The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3′ phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.
Activation of protein kinase B (PKB) by growth factors and hormones has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). In this report, we show that PKB can also be activated by PKA (cyclic AMP [cAMP]-dependent protein kinase) through a PI3-kinase-independent pathway. Although this activation required phosphorylation of PKB, PKB is not likely to be a physiological substrate of PKA since a mutation in the sole PKA consensus phosphorylation site of PKB did not abolish PKA-induced activation of PKB. In addition, mechanistically, this activation was different from that of growth factors since it did not require phosphorylation of the S473 residue, which is essential for full PKB activation induced by insulin. These data were supported by the fact that mutation of residue S473 of PKB to alanine did not prevent it from being activated by forskolin. Moreover, phosphopeptide maps of overexpressed PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant negative PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation.
p27kip1 (p27) is a multifunctional protein implicated in regulation of cell cycling, signal transduction, and adhesion. Its activity is controlled in part by Phosphatylinositol-3-Kinase (PI3K)/Akt1 signaling, and disruption of this regulatory connection has been identified in human breast cancers. The serine/threonine protein kinase Akt1 directly phosphorylates p27, so identifying the modified residue(s) is essential for understanding how it regulates p27 function. Various amino acids have been suggested as potential targets, but recent attention has focused on threonine 157 (T157) because it is located in a putative Akt1 consensus site. However, T157 is not evolutionarily conserved between mouse and human. We therefore re-evaluated Akt1 phosphorylation of p27 using purified proteins and in cells.
Here we show purified Akt1 phosphorylates human and mouse p27 equally well. Phospho-peptide mapping indicates Akt1 targets multiple sites conserved in both species, while phospho-amino acid analysis identifies the targeted residues as serine rather than threonine. P27 deletion mutants localized these sites to the N-terminus, which contains the major p27 phosphorylation site in cells (serine 10). P27 phosphorylated by Akt1 was detected by a phospho-S10 specific antibody, confirming this serine was targeted. Akt1 failed to phosphorylate p27S10A despite evidence of a second site from mapping experiments. This surprising result suggested S10 phosphorylation might be required for targeting the second site. We tested this idea by replacing S10 with threonine, which as expected led to the appearance of phospho-threonine. Phospho-serine was still present, however, confirming Akt1 sequentially targets multiple serines in this region. We took two approaches in an attempt to explain why different residues were previously implicated. A kinetic analysis revealed a putative Akt1 binding site in the C-terminus, which may explain why mutations in this region affect p27 phosphorylation. Furthermore, commercially available recombinant Akt1 preparations exhibit striking differences in substrate specificity and site selectivity. To confirm S10 is a relevant site, we first showed that full-length wild type Akt1 purified from mammalian cells phosphorylates both human and mouse p27 on S10. Finally, we found that in cultured cells under physiologically relevant conditions such as oxidative stress or growth factor deprivation, endogenous Akt1 causes p27 accumulation by phosphorylating S10.
Identifying where Akt1 phosphorylates p27 is essential for understanding its functional implications. We found that full-length wild type Akt1 – whether purified, transiently overexpressed in cells, or activated in response to cellular stress – phosphorylates p27 at S10, a noncanonical but evolutionarily conserved site known to regulate p27 activity and stability. Using recombinant Akt1 recapitulating this specificity, we showed modification of p27S10 also leads to phosphorylation of an adjacent serine. These results integrate PI3K/Akt1 signaling in response to stress with p27 regulation through its major phosphorylation site in cells, and thus identify new avenues for understanding p27 deregulation in human cancers.
We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKBβ, a dramatic increase in Akt1/PKBα protein and relocation of Akt1/PKBα from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27kip1, a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome.
The authors previously reported that physiological light induces the tyrosine phosphorylation of insulin receptors (IRs), which leads to the activation of the phosphoinositide 3-kinase (PI3K) and Akt (serine/threonine protein kinase B) survival pathway in rod photoreceptor cells. Tissue-specific deletion of IRs from photoreceptors resulted in stress-induced photoreceptor degeneration. Insulin growth factor 1 receptor (IGF-1R) is highly related in sequence and structure to the IR and shares 70% sequence identity overall and 84% identity within the tyrosine kinase domain. The role of IGF-1R in photoreceptor function is unknown. In this study the authors examined IGF-1R signaling in rod outer segment (ROS) membranes.
IGF-1R localization was examined in the plasma and disc membranes of ROS. Activation of the IGF-1R/PI3K/Akt pathway was analyzed using specific antibodies against phospho-tyrosine, IGF-1R, and phospho-Akt. PI3K activity was determined in the anti-phospho-tyrosine and anti-IGF-1R immunoprecipitates. Glutathione-S-transferase fusion proteins containing two Src homology 2 (SH2) domains of the p85 subunit of PI3K and their mutants were used to study the molecular interaction between IGF-1R and p85. In vivo IGF-1R signaling was studied in rats exposed to physiological light or to constant light.
IGF-1R is predominately localized to plasma membranes of ROS. These studies indicate that light stress results in an increase in tyrosine phosphorylation of IGF-1R and an increase in PI3K enzyme activity in anti-phosphotyrosine and anti-IGF-1R immunoprecipitates of ROS and retinal homogenates. The authors observed that light stress induces tyrosine phosphorylation of IGF-1R in ROS membranes, which leads to the binding of p85 through N-SH2 and C-SH2 domains. Finally, the authors observed a significant activation of Akt in light-stressed retinas, indicating activation of the Akt survival pathway downstream of IGF-1R activation.
Light stress induced the activation of PI3K through activation and binding of IGF-1R, which leads to activation of the Akt survival pathway in photoreceptors.
Insulin-like growth factor-1 (IGF-1) is a polypeptide growth factor with a variety of functions in both neuronal and non-neuronal cells. IGF-1 plays anti-apoptotic and other functions by activating multiple signaling pathways including Akt kinase, a serine/threonine kinase essential for cell survival. The nuclear transcription factor cAMP response element-binding protein (CREB) may also be involved although relationships between these two proteins in IGF-1 receptor signaling and protection is not clear, especially in neuronal cells.
IGF-1, in a concentration- and time-dependent manner, induces the activation/phosphorylation of Akt and CREB in PC12 cells by activating different signaling pathways. IGF-1 induced a sustained phosphorylation of Akt while only a transient one was seen for CREB. The phosphorylation of Akt is mediated by the PI3 kinase pathway while that of CREB is dependent on the activation of both MAPK kinase and p38 MAPK. Moreover, the stimulation of PKC attenuated the phosphorylation of Akt induced by IGF-1 while enhancing that of CREB. Survival assays with various kinase inhibitors suggested that the activation/phosphorylation of both Akt and CREB contributes to IGF-1 mediated cell survival in PC12 cells.
These data suggest that IGF-1 induced the activation of Akt and CREB using distinct pathways in PC12 cells.
We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.
In pediatric tumor xenograft models, tumor-derived IGF-2 results in intrinsic resistance to IGF-1R-targeted antibodies, maintaining continued tumor angiogenesis. We evaluated the anti-angiogenic activity of a ligand-binding antibody (MEDI-573) alone or in combination with IGF-1 receptor binding antibodies (MAB391, CP01-B02).
IGF-stimulated signaling was monitored by increased Akt phosphorylation in sarcoma and human umbilical chord vascular endothelial cells (HUVECs). Angiogenesis was determined in vitro using capillary tube formation in HUVECs and in vivo using a VEGF-stimulated Matrigel assay. Tumor growth delay was examined in four sarcoma xenograft models
The IGF-ligand binding antibody MEDI-573 suppressed Akt phosphorylation induced by exogenous IGF-1 and IGF-2 in sarcoma cells. Receptor binding antibodies suppressed IGF-1 stimulation of Akt phosphorylation, but IGF-2 circumvented this effect and maintained HUVEC tube formation. MEDI-573 inhibited HUVEC proliferation and tube formation in vitro, but did not inhibit angiogenesis in vivo, probably because MEDI-573 binds murine IGF-1 with low affinity. However, in vitro the anti-angiogenic activity of MEDI-573 was also circumvented by human recombinant IGF-1. The combination of receptor- and ligand-binding antibodies completely suppressed VEGF-stimulated proliferation of HUVECs in the presence of IGF-1 and IGF-2, prevented ligand-induced phosphorylation of IGF-1R/IR receptors, and suppressed VEGF/IGF-2 driven angiogenesis in vivo. The combination of CP1-BO2 plus MEDI-573 was significantly superior to therapy with either antibody alone against IGF-1 and IGF-2 secreting pediatric sarcoma xenograft models.
These results suggest that combination of antibodies targeting IGF receptor and ligands may be an effective therapeutic strategy to block angiogenesis for IGF-driven tumors.
The aim of the present study is to test the hypothesis that insulin-like-growth factor-1 (IGF-1) plays a role in the regulation of basolateral Cl channels in the thick ascending limb (TAL). The patch-clamp experiments demonstrated that application of IGF-I or insulin inhibited the basolateral 10-pS Cl channels. However, the concentration of insulin required for the inhibition of the Cl channels by 50% (K1/2) was ten times higher than those of IGF-1. The inhibitory effect of IGF-I on the 10-pS Cl channels was blocked by suppressing protein tyrosine kinase or by blocking phosphoinositide 3-kinase (PI3K). In contrast, inhibition of phospholipase C (PLC) failed to abolish the inhibitory effect of IGF-1 on the Cl channels in the TAL. Western blot analysis demonstrated that IGF-1 significantly increased the phosphorylation of phospholipid-dependent kinase (PDK) at serine residue 241 (Ser241) and AKT at Ser473 in the isolated medullary TAL. Moreover, inhibition of PI3K with LY294002 abolished the effect of IGF-1 on the phosphorylation of PDK and AKT. The notion that the effect of IGF-1 on the 10-pS Cl channels was induced by stimulation of PDK-AKT-mTOR pathway was further suggested by the finding that rapamycin completely abolished the effect of IGF-1 on the 10-pS Cl channels in the TAL. We conclude that IGF-1 inhibits the basolateral Cl channels by activating PI3K-AKT-mTOR pathways. The inhibitory effect of IGF-1 on the Cl channels may play a role in ameliorating the ischemia-induced renal injury through IGF-1 administration.
phosphoinositide 3-kinase; PKD; AKT; mTOR; Cl transport
Insulin-like growth factor I (IGF-I) stimulates smooth muscle cell (SMC) proliferation, and the mitogen-activated protein kinase (MAPK) pathway plays an important role in mediating IGF-I–induced mitogenic signaling. Our prior studies have shown that recruitment of Src homology 2 domain tyrosine phosphatase (SHP-2) to the membrane scaffolding protein Src homology 2 domain–containing protein tyrosine phosphatase substrate-1 (SHPS-1) is required for IGF-I–dependent MAPK activation. The current studies were undertaken to define the upstream signaling components that are required for IGF-I–stimulated MAPK activation and the role of SHPS-1 in regulating this process. The results show that IGF-I–induced Shc phosphorylation and its subsequent binding to Grb2 is required for sustained phosphorylation of MAPK and increased cell proliferation in SMCs. Furthermore, for Shc to be phosphorylated in response to IGF-I requires that Shc must associate with SHPS-1 and this association is mediated in part by SHP-2. Preincubation of cells with a peptide that contains a phospho-tyrosine binding motif sequence derived from SHPS-1 inhibited IGF-I–stimulated SHP-2 transfer to SHPS-1, the association of Shc with SHPS-1, and IGF-I–dependent Shc phosphorylation. Expression of an SHPS-1 mutant that did not bind to Shc or SHP-2 resulted in decreased Shc and MAPK phosphorylation in response to IGF-I. In addition, SMCs expressing a mutant form of the β3 subunit of the αVβ3, which results in impairment of SHP-2 transfer to SHPS-1, also showed attenuated IGF-I–dependent Shc and MAPK phosphorylation. Further analysis showed that Shc and SHP-2 can be coimmunoprecipitated after IGF-I stimulation. A cell-permeable peptide that contained a polyproline sequence from Shc selectively inhibited Shc/SHP-2 association and impaired Shc but not SHP-2 binding to SHPS-1. Exposure to this peptide also inhibited IGF-I–stimulated Shc and MAPK phosphorylation. Cells expressing a mutant form of Shc with the four prolines substituted with alanines showed no Shc/SHPS-1 association in response to IGF-I. We conclude that SHPS-1 functions as an anchor protein that recruits both Shc and SHP-2 and that their recruitment is necessary for IGF-I–dependent Shc phosphorylation, which is required for an optimal mitogenic response in SMCs.
Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor β subunit (PDGFRβ) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRβ mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cγ (PLCγ) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRβ. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCγ-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.
The insulin/IGF-1 pathway controls a number of physiological processes in the nematode worm Caenorhabditis elegans, including development, aging and stress response. We previously found that the Akt/PKB ortholog AKT-1 dampens the apoptotic response to genotoxic stress in the germline by negatively regulating the p53-like transcription factor CEP-1. Here, we report unexpected rearrangements to the insulin/IGF-1 pathway, whereby the insulin-like receptor DAF-2 and 3-phosphoinositide-dependent protein kinase PDK-1 oppose AKT-1 to promote DNA damage-induced apoptosis. While DNA damage does not affect phosphorylation at the PDK-1 site Thr350/Thr308 of AKT-1, it increased phosphorylation at Ser517/Ser473. Although ablation of daf-2 or pdk-1 completely suppressed akt-1-dependent apoptosis, the transcriptional activation of CEP-1 was unaffected, suggesting that daf-2 and pdk-1 act independently or downstream of cep-1 and akt-1. Ablation of the akt-1 paralog akt-2 or the downstream target of the insulin/IGF-1 pathway daf-16 (a FOXO transcription factor) restored sensitivity to damage-induced apoptosis in daf-2 and pdk-1 mutants. In addition, daf-2 and pdk-1 mutants have reduced levels of phospho-MPK-1/ERK in their germ cells, indicating that the insulin/IGF-1 pathway promotes Ras signaling in the germline. Ablation of the Ras effector gla-3, a negative regulator of mpk-1, restored sensitivity to apoptosis in daf-2 mutants, suggesting that gla-3 acts downstream of daf-2. In addition, the hypersensitivity of let-60/Ras gain-of-function mutants to damage-induced apoptosis was suppressed to wild-type levels by ablation of daf-2. Thus, insulin/IGF-1 signaling selectively engages AKT-2/DAF-16 to promote DNA damage-induced germ cell apoptosis downstream of CEP-1 through the Ras pathway.
C. elegans; PI3K; apoptosis; DNA damage; Ras
The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser487 phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr172 phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation.
Akt phosphorylates the α1 subunit of AMPK at Ser487, leading to reduced Thr172 phosphorylation and activation by LKB1. We establish a molecular mechanism for this effect and show that it causes down-regulation of AMPK in several PTEN-null tumour cell lines.
Akt; AMP-activated protein kinase (AMPK); cancer; cross-talk; tumour suppressor; ACC, acetyl-CoA carboxylase; AICAR, 5-amino-4-imidazolecarboxamide riboside; AMPK, AMP-activated protein kinase; BRSK, brain-specific kinase; CaMKK, calmodulin-dependent kinase kinase β; DMEM, Dulbecco’s modified Eagle’s medium; GSK3, glycogen synthase kinase 3; HEK, human embryonic kidney; IGF-1, insulin-like growth factor 1; LKB1, liver kinase B1; MEF, mouse embryonic fibroblast; MO25α, mouse protein-25α; mTORC1, mammalian (or mechanistic) target of rapamycin complex 1; NEAA, non-essential amino acid; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A (cAMP-dependent protein kinase); PTEN, phosphatase and tensin homologue deleted on chromosome 10; S6K1, S6 kinase 1; ST loop, serine/threonine-rich loop; STRADα, Ste20-related adapter protein-α; WT, wild-type
The serine/threonine protein kinase B (PKB/Akt) is involved in insulin signaling, cellular survival, and transformation. Carboxyl-terminal modulator protein (CTMP) has been identified as a novel PKB binding partner in a yeast two-hybrid screen, and appears to be a negative PKB regulator with tumor suppressor-like properties. In the present study we investigate novel mechanisms by which CTMP plays a role in apoptosis process.
CTMP is localized to mitochondria. Furthermore, CTMP becomes phosphorylated following the treatment of cells with pervanadate, an insulin-mimetic. Two serine residues (Ser37 and Ser38) were identified as novel in vivo phosphorylation sites of CTMP. Association of CTMP and heat shock protein 70 (Hsp70) inhibits the formation of complexes containing apoptotic protease activating factor 1 and Hsp70. Overexpression of CTMP increased the sensitivity of cells to apoptosis, most likely due to the inhibition of Hsp70 function.
Our data suggest that phosphorylation on Ser37/Ser38 of CTMP is important for the prevention of mitochondrial localization of CTMP, eventually leading to cell death by binding to Hsp70. In addition to its role in PKB inhibition, CTMP may therefore play a key role in mitochondria-mediated apoptosis by localizing to mitochondria.
L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBα)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Δp85α). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBα, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBα/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBα with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBα/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.
Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1). By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM), a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor) and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.
A critical protein in cell-signalling pathways, called protein kinase B, regulates many aspects of cell biology from metabolism to proliferation and survival, by modifying other proteins with the addition of a phosphate group. Hence, deregulation of its activity has acute consequences on cell function. Increased activity of a tumour-promoting form of protein kinase B or of upstream regulatory proteins has been observed in tumours, while impaired protein kinase B function has been linked to diabetes. Therefore, understanding the molecular mechanism of protein kinase B activation will help reveal how its activity might be regulated to limit disease progression. Toward this end, we studied how protein kinase B structure relates to its function, to identify molecular mechanisms regulating its kinase activity, modifying its cellular localization, and altering its binding to other proteins. By determining the spatial organization of different regions of the protein in inactive protein kinase B, we discovered a cavity at the interface of two distinct functional regions of the inactive form. We also localized the C-terminal end of the protein to the apex of the cavity, suggesting a role of this domain in regulating the inactive form of the protein. This represents a novel example of negative regulation by inhibition across these different regions of the protein. From these findings, we elucidated the mechanism of action of a highly specific protein kinase B inhibitor, AKT inhibitor VIII. We determined that simultaneous binding of the inhibitor to the two different functional regions, through the cavity, “locks” protein kinase B in an inactive conformation and prevents regulatory proteins from accessing the C-terminal domain.
The mechanism of inhibition of the activity of the tumor-promoter protein kinase B operates via interactions across its functional domains.
The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is critical for both normal mammary gland development and malignant transformation. It has been reported that the IGF-1 stimulates breast cancer cell proliferation and is upregulated in tumors with BRCA1/2 mutations. We report here that IGF-1 is negatively regulated by BRCA1 at the transcriptional level in human breast cancer cells. BRCA1 knockdown (BRCA1-KD) induces the expression of IGF-1 mRNA in MCF7 cells in an estrogen receptor α (ERα)-dependent manner. We found that both BRCA1 and ERα bind to the endogenous IGF-1 promoter region containing an estrogen-responsive element-like (EREL) site. BRCA1-KD does not significantly affect ERα binding on the IGF-1 promoter. Reporter analysis demonstrates that BRCA1 could regulate IGF-1 transcripts via this EREL site. In addition, enzyme-linked immunosorbent assay revealed that de-repression of IGF-1 transcription by BRCA1-KD increases the level of extracellular IGF-1 protein, and secreted IGF-1 seems to increase the phospho-IGF-1Rβ and activate its downstream signaling pathway. Blocking the IGF-1/IGF-1R/phosphoinositide 3-kinase (PI3K)/AKT pathway either by a neutralizing antibody or by small-molecule inhibitors preferentially reduces the proliferation of BRCA1-KD cells. Furthermore, the IGF-1-EREL-Luc reporter assay demonstrates that various inhibitors, which can inhibit the IGF-1R pathway, can suppress this reporter activity. These findings suggest that BRCA1 defectiveness keeps turning on IGF-1/PI3K/AKT signaling, which significantly contributes to increase cell survival and proliferation.
BRCA1; IGF-1; negative regulation; ERα; positive-feedback activation
The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels.
Electronic supplementary material
The online version of this article (doi:10.1007/s12013-010-9101-4) contains supplementary material, which is available to authorized users.
PKB; Akt; Phosphorylation; Protein phosphatase inhibitor
Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.
A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by ∼75% in CHO cells and ∼30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.
Backgound: Hexokinase II binds to mitochondria and promotes cell survival.
Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress.
Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection.
Significance: The Akt-HK-II signaling nexus is important in cell survival.
Hexokinase II (HK-II) is an enzyme that catalyzes the first step in glycolysis and localizes not only in the cytosol but also at mitochondria. Akt, activated by insulin-like growth factor 1 (IGF-1) treatment in neonatal rat ventricular myocytes, translocates to mitochondria and increases mitochondrial HK-II binding. Expression of an HK-II-dissociating peptide diminished IGF-1-induced increases in mitochondrial HK-II as well as protection against hydrogen peroxide treatment, suggesting an important role of mitochondrial HK-II in IGF-1/Akt-mediated protection. We hypothesized, on the basis of an Akt phosphorylation consensus sequence present in HK-II, that Thr-473 is the target of Akt kinase activity. Indeed, recombinant kinase-active Akt robustly phosphorylates WT HK-II, but not Thr-473 mutants. Phosphomimetic (T473D)HK-II, but not non-phosphorylatable (T473A)HK-II, constitutively increased mitochondrial binding compared with WT HK-II and concomitantly confers greater protection against hydrogen peroxide. Glucose 6-phosphate (G-6P), a product of the catalytic activity of HK-II, is well known to dissociate HK-II from mitochondria. Addition of G-6P to isolated mitochondria dose-dependently dissociates WT HK-II, and this response is inhibited significantly in mitochondria isolated from cardiomyocytes expressing T473D HK-II. Pretreatment with IGF-1 also inhibits G-6P-induced overexpressed or endogenous HK-II dissociation, and this response was blocked by Akt inhibition. These results show that Akt phosphorylation of HK-II at Thr-473 is responsible for the Akt-mediated increase in HK-II binding to mitochondria. This increase is, at least in part, due to the decreased sensitivity to G-6P-induced dissociation. Thus, phosphorylation-mediated regulation of mitochondrial HK-II would be a critical component of the protective effect of Akt.
Akt PKB; Cell Death; Hexokinase; Insulin-like Growth Factor (IGF); Mitochondria; Phosphorylation; Cardiomyocytes; Glucose 6-Phosphate
The eIF4E-binding proteins (4E-BPs) interact with translation initiation factor 4E to inhibit translation. Their binding to eIF4E is reversed by phosphorylation of several key Ser/Thr residues. In Drosophila, S6 kinase (dS6K) and a single 4E-BP (d4E-BP) are phosphorylated via the insulin and target of rapamycin (TOR) signaling pathways. Although S6K phosphorylation is independent of phosphoinositide 3-OH kinase (PI3K) and serine/threonine protein kinase Akt, that of 4E-BP is dependent on PI3K and Akt. This difference prompted us to examine the regulation of d4E-BP in greater detail. Analysis of d4E-BP phosphorylation using site-directed mutagenesis and isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the regulatory interplay between Thr37 and Thr46 of d4E-BP is conserved in flies and that phosphorylation of Thr46 is the major phosphorylation event that regulates d4E-BP activity. We used RNA interference (RNAi) to target components of the PI3K, Akt, and TOR pathways. RNAi experiments directed at components of the insulin and TOR signaling cascades show that d4E-BP is phosphorylated in a PI3K- and Akt-dependent manner. Surprisingly, RNAi of dAkt also affected insulin-stimulated phosphorylation of dS6K, indicating that dAkt may also play a role in dS6K phosphorylation.
Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.
Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCζ and PKCλ) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKCλ in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKCλ (λKD or λΔNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKCλ, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by λKD or λΔNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKCλ was ∼50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKCλ mutant that lacks the pseudosubstrate domain (λΔPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of λΔPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKCλ. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
Bcl-2 is a critical suppressor of apoptosis that is overproduced in many types of cancer. Phosphorylation of the Bcl-2 protein is induced on serine residues in tumor cells arrested by microtubule-targeting drugs (paclitaxel, vincristine, nocodazole) and has been associated with inactivation of antiapoptotic function through an unknown mechanism. Comparison of a variety of pharmacological inhibitors of serine/threonine-specific protein kinases demonstrated that the cyclin-dependent kinase inhibitor, flavopiridol, selectively blocks Bcl-2 phosphorylation induced by antimicrotubule drugs. Bcl-2 could also be coimmunoprecipitated with the kinase Cdc2 in M-phase-arrested cells, suggesting that Cdc2 may be responsible for phosphorylation of Bcl-2 in cells treated with microtubule-targeting drugs. Examination of several serine! alanine substitution mutants of Bcl-2 suggested that serine 70 and serine 87 represent major sites of Bcl-2 phosphorylation induced in response to microtubuletargeting drugs. Both these serines are within sequence contexts suitable for proline-directed kinases such as Cdc2. Phosphorylated Bcl-2 protein was discovered to associate in M-phase-arrested cells with Pin1, a mitotic peptidyl prolyl isomerase (PPIase) known to interact with substrates of Cdc2 during mitosis. In contrast, phosphorylation of Bcl-2 induced by microtubuletargeting drugs did not alter its ability to associate with Bcl-2 (homodimerization), Bax, BAG1, or other Bcl-2-binding proteins. Since the region in Bcl-2 containing serine 70 and serine 87 represents a proline-rich loop that has been associated with autorepression of its antiapoptotic activity, the discovery of Pin1 interactions with phosphorylated Bcl-2 raises the possibility that Pin1 alters the conformation of Bcl-2 and thereby modulates its function in cells arrested with antimicrotubule drugs.
Bcl-2; mitosis; phosphorylation; Cdc2; Pin1