(Received for publication, December 2, 1996, and in revised form, January 21, 1997)
From the Departments of Molecular Physiology and
Biological Physics, § Pharmacology, and ¶ Medicine,
University of Virginia School of Medicine, Charlottesville, Virginia
22908
Phosphorylation of PHAS-I by mitogen-activated
protein (MAP) kinase in vitro decreased PHAS-I binding to
eukaryotic initiation factor (eIF)-4E. The decrease in binding lagged
behind the phosphorylation of PHAS-I in Ser64, the
preferred site of MAP kinase. Binding of the Ala64 mutant
of PHAS-I to eIF-4E was abolished by MAP kinase, indicating that
phosphorylation of sites other than Ser64 control binding.
To identify such sites, PHAS-I was phosphorylated with MAP kinase and
[-32P]ATP and then cleaved proteolytically before the
resulting phosphopeptides were isolated by reverse phase chromatography
and directly identified by amino acid sequencing. Phosphorylated
residues were located by determining the cycles in which
32P was released when phosphopeptides were subjected to
sequential Edman degradation. With an extended incubation in
vitro, MAP kinase phosphorylated Thr36,
Thr45, Ser64, Thr69, and
Ser82. In rat adipocytes, the phosphorylation of all five
sites was increased by insulin and decreased by rapamycin although
there were differences in the magnitude of the effects. A form of
PHAS-I phosphorylated exclusively in Thr36 remained bound
to eIF-4E, indicating that phosphorylation of Thr36 is
insufficient for dissociation of the PHAS-I·eIF-4E complex. In
summary, our results indicate that multiple phosphorylation sites are
involved in the control of PHAS-I. All five sites identified fit a
(Ser/Thr)-Pro motif, suggesting that the phosphorylation of PHAS-I in
cells is mediated by a proline-directed protein kinase.
Insulin and growth factors act within minutes to stimulate protein
synthesis (1, 2). This rapid response is due to activation of mRNA
translation and involves phosphorylation of multiple translation factors. One of these factors is PHAS-I (also known as 4EBP-1) (3, 4),
a protein of Mr 12,500 that was first
identified in 32P-labeled adipocytes as a heat- and
acid-stable species that was markedly phosphorylated in response to
insulin (5). PHAS-I is now known to be expressed in a wide variety of
cell types and to act as a regulator of eIF-4E, the mRNA
cap-binding protein (6, 7).
eIF-4E is one of the least abundant of the known translation factors
(8, 9), and the amount of eIF-4E is believed to be limiting for
initiation, which is generally the rate-limiting phase of protein
synthesis (1, 2). Thus, increasing eIF-4E in cells increases mRNA
translation, particularly of those messages which possess a high degree
of secondary structure in their 5-untranslated regions (10). eIF-4E is
a key component of the eIF-4F complex, which catalyzes melting of
secondary structure in the 5
-untranslated region of the mRNA and
allows efficient binding and/or scanning by the 40 S ribosomal subunit
(1, 2, 8, 9). eIF-4F contains two other subunits, eIF-4A, an
ATP-dependent helicase, and eIF-4G, a relatively large
subunit that binds to both eIF-4A and eIF-4E (1, 2, 8, 9).
Nonphosphorylated PHAS-I binds tightly to eIF-4E (4, 11) and blocks
eIF-4E binding to eIF-4G (12, 13). Consequently, increasing PHAS-I
inhibits cap-dependent mRNA translation both in
vitro and in intact cells (4). However, when PHAS-I is
phosphorylated in response to insulin or certain growth factors, the
PHAS-I·eIF-4E complex dissociates (6, 7), thereby increasing eIF-4E
available to bind to eIF-4G. Increasing the eIF-4F complex by this
mechanism provides an explanation of the preferential stimulation by
insulin of the translation of messages, such as ornithine
decarboxylase, which have a high degree of secondary structure in their
5-untranslated region (14).
The immunosuppressive drug, rapamycin, promotes dephosphorylation of PHAS-I in adipocytes (15-17) and a variety of other cell types (18-21). Rapamycin is a potent inhibitor of the activation of p70S6K (22, 23), but this enzyme is almost certainly not a PHAS-I kinase in cells since it does not phosphorylate PHAS-I in vitro (6, 16, 24). PHAS-I can be phosphorylated in vitro by protein kinase C and casein kinase II (6, 24, 25), but whether either of these kinases phosphorylate PHAS-I in cells is not known. PHAS-I is an excellent substrate for MAP kinase1 in vitro (11, 24); and, Ser64, the site in PHAS-I preferred by MAP kinase, is phosphorylated in response to insulin in adipocytes (24). However, several lines of evidence indicate that MAP kinase is not the major mediator of insulin action on PHAS-I (6). For example, when bound to eIF-4E, the phosphorylation of PHAS-I by MAP kinase in vitro is relatively slow (6, 15, 16), and inhibiting activation of MAP kinase with the inhibitor of MEK activation, PD 098059, did not block the effects of insulin on PHAS-I in 3T3-L1 adipocytes (15).
The amino acid sequence surrounding a particular Ser or Thr residue contributes to the specificity of many protein kinases (26). Thus, identification of phosphorylation sites can provide clues as to which kinases phosphorylate a protein. PHAS-I isolated from adipocytes contains both phosphothreonine and phosphoserine (27-29), and results from peptide mapping studies (24) and two-dimensional electrophoretic analyses (15, 30) indicate that PHAS-I is phosphorylated in multiple sites in cells. Only one phosphorylation site (Ser64) has been identified directly (24), and the possibility exists that the sites most important in regulating the association of PHAS-I and eIF-4E has not been determined. The objectives of our experiments were to identify the sites of phosphorylation in PHAS-I in adipocytes, to investigate the role of these sites in modulating binding of PHAS-I to eIF-4E, and to determine the changes in phosphorylation produced by insulin and rapamycin.
Fat cells were isolated by collagenase digestion of
adipose tissue from male rats (Wistar, 120-140 g, 20 per preparation) (31) and then washed four times and suspended in low Pi
medium (0.2 mM sodium phosphate, 125 mM NaCl,
1.2 mM MgCl2, 4 mM KCl, 2.6 mM CaCl2, 0.5 mM glucose, 10 mg/ml
bovine serum albumin, and 24 mM sodium HEPES, pH 7.4).
Na32Pi (5 mCi/10-ml aliquot of cells) was
added, and the cells were incubated at 37 °C for 90 min, a time
sufficient to achieve steady-state labeling of intracellular ATP (32).
After treatments with insulin and rapamycin, the incubations were
terminated by homogenizing the cells as described previously (24) in
Homogenization Buffer (2 ml/ml packed cells), which
contained 100 mM NaF, 10 mM EDTA, 2 mM EGTA, 1 mM benzamidine, 0.2 mM
phenylmethylsulfonyl flouride, and 50 mM Tris, pH 7.8. The
homogenates were centrifuged at 28,000 × g for 30 min.
PHAS-I was immunoprecipitated from the supernatants by using an
affinity-purified antibody (3) coupled to protein A-Sepharose
(Pharmacia Biotech Inc.) as described previously (11). To elute PHAS-I
from the immune complexes, the beads were suspended in 300 µl of 1%
-mercaptoethanol, 1 mM EDTA, and 10 mM
Tris-HCl, pH 7.5, and incubated at 95 °C for 15 min. PHAS-I, which
is relatively stable to heat (3), was recovered in the supernatant
after centrifuging samples at 10,000 × g for 10 min.
Wild-type
PHAS-I (17), PHAS-I tagged at the NH2 terminus with a
hexahistidine sequence ([H6]PHAS-I) (24),
[H6]PHAS-I having a Ser to Ala mutation at position 64 ([H6]PHAS-IAla64) (11), the ERK2 isoform of
MAP kinase (33), and a constitutively active form of MEK1 (34) were
expressed in bacteria and purified as described in the references
indicated. MAP kinase was activated by incubation with MEK1 as
described by Scott et al. (34). Samples (160 µg/ml) of
PHAS-I, [H6]PHAS-I, or
[H6]PHAS-IAla64 were incubated at 23 °C in
a solution containing activated MAP kinase (34) (62 µg/ml), 1 mM dithiothreitol, 200 µM
[-32P]ATP (500-1000 cpm/pmol), 10 mM
MgCl2, and 50 mM Tris-HCl, pH 7.5. The
reactions were stopped by heating at 95 °C for 5 min. Samples of
[H6]PHAS-I and [H6]PHAS-IAla64
used in far-Western analyses of FLAG-4E binding (described later) were
phosphorylated in parallel incubations with unlabeled ATP, and the
reactions were stopped by adding SDS sample buffer.
[32P]PHAS-I samples (300 µl) from either in vitro phosphorylation reactions or immunoprecipitated from 32P-labeled adipocytes were incubated at 37 °C for 15 h with lysyl endopeptidase (17 µg/ml). The digests were acidified by adding 30 µl of 1% trifluoroacetic acid solution and applied at 30 °C to a reverse phase column (Waters Nova-Pak C18, 3.9 × 150 mm) that had been equilibrated in 0.1% trifluoroacetic acid (Buffer A). The flow rate was maintained at 1 ml/min. The column was washed for 5 min with Buffer A before peptides were eluted with a linear gradient of acetonitrile (0-60% in 120 min) produced by increasing the proportion of Buffer B (0.1% trifluoroacetic acid in acetonitrile). Fractions (1 ml) were collected, and peptides containing 32P were identified by measuring Cerenkov emissions. A second digestion was required to resolve phosphorylation sites present in phosphopeptides in fractions 62 through 72. These fractions were pooled and evaporated to dryness before the peptides were dissolved in 250 µl of solution containing 1 mM dithiothreitol, 1% hydrogenated Triton X-100 (Calbiochem), and 50 mM Tris-HCl, pH 8.0. The samples were incubated for 15 h at 37 °C with chymotrypsin (5 µg) and then acidified by adding 25 µl of 1% trifluoroacetic acid before peptides were resolved by reverse phase HPLC as described above.
The 32P-labeled peptides generated by phosphorylating
recombinant PHAS-I with [-32P]ATP and MAP kinase were
identified by using a vapor phase amino acid sequencer (Applied
Biosystems Procise 494). Phosphorylated residues within phosphopeptides
were located by determining the cycles in which 32P was
released when samples were subjected to sequential Edman degradation
under conditions that optimize recovery of 32P (35).
Protein samples were subjected to electrophoresis in 20% polyacrylamide gels in the presence of SDS by using the method of Laemmli (36). Dried gels were exposed to film to enable detection of 32P-labeled PHAS-I, and bands containing the protein were excised. The amounts of 32P in the gel slices were determined by measuring Cerenkov emissions. Binding of PHAS-I proteins to eIF-4E was assessed by far-Western blotting performed using a 32P-labeled recombinant eIF-4E fusion protein (FLAG-4E) as described previously (17). Phosphoamino acid analyses were performed by subjecting samples of phosphopeptides that had been purified by reverse phase HPLC to limited hydrolysis (5.7 N HCl for 2 h at 110 °C) (37). After removing the acid under vacuum, samples were subjected to high voltage electrophoresis at pH 1.9, which provides superior separation of phosphoserine and phosphothreonine. The fact that phosphothreonine and phosphotyrosine are not resolved at this pH was not a problem as PHAS-I from adipocytes is not phosphorylated on tyrosyl residues (27-29).2
Other Materials[-32P]ATP and
Na32Pi were purchased from DuPont NEN.
Chymotrypsin and lysyl endopeptidase were obtained from Boehringer
Mannheim and WAKO Pure Chemical Industries, respectively. HPLC grade
solvents were from Baker Chemical Co. Amino acid sequencing supplies
were from Applied Biosystems. Most commonly used chemicals were
from Sigma. Rapamycin was obtained from
Calbiochem-Novabiochem International, and human insulin was from Eli
Lilly.
PHAS-I contains seven Ser/Thr-Pro motifs
that represent potential sites of phosphorylation by MAP kinase (Fig.
1), the most effective of the protein kinases that have
been found to phosphorylate PHAS-I in vitro (6, 16, 24). In
previous experiments Ser64 was identified as the preferred
site of phosphorylation by MAP kinase (24). However, with extended
incubation with MAP kinase, the stoichiometry of PHAS-I phosphorylation
exceeded 1 mol/mol (24), and some phosphorylation of an
Ala64 mutant PHAS-I was observed (11), indicating that MAP
kinase phosphorylated more than one site in PHAS-I. Results from
peptide mapping experiments provide further evidence of multisite
phosphorylation (Fig. 2). Three peaks of
32P-labeled peptides, designated LE-P2, LE-P3, and LE-P4 in
order of elution, were resolved when a sample of recombinant
[H6]PHAS-I that had been phosphorylated in a 10-min
incubation with [-32P]ATP and MAP kinase was digested
with lysine endopeptidase and subjected to reverse phase HPLC (Fig.
2A, inset). LE-P2 was completely absent in
digests of [H6]PHAS-IAla64 that had been
phosphorylated by MAP kinase, indicating that LE-P2 represents the
32P-labeled Ser64 phosphopeptide. After 2 h with MAP kinase, relatively more 32P was found in sites
in LE-P3 plus LE-P4 than in Ser64 (Fig.
2A).3 In addition, a relatively
small peak, LE-P1, eluting very early in the acetonitrile gradient was
observed. The phosphorylation of other sites by MAP kinase was more
pronounced in Fig. 2 than in our previous studies (11, 24) because
approximately 10-fold higher concentrations of MAP kinase were used in
the present experiments. However, consistent with previous findings
(11, 24), the initial rate of phosphorylation of Ser64
(LE-P2) occurred more rapidly than the initial rate of phosphorylation of the other sites (Fig. 3A), which were
phosphorylated at least as rapidly in the Ala64 mutant as
in the wild-type protein (Fig. 3B).
The influence of Ser64 and other sites on eIF-4E binding was assessed by far-Western blotting using a 32P-labeled FLAG-4E. MAP kinase markedly decreased binding of [H6]PHAS-I to FLAG-4E (Fig. 3C, inset). However, the initial rate of decline in eIF-4E binding activity (Fig. 3C) produced by MAP kinase did not correlate well with the initial rate of Ser64 phosphorylation (Fig. 3A). For instance, almost no decrease in eIF-4E binding was observed after 5 min of incubation with MAP kinase, even though over half of the Ser64 in PHAS-I had been phosphorylated. The decrease in binding activity correlated much better with the phosphorylation of sites recovered in LE-P2 and LE-P3, indicating that phosphorylation of sites other than Ser64 control binding of PHAS-I to eIF-4E. Findings with [H6]PHAS-IAla64 provide additional support for this interpretation. The initial decrease in eIF-4E binding activity produced by phosphorylation of the mutant protein occurred with a time course that was similar to that obtained with [H6]PHAS-I (Fig. 3C), and extended incubation with MAP kinase abolished binding of the mutant PHAS-I to FLAG-4E (Fig. 3C, inset).
Identification of Sites in PHAS-I Phosphorylated by MAP Kinase in VitroThe first approach to identify the sites other than
Ser64 was to attempt to identify the phosphopeptides
derived from lysyl endopeptidase digests of PHAS-I. Except for the
4-amino acid peptide that would result from cleavage at
Lys68 and Lys72, all of the predicted peptides
contain at least 12 amino acids (Fig. 1) and would be expected to elute
relatively late in the acetonitrile gradient. For this reason, the
4-amino acid peptide containing Thr69 was considered the
most likely candidate for LE-P1. Phosphoamino acid analysis supported
this assignment, as LE-P1 was found to contain phosphothreonine but
little if any phosphoserine.2 Amino acid sequencing
indicated that the peptide contained Pro residues in the second and
third positions (Fig. 4). Other than the
Thr69 peptide, the only two other occurrences of adjacent
prolines in PHAS-I are Pro29-Pro30 and
Pro88-Pro89 (Fig. 1). Determining the position
of the phosphorylated residue within the LE-P1 phosphopeptide
solidified the assigment of Thr69 as the phosphorylation
site. This was accomplished in a separate sequencing run, where the
32P in LE-P1 was found to be released in cycle 1 (Fig. 4).
Even without the sequence data, finding release in the first cycle with
a peptide generated by lysyl endopeptidase would be indicative of
Thr69 as this residue is the only Ser or Thr adjacent to a
Lys in PHAS-I (Fig. 1).
The absence of LE-P2 in peptides derived from [H6]PHAS-IAla64 provided strong evidence that Ser64 was the site of phosphorylation in the LE-P2 phosphopeptide (Fig. 2A, inset). Proof that Ser64 was the phosphorylated residue was provided by amino acid sequencing which yielded a single sequence corresponding to the predicted Ser64 peptide (Fig. 4). As Ser64 is the only Ser/Thr in this peptide, this residue had to be the phosphorylated site. As expected, release of 32P from the phosphopeptide in LE-P2 occurred in cycle 8 (Fig. 4).
Amino acid sequencing indicated that LE-P3 contained the peptide generated by cleaving [H6]PHAS-I at Lys72 and Lys104. As this peptide contained multiple Ser and Thr, it was necessary to measure 32P release during sequential Edman degradation to determine the location of the phosphorylated residue. The surge in 32P release from LE-P3 occurred in cycle 10, identifying Ser82 as a phosphorylation site. The percentage of 32P released from LE-P3 samples was relatively low. This was probably due in part to the NH2-terminal Asp residue, as Asp may form a cyclic imide structure after reaction with the 1-ethyl-3-dimethylaminopropyl carbodiimide used to couple the peptides to the solid support (Sequalon-AA), thereby blocking Edman degradation (38). However, LE-P3 was also found to contain some of a relatively large peptide predicted to extend from the His-tag to Lys56 (Fig. 1). This peptide, which was the predominant species in LE-P4,2 contained multiple Ser/Thr residues that were too far from the NH2 terminus to be identified as phosphorylation sites by determining 32P release during Edman degradation. Therefore, an additional digestion was needed to resolve phosphorylation sites. Fractions containing LE-P3 and LE-P4 were pooled, and concentrated under vacuum to remove the acetonitrile and trifluoroacetic acid. After the peptides were digested with chymotrypsin, three peaks of radioactivity, designated CT-P1, CT-P2, and CT-P3, were resolved by reverse phase HPLC (Fig. 2B).
Amino acid sequencing indicated that CT-P1 contained a phosphopeptide derived from the more COOH-terminal portion of a region in PHAS-I that contains a repeat of the sequence, STTPGGT (Fig. 4). When a sample of CT-P1 was subjected to sequential Edman degradation, the surge in 32P release occurred in cycle 3, identifying Thr45 as a site of phosphorylation (Fig. 4). The more NH2-terminal portion of the region containing the repeat was found in CT-P2, and with this phosphopeptide, the surge of 32P also occurred in cycle 3, identifying Thr36 as a site of phosphoryation (Fig. 4). [32P]Phosphoserine was the only phosphoamino acid detected in CT-P3,2 consistent with the interpretation that Ser82 was present in this peak.
Identification of Sites in PHAS-I Controlled by Insulin and Rapamycin in VitroThe control of PHAS-I phosphorylation in cells
was investigated by incubating rat adipocytes in medium containing
32Pi. After treatments with insulin and
rapamycin, the cells were homogenized, and 32P-labeled
PHAS-I was immunoprecipitated from extracts. Samples were subjected to
SDS-PAGE, and autoradiograms were prepared (Fig. 5A, inset). Essentially all of the
32P-labeled protein migrated in bands corresponding to the
PHAS-I protein. Insulin not only increased the amount of
32P-labeled PHAS-I, but also increased the proportion of
the 32P-labeled protein that was found in the most slowly
migrating form, a finding that is consistent with the previous
demonstration that phosphorylation of the appropriate sites in PHAS-I
decreases its electrophoretic mobility (11). Rapamycin treatment caused a net decrease in the 32P-content of PHAS-I and increased
the proportion of 32P found in the forms of higher
electrophoretic mobility (Fig. 5A, inset).
Incubating cells with rapamycin prior to insulin attenuated the effects
of the hormone on increasing phosphorylation of PHAS-I.
To investigate the distribution of 32P among different
sites, the immunoprecipitated PHAS-I was cleaved with lysyl
endopeptidase, and samples of the digest were analyzed by HPLC under
conditions used to resolve the MAP kinase phosphorylation sites (Fig.
5A). The pattern of 32P-labeled peptides from
cellular PHAS-I was similar to that obtained with the recombinant
protein, although there were differences. Notably, the relative size of
LE-P1 was much larger with the adipocyte PHAS-I, indicating that the
site in this peak was highly phosphorylated in cells. The elution
positions of LE-P1 and LE-P2 from cellular PHAS-I (Fig. 5A)
were identical to those of the corresponding peaks derived from
recombinant PHAS-I (Fig. 2A). To verify that the peaks
contained the same sites of phosphorylation, 32P release
was measured following Edman degradation of the peptides (Fig.
6). Almost all of the 32P in the LE-P1
peptide was released in cycle 1, and phosphothreonine was the only
phosphoamino acid detected in this peak (Fig. 7). These
findings indicate that LE-P1 contains the Thr69 peptide.
With LE-P2 a single surge of 32P occurred in cycle 8 (Fig.
6), and phophoserine was the only phosphoamino acid detected in this
peak (Fig. 7). Thus, LE-P2 appears to contain the Ser64
peptide.
LE-P3 and LE-P4 were not as well resolved using PHAS-I from cells as when the phosphorylated recombinant protein was used. Likewise, LE-P3 and LE-P4 from non-tagged recombinant PHAS-I lacking the His-tag were not resolved.2 The better resolution obtained with [H6]PHAS-I is presumably because the additional amino acids in the His-tag region cause the large peptide in LE-P4 to elute at higher concentrations of acetonitrile. Subjecting peptides in LE-P3 to sequential Edman degradation resulted in release of 32P in cycle 10, indicative of Ser82 phosphorylation. LE-P3 also contains other sites found in the large NH2-terminal peptide, but residues from this peptide are not released during Edman degradation because the NH2 terminus of PHAS-I from adipocytes is blocked (3). To resolve sites in the large peptide, LE-P3 fractions were pooled and incubated with chymotrypsin. This treatment resulted in generation of peptides that eluted in sharp peaks having the same retention times as CT-P1, CT-P2, and CT-P3 (Fig. 5B). Thus, the elution pattern suggested that the peptides that were phosphorylated by MAP kinase in vitro were also phosphorylated in cells. The peptides in these peaks were found to contain phosphothreonine, phosphothreonine, and phosphoserine (Fig. 7), respectively, as would be expected if Thr45, Thr36, and Ser82 were the phosphorylated residues. Surges of 32P release occurred in cycle 3 when the phosphopeptides in either CT-P1 or CT-P2 were subjected to sequential Edman degradation (Fig. 6). Thus, determinations of the positions of the phosphorylated residues in CT-P1 and CT-P2 supported the assignments of Thr45 and Thr36 as phosphorylation sites.
The effects of insulin and rapamycin on the 32P contents of
the five sites are summarized in Fig. 8. Insulin
increased the amount of 32P in all five sites, but the
effects of insulin on the different sites differed both in the extent
of 32P introduced into the sites and in the percentage
change in 32P. Relatively little 32P was
recovered in the Ser82 peptide, and Ser82 was
least affected by insulin, which produced only a 2-fold increase in
32P. The effects of insulin on the other four sites ranged
from approximately 2.5-fold with Thr69 to more than 4-fold
with Thr45. However, the different fold increases in
32P in Thr36, Thr45,
Ser64, and Thr69 produced by the hormone were a
function of the basal state of phosphorylation, as the four sites
contained approximately the same amount of 32P after
insulin treatment.
Incubating cells with rapamycin alone decreased the amount of 32P in all of the sites except Ser82 (Fig. 8). Differences among the sites in sensitivity to rapamycin emerged in the presence of insulin. Rapamycin markedly inhibited the insulin-stimulated phosphorylation of Thr45 and Thr69 (Fig. 8). In contrast, the increases in the 32P content of Ser64 produced by insulin in the presence and absence of rapamycin were approximately equal. Similarly, rapamycin had little if any effect on the increment in the 32P content of Thr36 produced by insulin.
Identification of a Phosphorylated Form of PHAS-I That Binds eIF-4EA partially phosphorylated form of PHAS-I has been
previously shown to bind labeled eIF-4E in far-Western analyses (17). Likewise, some phosphorylated PHAS-I was recovered with eIF-4E when
PHAS-I·eIF-4E complexes were purified from adipocyte extracts by
using m7GTP-Sepharose (15, 16). To identify the
phosphorylated sites in the eIF-4E-bound form of PHAS-I,
PHAS-I·eIF-4E complexes were purified from extracts of
32P-labeled adipocytes that had been incubated with
rapamycin plus insulin. After digesting the PHAS-I with lysyl
endopeptidase, essentially all of the 32P was recovered in
LE-P3.2 When the peptides in this peak were digested with
chymotrypsin and subjected to HPLC, the 32P was found in
CT-P2 (Fig. 9, lower panel), indicating that
the eIF-4E bound form was phosphorylated exclusively in
Thr36. For comparison, note that the PHAS-I protein that
did not bind to the cap affinity resin contained 32P in
both Thr36 and Thr45 (Fig. 9, upper
panel).
Our results provide definitive evidence that in rat adipocytes insulin stimulates the phosphorylation of PHAS-I in five sites, all of which fit a Ser/Thr-Pro motif (Fig. 1). The existence of multiple sites raises the possibility that more than one site is involved in the control of PHAS-I binding to eIF-4E. Moreover, identification of the sequence of amino acids surrounding the different phosphorylated residues has provided information needed to identify the protein kinases responsible for phosphorylating PHAS-I in cells. Thus, the results have important implications with respect to not only the control of PHAS-I binding to eIF-4E but also to the mechanisms of action of insulin and rapamycin.
Multisite Phosphorylation and the Control of PHAS-I Binding to eIF-4EAll five of the phosphorylation sites identified in rat PHAS-I are conserved in not only the mouse (15) and human proteins (4), but also in PHAS-II, another eIF-4E binding protein that is approximately 60% identical to PHAS-I (4, 17). The conservation of sites suggests that all may be important for the function of the protein. However, our results indicate that phosphorylation of certain sites can have a much greater impact on the binding of PHAS-I to eIF-4E than phosphorylation of others. For example, phosphorylation of Ser64, the only site that had been previously identified in PHAS-I (24), did not attenuate binding of PHAS-I to eIF-4E as assessed by far-Western blotting (Fig. 3). Indeed, in contrast to our previous hypothesis (4, 11) phosphorylation of this site was neither necessary nor sufficient for inhibiting binding of PHAS-I to eIF-4E, at least in far-Western analyses. Examining the phosphorylation patterns of PHAS-I in cells provided clues concerning the relative importance of the other sites. For example, the finding that PHAS-I phosphorylated in Thr36 remained bound to eIF-4E in extracts of rat adipocytes (Fig. 9) indicates that phosphorylation of Thr36 is not sufficient for dissociation of the PHAS-I·eIF-4E complex. It would also seem unlikely that Ser82 phosphorylation could explain the near complete inhibition of PHAS-I binding to eIF-4E produced by insulin (4, 11) since Ser82 appeared to contain much less 32P than any of the other four sites (Fig. 8). However, the apparently low stoichiometry of phosphorylation of Ser82 may have been due to poor recovery of CT-P3. Therefore, a role for Ser82 phosphorylation in regulating PHAS-I is still possible. Moreover, it should be stressed that with five sites of phosphorylation, complex interactions are possible, and phosphorylation of sites such as Thr36 and Ser64 could increase or decrease the rate of phosphorylation and/or the influence of other sites on eIF-4E binding. Nevertheless, through the process of elimination, Thr45 and Thr69 have emerged as candidates for important regulatory sites.
Implications of Multisite Phosphorylation With Respect to Signaling PathwaysThe protein kinases responsible for phosphorylating PHAS-I in adipocytes have not been determined. However, it is now possible to exclude certain protein kinases that are capable of phosphorylating PHAS-I in vitro. Diggle et al. (25) first demonstrated that casein kinase II phosphorylated PHAS-I purified from adipocyte extracts. Based on evidence that insulin activated casein kinase II in adipocytes, it was proposed that casein kinase II mediated the phosphorylation of PHAS-I by insulin (25). None of the five sites phosphorylated in rat adipocytes meet the minimum consensus requirement for phosphorylation by casein kinase II (26). We have recently found that casein kinase II preferentially phosphorylates Ser111, which is not phosphorylated in cells.2 Thus, it is clear that casein kinase II cannot be the major mediator of the action of insulin on PHAS-I. Similarly, none of the sites phosphorylated in vitro meet the requirements for phosphorylation by protein kinase C (26), which phosphorylates recombinant PHAS-I (6, 24).
MAP kinase is by far the most effective kinase that has been described for phosphorylating PHAS-I in vitro (6, 16, 24). MAP kinase is activated by insulin in adipocytes (39), and all five sites phosphorylated by MAP kinase in vitro were phosphorylated in response to the hormone (Fig. 6). Despite this finding, neither the ERK1 nor ERK2 isoform is the sole mediator of the phosphorylation of PHAS-I in response to insulin in adipocytes as blocking MAP kinase activation with the MEK inhibitor, PD 098059, did not attenuate the effect of insulin in these cells (15). As discussed previously (15), this result does not eliminate the possibility that MAP kinase contributes to the control of PHAS-I phosphorylation as more than one pathway may be involved. Interestingly, PD 098059 was recently found to promote dephosphorylation of PHAS-I in CHO cells (40). Thus, there is reason to suspect that MAP kinase, or another kinase regulated by MEK, is involved in the control of PHAS-I in certain cell types.
PHAS-I phosphorylation is controlled by a rapamycin-sensitive pathway that is distinct from the MAP kinase signaling pathway (15, 16, 19, 21). The most extensively characterized enzyme regulated by rapamycin is p70S6K, a protein kinase that is activated by insulin and a variety of mitogenic stimuli (23). Full activation of the kinase appears to require phosphorylation of two classes of sites. One class consists of three Ser/Thr residues (Thr229, Thr389, and Ser404) that are flanked by aromatic residues (41). The other consists of four Ser/Thr-Pro residues found in a 14-amino acid stretch located near the COOH terminus of the kinase in a region referred to as the autoinhibitory domain (42, 43). Rapamycin is a potent inhibitor of the activation of p70S6K (44, 45), and an obvious possibility was that p70S6K was responsible for phosphorylating PHAS-I in cells. However, this hypothesis was eliminated by the finding that PHAS-I was not a substrate for p70S6K (6, 16, 24).
The five phosphorylation sites in PHAS-I resemble the four sites in the autoinhibitory domain of p70S6K (42). Both sets of sites are markedly increased in response to insulin or mitogenic stimulation (6, 23), and it is an intriguing possibility that the Ser/Thr-Pro sites in the two proteins are phosphorylated by the same protein kinase. Phosphorylation of PHAS-I and p70S6K by a common kinase would provide a mechanism linking capped mRNA translation, which is dependent on eIF-4E (9), and polypyrimidine tract mRNA translation, which appears to be regulated by phosphorylation of ribosomal protein S6 (46). However, circumstances might exist in which it would not be advantageous for a cell to up-regulate translation of both classes of mRNA, and the potential exists for selective regulation of the phosphorylation of PHAS-I and p70S6K. PHAS-I lacks the hydrophobic class of sites found in p70S6K as none of the Ser/Thr residues in PHAS-I are flanked by aromatic residues. The closest resemblance to the hydrophobic sites are two Ser that are adjacent to Phe and Tyr in a tandem repeat of the sequence, (Tyr/Phe)-Ser-Thr-Thr-Pro-Gly-Gly (Fig. 1). Neither of these Ser residues were phosphorylated to a significant extent in adipocytes as relatively little 32P released in cycle 1 when the CT-P1 and CT-P2 peptides were subjected to sequential Edman degradation (Fig. 6). In view of the differences in sequence surrounding the two classes of sites, it seems clear that a single protein kinase is not responsible for phosphorylating PHAS-I and the hydrophobic sites in p70S6K. This is an important point in considering the action of rapamycin, which decreases four of the Ser/Thr-Pro sites in PHAS-I (Fig. 8) and all three hydrophobic sites in p70S6K (41). However, it should be noted that rapamycin does promote dephosphorylation of p70S6K in Ser411, a site having Pro in the +1 position (41).
It is well established that rapamycin inhibits the mTOR signaling
pathway, but how mTOR signals is still a mystery (22). A region of mTOR
has homology with the catalytic domain of phosphatidyl inositol 3-OH
kinase, but it is not clear that mTOR functions as a lipid kinase.
Indeed, there is good reason to suspect that mTOR signals as a protein
kinase as it is homologous to the new class of protein kinases, which
include the catalytic subunit of DNA-dependent protein
kinase and the ATM gene product that is mutated in ataxia
telangiectasia (47). In addition, the protein undergoes
autophosphorylation in a reaction that is subject to inhibition by
rapamycin (48). The effect of rapamycin on autophosphorylation, as well
as the inhibitory effect of rapamycin on mTOR in cells, requires an
additional receptor protein, FKBP12, as it is the rapamycin·FKBP12
complex that actually binds to mTOR (22). Rapamycin binding to FKBP12
is competitively inhibited by FK506, and because the FK506/rapamycin
complex does not bind mTOR, FK506 competitively inhibits the effects of
rapamycin on mTOR-dependent pathways (22).
The findings that FK506 blocks the effects of rapamycin on PHAS-I (15, 19) and p70S6K (44, 45) provide strong evidence that the mTOR signaling pathway regulates both proteins. Results of recent experiments in T lymphoma cells (YAC-1) provide additional evidence implicating mTOR in the control of PHAS-I (20). In wild-type lymphoma cells rapamycin potently decreased PHAS-I phosphorylation, but rapamycin had little, if any, effect on PHAS-I in mutant lymphoma cells that had been selected on the basis of resistance to the antiproliferative effects of rapamycin (20). There is strong evidence that the rapamycin-resistant phenotype is due to a mutation which decreases the affinity of mTOR for rapamycin·FKBP-12 (49). Thus, there is both pharmacologic and genetic evidence supporting the conclusion that the mTOR signaling pathway is involved in the control of PHAS-I. The challenge now is to identify the PHAS-I kinases and/or phosphatases involved in the mTOR-dependent control of PHAS-I.