Phosphorylation Of PHAS-I Research Articles

Rapamycin inhibits substance P-induced protein synthesis and phosphorylation of PHAS-I (4E-BP1) and p70 S6 kinase (p70(S6K)) in human astrocytoma cells

The mitogenic substance P receptor (NK-1 subtype) is expressed in many primary human tumors with the highest frequency of expression appearing in astrocytomas and glioblastomas (75% and 100%, respectively). Recently, we showed that substance P neuropeptide induces DNA synthesis in the human astrocytoma U-373MG cells by activating the mitogen-activated protein (MAP) kinase pathway leading to the induction of c-Fos and c-Myc expression. The induction of these immediate early genes is necessary for the progression of cells form G1 to S phase of the cell cycle. In this study, we demonstrate that U-373MG cells are highly sensitive to the growth-inhibitory action of rapamycin at nanomolar concentrations (IC50 <1 ng/ml). We also show that SP peptide stimulates protein synthesis in the U-373MG cell line by activating a rapamycin-sensitive signaling pathway. Further, we demonstrate that SP is potent in stimulating PHAS-I protein (also known as 4E-BP1) phosphorylation and p70 S6 kinase (p70(S6K)) phosphorylation and enzymatic activity, and that this stimulation is inhibited by subnanomolar concentrations of rapamycin. In contrast, rapamycin was not at all effective in repressing SP-induced activation of MAP kinase pathway, c-Fos phosphoprotein expression, and DNA synthesis in U-373MG astrocytoma cells.

Inactivation of eIF2B and Phosphorylation of PHAS-I in Heat-shocked Rat Hepatoma Cells

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43 degrees C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.

Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin.

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.

Insulin activates a PD 098059-sensitive kinase that is involved in the regulation of p70 S6K and PHAS-I

Incubating either Chinese hamster ovary (CHO) cells or 3T3-L1 adipocytes with insulin increased the phosphorylation of the eIF-4E-binding protein, PHAS-I. Insulin also activated p70 S6K and the Erk-1 and Erk-2 isoforms of mitogen-activated protein kinase (MAP kinase). However, the concentrations of the hormone needed to activate MAP kinase were 10–100 times higher than those needed to increase PHAS-I phosphorylation and p70 S6K activity. Incubating cells with the inhibitor of MAP kinase kinase (MEK) activation, PD 098059, blocked the effects of low concentrations of insulin on PHAS-I and p70 S6K. The effects of the inhibitor were overcome by increasing concentrations of insulin. The results indicate that insulin activates a PD 098059-sensitive kinase that is involved in the regulation of both p70 S6K and PHAS-I.

PHAS-I phosphorylation in response to foetal bovine serum (FBS) is regulated by an ERK1/ERK2-independent and rapamycin-sensitive pathway in 3T3-L1 adipocytes

The phosphorylation state of PHAS-I is thought to be important in the regulation of protein synthesis initiation. PHAS-I phosphorylation significantly increases in response to growth factors and insulin. ERK1/ERK2 have previously been implicated as PHAS-I kinases. Present work utilised a specific phosphorothioate oligonucleotide antisense strategy against ERK1/ERK2 to determine whether ERK1/ERK2 mediate FBS-stimulated PHAS-I phosphorylation in vivo. Depleting >90% of cellular ERK1/ERK2 had no effect on FBS-stimulated PHAS-I phosphorylation. However, treatment of cells with a specific p70 S6k pathway inhibitor, rapamycin, markedly attenuated FBS-stimulated PHAS-I phosphorylation. These results indicate that PHAS-I phosphorylation in response to FBS occurs through an ERK1/ERK2-independent and rapamycin-sensitive pathway in 3T3-L1 adipocytes.

Identification of Phosphorylation Sites in the Translational Regulator, PHAS-I, That Are Controlled by Insulin and Rapamycin in Rat Adipocytes

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 [gamma-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 stimulates protein synthesis in skeletal muscle by enhancing the association of eIF-4E and eIF-4G.

Insulin stimulated protein synthesis in gastrocnemius muscle of perfused rat hindlimb preparations by approximately twofold. The stimulation of protein synthesis was associated with a 12-fold increase in the amount of eukaryotic initiation factor eIF-4G bound to the mRNA cap-binding protein eIF-4E. In part, the increased binding of eIF-4G to eIF-4E was a result of release of eIF-4E bound to the translational regulator, PHAS-I, through a mechanism involving enhanced phosphorylation of PHAS-I. However, the insulin-induced association of eIF-4E and eIF-4G was not due to increased net phosphorylation of eIF-4E because insulin decreased the amount present in the phosphorylated form from 86 to 59% of total eIF-4E. Overall, the results suggest that insulin stimulates protein synthesis in gastrocnemius muscle through a mechanism involving increased binding of eIF-4G to eIF-4E, which is in part due to phosphorylation of PHAS-I, resulting in a release of eIF-4E from the inactive PHAS-I x eIF-4E complex.

Phosphorylation of the eIF4E-binding protein PHAS-I after exposure of PC12 cells to EGF and NGF

PHAS-I or the eIF4E-binding protein 1 regulates the cap-binding activity of eIF4E by sequestering eIF4E. Binding of eIF4E to PHAS-I is regulated by phosphorylation of PHAS-I. PC12 cells were used to study the signal transduction pathway leading to phosphorylation of PHAS-I. Both EGF and NGF induced phosphorylation of PHAS-I. Wortmannin, a PI-3 kinase inhibitor, staurosporine, a PKC inhibitor, and rapamycin, a FRAP inhibitor all blocked the phosphorylation of PHAS-I. Of the three inhibitors, only wortmannin was able to inhibit MAPK phosphorylation. This excludes a role for MAPK in NGF- and EGF-induced PHAS-I phosphorylation in PC12 cells. Apparently, PHAS-I was phosphorylated in a PI-3 kinase-, PKC-, and FRAP-dependent manner after EGF or NGF stimulation. Only PI-3 kinase and FRAP are involved in the regulation of the basal level of PHAS-I phosphorylation.

Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase.

Insulin rapidly stimulates protein synthesis in a wide variety of tissues. This stimulation is associated with phosphorylation of several translational initiation and elongation factors, but little is known about the signaling pathways to these events. To study these pathways, we have used a myeloid progenitor cell line (32D) which is dependent on interleukin 3 but insensitive to insulin because of the very low levels of insulin receptor (IR) and the complete lack of insulin receptor substrate (IRS)-signaling proteins (IRS-1 and IRS-2). Expression of more IR permits partial stimulation of mitogen-activated protein kinase by insulin, and expression of IRS-1 alone mediates insulin stimulation of the 70-kDa S6 kinase (pp70S6K) by the endogenous IR. However, expression of both IR and IRS-1 is required for stimulation of protein synthesis. Moreover, this effect requires activation of phosphatidylinositol 3-kinase (PI3K), as determined by wortmannin inhibition and the use of an IRS-1 variant lacking all Tyr residues except those which activate PI3K. Stimulation of general protein synthesis does not involve activation by IRS-1 of GRB-2-SOS-p21ras or SH-PTP2, since IRS-1 variants lacking the SH2-binding Tyr residues for these proteins are fully active. Nor does it involve pp70S6K, since rapamycin, while strongly inhibiting the synthesis of a small subset of growth-regulated proteins, only slightly inhibits total protein synthesis. Recruitment of mRNAs to the ribosome is enhanced by phosphorylation of eIF4E, the cap-binding protein, and PHAS-I, a protein that specifically binds eIF4E. The behavior of cell lines containing IRS-1 variants and inhibition by wortmannin and rapamycin indicate that the phosphorylation of both proteins requires IRS-1-mediated stimulation of PI3K and pp70S6K but not mitogen-activated protein kinase or SH-PTP2.

Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle.

We have investigated the roles of eukaryotic initiation factor 4E (eIF-4E), the cap-binding protein, and the translational regulator, PHAS-I, in the effects of insulin and alloxan-induced diabetes on protein synthesis in rat skeletal muscle. Diabetes increased the amount of eIF-4E found in the inactive PHAS-I.eIF-4E complex by threefold, explaining in part the inhibitory effect of insulin deficiency on translation initiation. Insulin treatment of diabetic rats caused dissociation of the complex, consistent with the action of the hormone on reversing the inhibitory effect of diabetes on translation initiation. The effects of both insulin and diabetes on PHAS-I binding to eIF-4E appeared to be due to changes in PHAS-I phosphorylation. Neither insulin nor diabetes changed the phosphorylation state of eIF-4E. The results indicate that the effects of both insulin and diabetes on protein synthesis in skeletal muscle involve modulation of the interaction of PHAS-I and eIF-4E.

CAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells.

Incubating rat aortic smooth muscle cells with either platelet-derived growth factor BB (PDGF) or insulin-like growth factor I (IGF-I) increased the phosphorylation of PHAS-I, an inhibitor of the mRNA cap binding protein, eukaryotic initiation factor (eIF) 4E. Phosphorylation of PHAS-I promoted dissociation of the PHAS-I-eIF-4E complex, an effect that could partly explain the stimulation of protein synthesis by the two growth factors. Increasing cAMP with forskolin decreased PHAS-I phosphorylation and markedly increased the amount of eIF-4E bound to PHAS-I, effects consistent with an action of cAMP to inhibit protein synthesis. Both PDGF and IGF-I activated p70S6K, but only PDGF increased mitogen-activated protein kinase activity. Forskolin decreased by 50% the effect of PDGF on increasing p70S6K, and forskolin abolished the effect of IGF-I on the kinase. The effects of PDGF and IGF-I on increasing PHAS-I phosphorylation, on dissociating the PHAS-I-eIF-4E complex, and on increasing p70S6K were abolished by rapamycin. The results indicate that IGF-I and PDGF increase PHAS-I phosphorylation in smooth muscle cells by the same rapamycin-sensitive pathway that leads to activation of p70S6K.

Control of PHAS-I by Insulin in 3T3-L1 Adipocytes: SYNTHESIS, DEGRADATION, AND PHOSPHORYLATION BY A RAPAMYCIN-SENSITIVE AND MITOGEN- ACTIVATED PROTEIN KINASE-INDEPENDENT PATHWAY

PHAS-I levels increased 8-fold as 3T3-L1 fibroblasts differentiated into adipocytes and acquired sensitivity to insulin. Insulin increased PHAS-I protein (3.3-fold after 2 days), the rate of PHAS-I synthesis (3-fold after 1 h), and the half-life of the protein (from 1.5 to 2.5 days). Insulin also increased the phosphorylation of PHAS-I and promoted dissociation of the PHAS-I eukaryotic initiation factor-4E (eIF-4E) complex, effects that were maximal within 10 min. With recombinant [H6]PHAS-I as substrate, mitogen-activated protein (MAP) kinase was the only insulin-stimulated PHAS-I kinase detected after fractionation of extracts by Mono Q chromatography; however, MAP kinase did not readily phosphorylate [H6]PHAS-I when the [H6]PHAS-I.eIF-4E complex was the substrate. Thus, while MAP kinase may phosphorylate free PHAS-I, it is not sufficient to dissociate the complex. Moreover, rapamycin attenuated the stimulation of PHAS-I phosphorylation by insulin and markedly inhibited dissociation of PHAS-I.eIF-4E, without decreasing MAP kinase activity. Rapamycin abolished the effects of insulin on increasing phosphorylation of ribosomal protein S6 and on activating p70S6K. The MAP kinase kinase inhibitor, PD 098059, markedly decreased MAP kinase activation by insulin, but it did not change PHAS-I phosphorylation or the association of PHAS-I with eIF-4E. In summary, insulin increases the expression of PHAS-I and promotes phosphorylation of multiple sites in the protein via multiple transduction pathways, one of which is rapamycin-sensitive and independent of MAP kinase. Rapamycin may inhibit translation initiation by increasing PHAS-I binding to eIF-4E.

Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes.

PHAS-I is a heat- and acid-stable protein that is phosphorylated on Ser/Thr residues in response to insulin and growth factors. To investigate the phosphorylation of PHAS-I, the protein was expressed in bacteria and purified for use as substrate in protein kinase reactions in vitro. Recombinant PHAS-I was rapidly and stoichiometrically phosphorylated by mitogen-activated protein (MAP) kinase. At saturating MgATP, the Km and Vmax observed with PHAS-I were almost identical to those obtained with myelin basic protein, one of the best MAP kinase substrates. PHAS-I was also phosphorylated at a significant rate by casein kinase II and protein kinase C. To investigate sites of phosphorylation, PHAS-I was digested with collagenase and phosphopeptides were resolved by reverse phase high performance liquid chromatography. Almost all of the phosphate introduced by MAP kinase was recovered in the peptide, Leu-Met-Glu-Cys-Arg-Asn-Ser-Pro-Val-Ala-Lys-Thr. 32P was released in the seventh cycle of Edman degradation, identifying the Ser (Ser64) as the phosphorylated residue. Ser64 was also phosphorylated in response to insulin in rat adipocytes. We conclude that PHAS-I is a substrate for MAP kinase both in vivo and in vitro. As PHAS-I is one of the most prominent insulin-stimulated phosphoproteins in adipocytes, it may qualify as the major MAP kinase substrate in these cells.