Characterization of Phosphopeptide Positional Isomers on the Transcriptional Co-activator TAZ.

The transcriptional co-activator with the PDZ binding motif (TAZ) is a critical regulator of numerous cellular processes such as cell differentiation, development, proliferation, and cell growth. Aberrant expression and activity of TAZ are also featured in many human malignancies. A hallmark of TAZ biology is its cytoplasmic retention mediated by 14-3-3 isoforms in response to phosphorylation of Ser89 by members of the LATS family of kinases. Following the observation that TAZ is a highly phosphorylated protein even when Ser89 is mutated, high-resolution mass spectrometry employing data-independent acquisition and ion mobility separation was conducted to elucidate additional TAZ phosphorylation sites that may play a role in regulating this critical transcriptional rheostat. Numerous phosphorylation sites on TAZ were identified, including several novel modifications. Of notable interest was the identification of positional phosphoisomers on a phosphopeptide containing Ser89. Optimized use of a so-called wideband enhancement acquisition technique yielded higher-quality fragmentation data that confirmed the detection of Ser93 as the positional phosphoisomer partner of Ser89 and identified diagnostic fragment ions for the phosphorylation events. Functional analysis indicated that Ser93 phosphorylation reduces the level of 14-3-3 association and increases the level of nuclear translocation, indicating this phosphorylation event attenuates the 14-3-3-mediated TAZ cytoplasmic retention mechanism. These findings suggest that the biological activities of TAZ are likely dynamically regulated by multisite phosphorylation.

The Atypical Dual Specificity Phosphatase hYVH1 Associates with Multiple Ribonucleoprotein Particles.

Human YVH1 (hYVH1), also known as dual specificity phosphatase 12 (DUSP12), is a poorly characterized atypical dual specificity phosphatase widely conserved throughout evolution. Recent findings have demonstrated that hYVH1 expression affects cellular DNA content and is a novel cell survival phosphatase preventing both thermal and oxidative stress-induced cell death, whereas studies in yeast have established YVH1 as a novel 60S ribosome biogenesis factor. In this study, we have isolated novel hYVH1-associating proteins from human U2OS osteosarcoma cells using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Numerous ribosomal proteins were identified, confirming the work done in yeast. Furthermore, proteins known to be present on additional RNP particles were identified, including Y box-binding protein 1 (YB-1) and fragile X mental retardation protein, proteins that function in translational repression and stress granule regulation. Follow-up studies demonstrated that hYVH1 co-localizes with YB-1 and fragile X mental retardation protein on stress granules in response to arsenic treatment. Interestingly, hYVH1-positive stress granules were significantly smaller, whereas knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, indicating a possible role for hYVH1 in stress granule disassembly. These results propagate a role for dual specificity phosphatases at RNP particles and suggest that hYVH1 may affect a variety of fundamental cellular processes by regulating messenger ribonucleoprotein (mRNP) dynamics.

Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics.

Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Previously, we have demonstrated that RME-8 associates with early endosomes in a phosphatidylinositol 3-phosphate (PI(3)P)-dependent fashion. In this study, we have now identified amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys(17), Trp(20), Tyr(24), or Arg(26) are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates early endosomal clathrin dynamics and alters the steady state localization of the cation-independent mannose 6-phosphate receptor. Interestingly, RME-8 endosomal association is also regulated by the PI(3)P-binding protein SNX1, a member of the retromer complex. Wild type SNX1 restores endosomal localization of RME-8 W20A, whereas a SNX1 variant deficient in PI(3)P binding disrupts endosomal localization of wild type RME-8. These results further highlight the critical role for PI(3)P in the RME-8-mediated organizational control of various endosomal activities, including retrograde transport.

The dual-specificity phosphatase hYVH1 (DUSP12) is a novel modulator of cellular DNA content.

The dual-specificity phosphatase hYVH1 (DUSP12) is an evolutionary conserved phosphatase that also contains a unique zinc-binding domain. Recent evidence suggests that this enzyme plays a role in cell survival and ribosome biogenesis. Here, we report that hYVH1 expression also affects cell cycle progression. Overexpression of hYVH1 caused a significant increase in polyploidy and in the G 2/M cell population, with a subsequent decrease in the G 0/G 1 population. Phosphatase activity is dispensable, while the zinc-binding domain is necessary and sufficient for hYVH1-mediated cell cycle changes. In agreement with this, siRNA-mediated silencing of hYVH1 expression resulted in a dramatic increase in the G 0/G 1 population and susceptibility to cellular senescence. Additionally, mass spectrometry-based methods identified novel hYVH1 phosphorylation sites, including a C-terminal modification at position Ser ( 335) in the zinc-binding domain. Interestingly, phosphorylation at Ser335 regulates subcellular targeting of hYVH1 and augments the hYVH1 G 2/M phenotype. Collectively we demonstrate that hYVH1 is a novel modulator of cell cycle progression; a function mainly mediated by its C-terminal zinc-binding domain.

Gold nanoparticle enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins.

We present a simple method by which gold nanoparticles (AuNPs) are used to simultaneously isolate and enrich for free or modified thiol-containing peptides, thus facilitating the identification of protein S-modification sites. Here, protein disulfide isomerase (PDI) and dual specificity phosphatase 12 (DUSP12 or hYVH1) were S-nitrosylated or S-glutathionylated, their free thiols differentially alkylated, and subjected to proteolysis. AuNPs were added to the digests, and the AuNP-bound peptides were isolated by centrifugation and released by thiol exchange. These AuNP-bound peptides were analyzed by MALDI-TOF mass spectrometry revealing that AuNPs result in a significant enrichment of free thiol-containing as well as S-nitrosylated, S-glutathionylated, and S-alkylated peptides, leading to the unequivocal assignment of thiols susceptible to modification.

Redox regulation of the human dual specificity phosphatase YVH1 through disulfide bond formation.

YVH1 was one of the first eukaryotic dual specificity phosphatases cloned, and orthologues poses a unique C-terminal zinc-coordinating domain in addition to a cysteine-based phosphatase domain. Our recent results revealed that human YVH1 (hYVH1) protects cells from oxidative stress. This function requires phosphatase activity and the zinc binding domain. This current study provides evidence that the thiol-rich zinc-coordinating domain may act as a redox sensor to impede the active site cysteine from inactivating oxidation. Furthermore, using differential thiol labeling and mass spectrometry, it was determined that hYVH1 forms intramolecular disulfide bonds at the catalytic cleft as well as within the zinc binding domain to avoid irreversible inactivation during severe oxidative stress. Importantly, zinc ejection is readily reversible and required for hYVH1 activity upon returning to favorable conditions. This inimitable mechanism provides a means for hYVH1 to remain functionally responsive for protecting cells during oxidative stimuli.

Characterization of a group 1 late embryogenesis abundant protein in encysted embryos of the brine shrimp Artemia franciscana.

Late embryogenesis abundant (LEA) proteins are hydrophilic molecules that are believed to function in desiccation and low-temperature tolerance in some plants and plant propagules, certain prokaryotes, and several animal species. The brine shrimp Artemia franciscana can produce encysted embryos (cysts) that enter diapause and are resistant to severe desiccation. This ability is based on biochemical adaptations, one of which appears to be the accumulation of the LEA protein that is the focus of this study. The studies described herein characterize a 21 kDa protein in encysted Artemia embryos as a group 1 LEA protein. The amino acid sequence of this protein and its gene have been determined and entered into the NCBI database (no. EF656614). The LEA protein consists of 182 amino acids and it is extremely hydrophilic, with glycine (23%), glutamine (17%), and glutamic acid (12.6%) being the most abundant amino acids. This protein also consists of 8 tandem repeats of a 20 amino acid sequence, which is characteristic of group 1 LEA proteins from non-animal species. The LEA protein and its gene are expressed only in encysted embryos and not in larvae or adults. Evidence is presented to show that the LEA protein functions in the prevention of drying-induced protein aggregation, which supports its functional role in desiccation tolerance. This report describes, for the first time, the purification and characterization of a group 1 LEA protein from an animal species.

Identification of redox sensitive thiols of protein disulfide isomerase using isotope coded affinity technology and mass spectrometry.

Regulation of the redox state of protein disulfide isomerase (PDI) is critical for its various catalytic functions. Here we describe a procedure utilizing isotope-coded affinity tag (ICAT) technology and mass spectrometry that quantitates relative changes in the dynamic thiol and disulfide states of human PDI. Human PDI contains six cysteine residues, four present in two active sites within the a and a' domains, and two present in the b' domain. ICAT labeling of human PDI indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an approximately 80% decrease in available thiols within the a domain was detected. Surprisingly, the redox state of one of the two cysteines, Cys-295, within the b' domain was altered between the fully reduced and the auto-oxidized state of PDI while the other b' domain cysteine remained fully reduced. An interesting mono- and dioxidation modification of an invariable tryptophan residue, Trp-35, within the active site was also mapped by tandem mass spectrometry. Our findings indicate that ICAT methodology in conjunction with mass spectrometry represents a powerful tool to monitor changes in the redox state of individual cysteine residues within PDI under various conditions.

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry.

Yersinia encodes a protein kinase, YpkA, which disrupts the actin cytoskeleton. Using an approach termed chemical genetics, we identified a 36-kDa substrate for YpkA in both J774 lysates and bovine brain cytosol. Mass spectrometry analysis identified this substrate as FLJ20113, an open reading frame that corresponds to otubain 1, a deubiquitinating enzyme implicated in immune cell clonal anergy. We demonstrate that otubain 1 is phosphorylated by YpkA in vitro and interacts with YpkA and actin in vivo. Identification of otubain 1 as a YpkA substrate suggests that regulation of immune cell anergy may be a survival mechanism for Yersinia.

A direct, continuous, sensitive assay for protein disulphide-isomerase based on fluorescence self-quenching.

PDI (protein disulphide-isomerase) activity is generally monitored by insulin turbidity assay or scrambled RNase assay, both of which are performed by UV-visible spectroscopy. In this paper, we present a sensitive fluorimetric assay for continuous determination of disulphide reduction activity of PDI. This assay utilizes the pseudo-substrate diabz-GSSG [where diabz stands for di-(o-aminobenzoyl)], which is formed by the reaction of isatoic anhydride with the two free N-terminal amino groups of GSSG. The proximity of two benzoyl groups leads to quenching of the diabz-GSSG fluorescence by approx. 50% in comparison with its non-disulphide-linked form, abz-GSH (where abz stands for o-aminobenzoyl). Therefore the PDI-dependent disulphide reduction can be monitored by the increase in fluorescence accompanying the loss of proximity-quenching upon conversion of diabz-GSSG into abz-GSH. The apparent K(m) of PDI for diabz-GSSG was estimated to be approx. 15 muM. Unlike the insulin turbidity assay and scrambled RNase assay, the diabz-GSSG-based assay was shown to be effective in determining a single turnover of enzyme in the absence of reducing agents with no appreciable blank rates. The assay is simple to perform and very sensitive, with an estimated detection limit of approx. 2.5 nM PDI, enabling its use for the determination of platelet surface PDI activity in crude sample preparations.

The yersinia virulence factor YopM forms a novel protein complex with two cellular kinases.

Pathogenic Yersinia contain a virulence plasmid that encodes genes for intracellular effectors, which neutralize the host immune response. One effector, YopM, is necessary for Yersinia virulence, but its function in host cells is unknown. To identify potential cellular pathways affected by YopM, proteins that co-immunoprecipitate with YopM in mammalian cells were isolated and identified by mass spectrometry. Results demonstrate that two kinases, protein kinase C-like 2 (PRK2) and ribosomal S6 protein kinase 1 (RSK1), interact directly with YopM. These two kinases associate only when YopM is present, and expression of YopM in cells stimulates the activity of both kinases. RSK1 is activated directly by interaction with YopM, and RSK1 kinase activity is required for YopM-stimulated PRK2 activity. YopM activation of RSK1 occurs independently of the actions of YopJ on the MAPK pathway. YopM is also required for Yersinia-induced changes in RSK1 mobility in infected macrophage cells. These results identify the first intracellular targets of YopM and suggest YopM acts to stimulate the activity of PRK2 and RSK1.

Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases.

The Gram-negative bacterial pathogen Yersinia delivers six effector proteins into the host cells to thwart the host innate immune response. One of the effectors, YopT, causes the disruption of the actin cytoskeleton and contributes to the inhibition of phagocytosis of the pathogen. YopT functions as a cysteine protease to cleave Rho family GTPases. We have analyzed the YopT cleavage products of Rho GTPases by TLC and determined their chemical structure by MS. Amino acid labeling experiments were performed to locate the exact site in RhoA where the YopT cleavage occurs. Our data unambiguously demonstrate that YopT cleaves N-terminal to the prenylated cysteine in RhoA, Rac, and Cdc42 and that the cleavage product of the GTPases is geranylgeranyl cysteine methyl ester. YopT cleaves GTP- and GDP-bound forms of RhoA equally, suggesting that the cleavage does not depend upon the conformation status of the GTPases. YopT also cleaves both farnesylated and geranylgeranylated forms of RhoA. The polybasic sequence in the C terminus of RhoA is essential for YopT substrate recognition and cleavage.