Phosphorylation of TSC2 by PKC-δ reveals a novel signaling pathway that couples protein synthesis to mTORC1 activity.

Downstream of insulin-like growth factor receptor, the TSC1/2/ TCB1D7 (tuberous sclerosis complex) and mTOR (mechanistic target of rapamycin) pathways are implicated in many human diseases, including cancer and diabetes. Targeting this pathway is currently an important approach for palliating or eradicating cancer. Downstream of mTOR, translational machinery targeting holds great promise for anticancer drug development. Therefore, we investigated whether the protein synthesis machinery that is regulated by mTORC1 (mTOR complex 1) signaling can in turn regulate mTORC1 activity. We found that inhibition of protein synthesis results in rapid activation of mTORC1 signaling, thereby uncovering a feedback loop between mTOR and the translation machinery. This mTORC1 activation requires tuberous sclerosis complex (TSC) but is independent of AKT. In addition, by using a PKC-δ (protein kinase c delta)-specific inhibitor and PKC-δ siRNA knockdown, we found that PKC-δ kinase activity is required for mTORC1 activation in response to translation inhibitors. Furthermore, translation inhibition activates PKC-δ. Subsequently, we investigated whether PKC-δ can phosphorylate and inactivate TSC1/2, leading to mTORC1 activation. In vitro kinase assays showed direct phosphorylation of TSC2 (S932 and S939) by PKC-δ, which was confirmed by mass spectrometry. In vivo kinase analysis further indicated that both S932 and S939 are phosphorylated in response to translation inhibitors. Finally, phosphorylation defective TSC2 mutants (S932A and S939A single mutants and a S932A/S939A double mutant) failed to upregulate mTORC1 activity in the presence of translation inhibitors, suggesting that activation of mTORC1 by translation inhibitors is mediated by PKC-δ phosphorylation of TSC2 at S932/S939, which inactivates TSC.

A distinct H2A.X isoform is enriched in Xenopus laevis eggs and early embryos and is phosphorylated in the absence of a checkpoint.

Histone H2A.X is an H2A variant present in multicellular organisms that is specifically phosphorylated on the serine in the C-terminal consensus sequence, canonically "SQEY," in response to DNA damage. We have recently shown the significance of phosphorylation of the penultimate tyrosine for maintenance and processing of the DNA damage response in mammalian cells. Here, we report the identification of distinct H2A.X variants in the eggs and early embryos of the frog Xenopus laevis that contain a C-terminal SQEF, among other changes; we have denoted these proteins as "H2A.X-F." H2A.X-F is present only in late-staged oocytes, eggs, and premidblastula transition embryos and is not present in somatic cells. Similar unannotated isoforms were identified in other rapidly developing aquatic species, such as Xenopus tropicalis, goldfish, and zebrafish, and in Arabidopsis and chickpea. Furthermore, we demonstrate by mass spectrometry and phospho-specific antibodies that H2A.X-F is phosphorylated in the absence of exogenous DNA damage, in both actively dividing, unperturbed embryos and cell-free egg extract in the absence and presence of DNA damage and S-phase checkpoint conditions. We propose that this isoform may be involved in modulating the cellular response to the rapid early cell cycles in externally developing species.

Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery.

TDP-43 is a highly conserved and ubiquitously expressed member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins. Recently, TDP-43 was shown to be a major disease protein in the ubiquitinated inclusions characteristic of most cases of amyotrophic lateral sclerosis (ALS), tau-negative frontotemporal lobar degeneration (FTLD), and inclusion body myopathy. In these diseases, TDP-43 is redistributed from its predominantly nuclear location to ubiquitin-positive, cytoplasmic foci. The extent to which TDP-43 drives pathophysiology is unknown, but the identification of mutations in TDP-43 in familial forms of ALS and FTLD-U suggests an important role for this protein in pathogenesis. Little is known about TDP-43 function and only a few TDP-43 interacting proteins have been previously identified, which makes further insight into both the normal and pathological functions of TDP-43 difficult. Here we show, via a global proteomic approach, that TDP-43 has extensive interaction with proteins that regulate RNA metabolism. Some interactions with TDP-43 were found to be dependent on RNA-binding, whereas other interactions are RNA-independent. Disease-causing mutations in TDP-43 (A315T and M337V) do not alter its interaction profile. TDP-43 interacting proteins largely cluster into two distinct interaction networks, a nuclear/splicing cluster and a cytoplasmic/translation cluster, strongly suggesting that TDP-43 has multiple roles in RNA metabolism and functions in both the nucleus and the cytoplasm. Finally, we found numerous TDP-43 interactors that are known components of stress granules, and indeed, we find that TDP-43 is also recruited to stress granules.

Identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (MAK)-related kinase.

MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position -3 (P-3) being more stringent than for prolines at P-2 and P+1. Using the consensus, we identified a putative phosphorylation site (RPLT(1080)S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.

Application of SIMSTEX to oligomerization of insulin analogs and mutants.

The propensity of various insulins and their analogs to oligomerize was investigated by mass spectrometric methods including measurement of the relative abundances of oligomers in the gas phase and the kinetics of H/D amide exchange. The kinetics of deuterium uptake show a good fit when the exchanging amides are placed in three kinetic groups: fast, intermediate, and slow. r-Human insulin, of the insulins investigated, has fewer amides that exchange at intermediate rates and more that exchange at slow rates, in accord with its higher extent of association in solution. We adapted PLIMSTEX (protein ligand interactions by mass spectrometry, titration, and H/D exchange) to determine protein/ligand affinities in solution, to determine self-association equilibrium constants for proteins, and to apply them to various insulin analogs. We term this adaptation SIMSTEX (self-association interactions using mass spectrometry, self-titration and H/D exchange); it gives affinity constants that compare well with the literature results. The results from SIMSTEX show that some mutants (e.g., GlnB13) have an increased tendency to self-associate, possibly slowing down their action in vivo. Other mutants (e.g., lispro and AspB9) have lower propensities for self-association, thus providing potentially faster-acting analogs for use in controlling diabetes.

Determination of affinity constants and response factors of the noncovalent dimer of gramicidin by electrospray ionization mass spectrometry and mathematical modeling.

The dimerization of gramicidin, a 15-residue membrane peptide, in solution can be viewed as a model for protein-protein interactions. We reported previously that the dimer can be observed when electrosprayed from organic solvents and that the abundances of the dimer depends on the dielectric constant of the solvent. Here, we report an effort to determine an affinity constant for the dimerization of gramicidin by using gas-phase abundance. Two issues affecting the determination are the electrospray-induced dissociation of the dimer and discrimination in the electrospray of the dimer compared with the monomer. Other methods developed for the purpose of determining affinity from mass spectral abundance do not address the dissociation of the complex in the gas phase or can not be applied for cases of low affinity constant, K(a). We present a mathematical model that uses the ratio of the signal intensities of the dimer and the monomer during a titration. The model also incorporates the dissociation and an electrospray ionization-response factor of the dimer for extracting the affinity constant for the dimerization of gramicidin. The dimerization constants from the new method agree within a factor of two with values reported in the literature.

The gramicidin dimer shows both EX1 and EX2 mechanisms of H/D exchange.

We describe the use of H/D amide exchange and electrospray ionization mass spectrometry to study, in organic solvents, the pentadecapeptide gramicidin as a model for protein self association. In methanol-OD, all active H's in the peptide exchange for D within 5 min, indicating a monomer/dimer equilibrium that is shifted towards the fast-exchanging monomer. H/D exchange in n-propanol-OD, however, showed a partially protected gramicidin that slowly converts to a second species that exchanges nearly all the active hydrogens, indicating EX1 kinetics for the H/D exchange. We propose that this behavior is the result of the slower rate of unfolding in n-propanol compared with that in methanol. The rate constant for the unfolding of the dimer is the rate of disappearance of the partially protected species, and it agrees within a factor of two with a value reported in literature. The rate constant of dimer refolding can be determined from the ratio of the rate constant for unfolding and the affinity constant for the dimer, which we determined in an earlier study. The unfolding activation energy is 20 kcal mol(-1), determined by performing the exchange experiments as a function of temperature. To study gramicidin in an even more hydrophobic medium than n-propanol, we measured its H/D exchange kinetics in a phospholipids vesicle and found a different H/D amide exchange behavior. Gramicidin is an unusual peptide dimer that can exhibit both EX1 and EX2 mechanisms for its H/D exchange, depending on the solvent.

Analysis of histones in Xenopus laevis. II. mass spectrometry reveals an index of cell type-specific modifications on H3 and H4.

Epigenetic information is hypothesized to be encoded in histone variants and post-translational modifications. Varied cell- and locus-specific combinations of these epigenetic marks are likely contributors to regulation of chromatin-templated transactions, including transcription, replication, recombination, and repair. Therefore, the relative abundance of histone modifications in a given cell type is a potential index of cell fate and specificity. Here, we utilize mass spectrometry techniques to characterize the relative abundance index of cell type-specific modifications on histones H3 and H4 in distinct cell types from the frog Xenopus laevis, including the sperm, the stored predeposition histones in the egg, the early embryo equivalent pronuclei, cultured somatic cells, and erythrocytes. We used collisionally associated dissociation to identify the modifications present on histone H3 in a variety of cell types, resolving 26 distinctly modified H3 peptides. We employed the electron transfer dissociation fragmentation technique in a "middle-down" approach on the H4 N-terminal tail to explore the overlap of post-translational modifications. We observed 66 discrete isoforms of the H4 1-23 fragment in four different cell types. Isolation of the stored, predeposition histone H4 from the frog egg also revealed a more varied pattern of modifications than the previously known diacetylation on Lys(5) and Lys(12). The developmental transitions of modifications on H3 and H4 were strikingly varied, implying a strong correlation of the histone code with cell type and fate. Our results are consistent with a histone code index for each cell type and uncover potential cross-talk between modifications on a single tail.

Analysis of histones in Xenopus laevis. I. A distinct index of enriched variants and modifications exists in each cell type and is remodeled during developmental transitions.

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.

Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila.

Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.

Electrospray ionization-mass spectrometry and tandem mass spectrometry reveal self-association and metal-ion binding of hydrophobic peptides: a study of the gramicidin dimer.

Gramicidin is a membrane pentadecapeptide that acts as a channel, allowing the passage of monovalent metal ions and assisting in bacterial cell death. The active form is a noncovalently bound dimer. One means to study the self-assembly of this peptide has been to compare the state of the peptide in various solvents ranging from hydrophilic (e.g., trifluoroethanol) to hydrophobic (e.g., n-propanol). In this article, we report the use of electrospray mass spectrometry to study the self-association of gramicidin in various organic and mixed solvents that are introduced directly into the mass spectrometer. The dimer (both homo and hetero) can survive the introduction into the gas phase, and the amount in the gas phase increases with the decreasing dielectric constant of the solvent, reflecting solution-phase behavior. Tandem mass spectrometry data reveal that the stability of dimer in the gas phase decreases with increasing metal ion size, strongly suggesting that the metal ion binds inside the dimer between the monomers.