Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the paper will be accessible via the Research Library.
Quantitative proteomics: A new approach to an old pathway
The mitogen epidermal growth factor (EGF) induces auto-phosphorylation of the EGF receptor (EGFR) followed by tyrosine phosphorylation of a plethora of downstream targets. Phospho-proteomic approaches provide an unbiased view of early signaling events; however they are currently unable to provide a dynamic view of signaling cascades. Blagoev et al. now report the development of a new quantitative mass spectrometry method that converts temporal changes to differences in peptide isotopic abundance, and use this to characterize the temporal activation of the EGFR phosphotyrosine proteome.
The authors previously developed a method, termed stable isotope labeling by amino acids in cell culture, or SILAC, which uniformly labels cellular proteins with two distinct arginine isotopes. Using mass spectrometry, this label provides a handle to quantify the relative abundance of protein in distinct cell populations. In this report, the authors extend the method to allow the incorporation of a third isotopic label for arginine. They investigate EGF signaling kinetics by collecting cells at multiple time points after EGF stimulation and isolating the phosphotyrosine signaling components by immunoprecipitation.
An "activation profile" of 202 proteins is presented, of which 81 had at least a 1.5-fold change in activation during the time course of the experiment. Fifty previously known effectors of early EGF signaling events were validated in this screen. The set of activated proteins included those that were directly phosphorylated by the EGFR, substrates of downstream kinases and proteins that interact with tyrosine-phosphorylated targets. The EGFR was shown to be substantially activated within one minute of EGF stimulation. A number of other proteins were activated with similar kinetics to EGFR, including the E3 ubiquitin ligase c-Cbl, which plays an early role in receptor internalization. As expected, downstream components of the signaling pathway, such as adaptors recruited to the receptor and downstream kinases, were sequentially activated. Nine Arp2/3 members — a complex involved in actin microfilament branching — displayed essentially identical activation profiles consistent with cytoskeletal remodeling in response to growth factors.
In addition to known members of the EGFR signaling pathway, six proteins with no known function were identified. For instance, Swiprosin 1 was found to have similar dynamics to the Arp2/3 proteins, suggesting an involvement in EGF-induced actin rearrangement. Other identified proteins had known functions unrelated to EGF signaling, such as CYLD, a tumor suppressor that regulates apoptosis.
This quantitative and temporal global picture of tyrosine phosphorylation-dependent changes in EGF signaling is an important step forward in efforts to model signaling pathways. This approach can now be applied to a broader set of signaling pathways to characterize their activation by endogenous ligands as well as pharmacological agents.
Joanne Kotz, Assistant Editor Nature Chemical Biology
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Blagoy Blagoev, Shao-En Ong, Irina Kratchmarova & Matthias Mann Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics Nature Biotechnology, 22, 1139 – 1145 (2004); doi:10.1038/nbt1005
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Sam A Johnson & Tony Hunter Phosphoproteomics finds its timing Nature Biotechnology, 22, 1093 – 1094 (2004); doi:10.1038/nbt0904-1093
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Voltage-gated calcium channels (Cav channels) open upon membrane depolarization to allow entry of Ca2+ into excitable cells. The magnitude of the Ca2+ influx is controlled by growth factors that signal via effectors such as phosphatidylinositol-3-kinases (PI3Ks). However, the downstream pathways that lead to increased Ca2+ influxes are not well established. Viard et al. now report that a signaling cascade involving Akt/PKB induces translocation of functional Cav channels to the cell surface.
Expression of functional PI3Kγ and the Cavβ2 subunit in COS-7 cells caused an increase in calcium channel current densities. PI3Kγ acts through the phosphorylation of either mitogen-activated protein kinase (MAPK) or phosphoinositides (PIs) to generate PIP3. Coexpression of PI3Kγ and a GFP-tagged pleckstrin homology domain from a general PI receptor (PHGRP1-GFP), to scavenge PIP3, resulted in the elimination of PI3Kγ-induced calcium current. This suggests that PIP3 is a mediator of calcium current induction.
PI3Kγ expression induced concentration of Cav channels at the plasma membrane. Akt/PKB is known to be recruited to the plasma membrane through the interaction of its PH domain with PIP3. Co-transfection of a dominant-negative mutant of Akt/PKB with PI3Kγ prevented calcium current increase and decreased membrane localization of Cav channel subunits. In contrast, expression of myristoylated Akt/PKB, which is constitutively membrane-localized, promoted PI3Kγ-independent calcium current increase and Cav channel translocation. Based on these results, the authors conclude that PI3Kγ-induced Cav channel trafficking is mediated through Akt/PKB.
The authors identified a putative Akt/PKB phosphorylation site specific to the Cavβ2 subunit. Mutation of Ser 574 to Ala prevented PI3Kγ-induced phosphorylation and Cav channel trafficking. In contrast, the phosphomimic mutation S574E resulted in constitutive targeting of the Cav channel to the plasma membrane and increased calcium currents. These results indicate that S574 phosphorylation is sufficient to mediate the increase in functional Cav channels.
Rat dorsal root ganglia neurons were transfected with PHGRP1-GFP to test if the PI3Kγ pathway was active in neurons. Both Akt/PKB phosphorylation and plasma membrane localization of PHGRP1-GFP occurred upon exposure to insulin-like growth factor (IGF-1). Also, in neurons transfected with GFP-Cav2.2α1/β2a channels, IGF-1 exposure triggered membrane localization of the channel. Therefore, the authors conclude that the PI3K/Akt/PKB pathway is functional in neurons.
Ca2+ entry is known to protect neuronal cells from apoptosis by activation of Akt/PKB. Thus, the PI3Kγ/Akt/PKB pathway, by increasing calcium influx, could be functioning to promote cell survival in a positive feedback cycle. PI3K may also modulate the excitability of neurons through the regulation of Cav trafficking, with possible implications for epilepsy.
Joanne Kotz, Assistant Editor Nature Chemical Biology
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Patricia Viard, Adrian J Butcher, Guillaume Halet, Anthony Davies, Bernd Nürnberg, Fay Heblich & Annette C Dolphin PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane Nature Neuroscience, 7, 939 - 946 (2004); doi:10.1038/nn1300
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TGF-beta signaling: cPML holds the key
A hallmark of acute promyelocytic leukemia (APL) is a chromosomal translocation that results in the formation of a promyelocytic leukemia-retinoic acid receptor α (PML-RARα) fusion protein. PML functions as a tumor suppressor by enhancing transcription of the tumor suppressors p53 and Rb. PML-RARα interacts with nuclear PML (nPML), causing its delocalization from PML nuclear bodies and inhibiting its activity. Functions of cytoplasmic PML (cPML) have remained unclear. Lin et al. now report that cPML is a critical modulator of TGF-β signaling, explaining the unresponsiveness of APL to TGF-β-mediated tumor suppression.
Mouse embryonic fibroblasts (MEFs) from Pml-/- mice were significantly impaired in TGF-β1 induced gene expression and biological responses, including growth inhibition, induction of apoptosis, and induction of cellular senescence. However, TGF-β-mediated signaling could be fully rescued either by re-expression of cPML isofoms or cytoplasmically localized mutants of an nPML isoform. The cytokine TGF-β1 induced expression of cPML, resulting in a punctate cytoplasmic localization characteristic of the protein. Based on these results, the authors conclude that cPML plays a role in TGF-β signaling, and that TGF-β1 induces cPML expression.
In dissecting the TGF-β1 signaling pathway impaired in Pml-/- MEFs, Lin et al. found reductions in Smad2 and Smad3 phosphorylation. Consequently, phosphorylation-dependent Smad3 nuclear localization was impaired in knockout cells and could be restored by ectopic cPML expression. Agonist sensitive interaction with the TGF-beta effector proteins Smad2, Smad3 and SARA (Smad anchor for receptor activation) was demonstrated by co-immunoprecipitation with endogenous cPML. The association of SARA and Smad2/3 was absent in Pml-/- MEFs, but was restored by the expression of cPML. The cPML-dependent association of SARA and Smad2/3 did not require the Smad binding domain of SARA, suggesting that cPML acts as a bridge between the two. The authors conclude that in vivo cPML promotes the formation of a SARA/Smad2/3 complex.
APL blasts are known to be impaired in their response to TGF-β and TGF-β1-induced Smad2/3 phosphorylation, as well as cPML induction, was reduced in the APL cell line NB4. Retinoic acid induced degradation of PML-RARα restored Smad2/3 phosphorylation, cPML induction and the association of cPML with Smad3. These results suggest a molecular mechanism in which heterodimerization of PML-RARα and cPML inhibits the latter's function, resulting in a loss of responsiveness to TGF-β.
PML is frequently lost in tumors, including colon cancers, which have been observed to be refractile to TGF-β. The authors suggest that loss of cPML may play a role in such cancers. Future experiments will be directed toward understanding the role of cPML in other TGF-β-dependent events including wound healing.
Joanne Kotz, Assistant Editor Nature Chemical Biology
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Hui-Kuan Lin, Stephan Bergmann & Pier Paolo Pandolfi Cytoplasmic PML function in TGF-β signalling Nature, 431, 205 - 211 (09 September 2004); doi:10.1038/nature02783
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Christine Le Roy & Jeffrey L. Wrana Cell biology: An unexpected social servant Nature, 431, 142 (09 September 2004)
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Translation: Building with new blocks
Proteins are typically assembled from twenty naturally occurring amino acids, each of which is specified by one or more codons in the genetic code. It is known that two additional amino acids, selenocysteine and pyrrolysine, can be genetically coded for. In both cases, the unusual amino acid is specified by a canonical stop codon, UGA for selenocysteine and UAG for pyrrolysine. For selenocysteine, it has been shown that the appropriate tRNA is initially charged with serine, which is subsequently modified to selenocysteine. With the discovery of pyrrolysine in certain Archea and eubacteria, an analogous mechanism was hypothesized, in which unmodified lysine would be attached to the tRNA and later enzymatically modified to the pyrrolysyl tRNA. However, Blight et al. have now demonstrated that tRNA can be directly charged with pyrrolysine.
Incubation of uncharged tRNA with pyrrolysine, ATP, and the pyrrolysyl-tRNA synthetase (PylS), resulted in charging of tRNACUA. In contrast, no reaction occurred if pyrrolysine was replaced by lysine or a mixture of the twenty canonical amino acids. tRNA synthetase activity can be measured by monitoring isotopic incorporation of 32P-pyrophosphate into ATP during the aminoacylation reaction. PylS catalysis of this exchange was only observed with the addition of pyrrolysine, and not lysine or any other amino acid. Thus, the authors conclude that pyrrolysine is directly attached to its cognate tRNA in vitro.
Pyrrolysine does not occur in E. coli, making it a good system for testing the requirements for reconstituting pyrrolysine incorporation into proteins in vivo. Methylamine methyltransferase, a protein from methanogenic Archea, contains a UAG codon coding for pyrrolysine. When the protein is heterologously expression in E. coli, the UAG is read as a stop, and a truncated protein is produced. The authors were only able to reconstitute production of the full-length, pyrrolysine-containing protein when synthetic pyrrolysine was added and pylS and pylT, which encodes tRNACUA, were heterologously expressed. This supports an in vivo mechanism of direct aminoacylation of tRNA with pyrrolysine and represents the first case of tRNA charging with a non-canonical amino acid.
The reconstitution in E. coli of pyrrolysine incorporation into methylamine methyltransferse suggests that it will be possible to expand the genetic code to add this unusual amino acid site-specifically into proteins. The authors are now directing their efforts toward engineering a pathway for the biosynthesis of pyrrolysine in E. coli.
Joanne Kotz, Assistant Editor Nature Chemical Biology
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Sherry K. Blight, Ross C. Larue, Anirban Mahapatra, David G. Longstaff, Edward Chang, Gang Zhao, Patrick T. Kang, Kari B. Green-Church, Michael K. Chan & Joseph A. Krzycki Direct charging of tRNACUA with pyrrolysine in vitro and in vivo Nature, 431, 333 - 335 (16 September 2004); doi:10.1038/nature02895
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