphoamino acid analysis of p25 and ~220 isolated from insulinstimulated cells shows only phosphoserine, even when extracts are prepared in the presence of 1 mM vanadate (data not shown). In addition to eIF-4F, other proteins involved in the mRNA binding step of protein synthesis initiation are phosphorylated in response to insulin. Exposure of 3T3-Ll cells to insulin promotes the phosphorylation of eIF-4B, eIF-3 ~120, and ribosomal protein S6, typically with a 2.5-3-fold increase in phosphorylation (summarized in Table I). In studies with reticulocytes, purification of eIF-4B and eIF-3 have suggested that the increased phosphorylation observed reflects the total cellular pools of these factors (data not shown).
Treatment of 3T3-Ll cells with 1 X 10e6 M PMA promotes the phosphorylation of eIF-4F ~25 and ~220 by 4-fold, with a 2-2.5-fold increase in phosphorylation of eIF-4B and eIF-3  Table I, and indicate that these components can be phosphorylated in 3T3-Ll cells by a protein kinase C-dependent mechanism. However, in reticulocytes, ~170 is the major phosphorylated subunit of eIF-3 which copurifies with eIF-4F. The reasons for this difference are unclear. Cell extracts derived from PMA-stimulated cells also retain the ability to phosphorylate eIF-4F, eIF-4B, and eIF-3 in vitro (data not shown).
To determine the relative contributions of protein kinase C-dependent and independent pathways involved in initiation factor phosphorylation in response to insulin, 3T3-Ll cells have been down-regulated by prolonged exposure to PMA. When further challenged with PMA, no increase in phosphorylation of any initiation factor studied or S6 can be detected, indicating that the cells are depleted of protein kinase C. Upon addition of insulin to such cells, phosphorylation of eIF-4F ~220 is stimulated &fold, a degree similar to that observed by addition of insulin to quiescent cells (Table  I). A 3-fold stimulation of S6 is the same as that observed in the absence of down-regulation.
Phosphorylation of eIF-4B is increased 2-fold in the down-regulated cells in response to insulin. Similar results for eIF-4B have been described in phorbol ester down-regulated, serum-stimulated HeLa cells.' Little stimulation of phosphorylation of eIF-3 ~120 and eIF-4F ~25 is observed under these conditions.
When eIF-4F is analyzed by two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis (15), approximately 25% of ~2.5 is in the phosphorylated form following down-regulation with PMA (data not shown). Therefore, the lack of phosphorylation of ~25 in response to insulin is not due to the fact that p25 is fully phosphorylated during the down-regulation process. The data indicate that phosphorylation of eIF-4F ~220, eIF-4B, and S6 appears to be through a protein kinase Cindependent mechanism in PMA down-regulated, insulinstimulated 3T3-Ll cells. Radioactive subunits of eIF-4F ~25 and ~220, isolated from insulin and PMA-stimulated cells, have been subjected to tryptic digestion and two-dimensional phosphopeptide mapping. In all cases, mapping of labeled ~25 gives rise to a single phosphopeptide, the intensity of which is increased upon insulin or PMA treatment of cells. This is consistent with previous studies in reticulocytes stimulated with PMA (15) and with phosphorylation of eIF-4F ~25 in vitro by protein kinase C (15). The phosphopeptides generated from the ~220 subunit showed a more complex pattern. Upon insulin stimulation, new species of labeled peptides can be seen, in addition to an increase in the intensity of existing peptides. The pattern generated includes some phosphopeptides visualized following phosphorylation of ~220 by protease-activated kinase II, but is distinct from those obtained upon PMA treatment or phosphorylation of eIF-4F by protein kinase C in uitro. eIF-4F ~220 isolated from down-regulated cells treated with insulin contains the same novel phosphopeptides observed with insulin alone. This suggests that protease-activated kinase II and at least one other protein kinase is involved in phosphorylation of eIF-4F ~220 in viva in response to insulin. Tuazon et al. (36) have shown that eIF-3, eIF-4B, and eIF-4F can be phosphorylated in vitro by three or more protein kinases. Protein kinase C isolated from bovine brain, phosphorylates eIF-3 ~120 and ~170, eIF-4B, and eIF-4F ~25 and ' R. Duncan and S. J. Morley, unpublished data. ~220 (15,36). These data, in conjunction with the experiments described herein, suggest that protein kinase C directly modulates these initiation factors in response to PMA. Two other protein kinases, protease-activated kinase II (the multipotential S6 kinase) and protease-activated kinase I, phosphorylate eIF-3 ~120 and/or ~170, eIF-4B, and eIF-4F ~220 in uitro, while casein kinase II modifies eIF-3 ~120 and ~170 and eIF-4B (36). The classical mitogen-stimulated S6 kinase from 3T3-Ll cells activated in response to insulin, does not phosphorylate any of the initiation factors directly (36). Similarly, the insulin-stimulated microtubule-associated protein-2 kinase which phosphorylates an S6 kinase in Xenopus eggs (7,8), does not appreciably modify eIF-4F ~220 in vitro (data not shown). Additional studies have indicated that eIF-4F ~220 can be phosphorylated in vitro by purified insulin receptor preparations.
Phosphoamino acid analysis shows the presence of phosphoserine and phosphothreonine in addition to phosphotyrosine (data not shown). This probably reflects the presence of membrane-associated insulin-sensitive serine/ threonine kinases in the receptor preparation (1). The physiological relevance of this remains unclear as ~220 labeled in vivo in response to mitogens showed only the presence of phosphoserine.
These data show that eIF-4F ~220, eIF-4B, and S6 are phosphorylated by both a PMA-dependent and insulin-dependent pathway, whereas phosphorylation of eIF-4F ~25 and eIF-3 ~120 is stimulated primarily upon activation of protein kinase C. In addition, these studies suggest that in 3T3-Ll cells, insulin may increase the activity of protein kinase C, either directly or indirectly.
In light of the data presented above, insulin also activates protease-activated kinase II, which appears to be one of the protein kinases responsible for the insulin-induced phosphorylation of these initiation factors but is not the only protein kinase modifying eIF-4F ~220 in uiuo. Studies are currently underway to determine how these phosphorylation events lead to stimulation of protein synthesis in these cells. Coordinate phosphorylation of eIF-4F, eIF-4B, eIF-3, and S6 could be responsible for stimulation of protein synthesis and for altered translation of specific classes of mRNA observed upon stimulation of quiescent cells by hormones and growth factors (4, 17).