193 nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1- through 3-) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge-reduced radical products generated by UV photoexcitation were also subjected to collision induced dissociation (termed, activated – electron photodetachment dissociation (a-EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH ~11.5), piperidine-modified mobile phase, LC-MS/UVPD was implemented and resulted in the successful analysis of mitogen-activated pathway kinases (MAPKs) using ultrafast activation times (5 nanoseconds).

Non‐visual arrestins‐2 and‐3 interact with numerous GPCRs and dozens of non‐receptors partners, such as MAP kinases. Arrestin‐3 promotes JNK3 activation, scaffolding ASK1‐MKK4‐JNK3 cascade. Full activation of JNKs, unlike other MAP kinases, requires two upstream kinases, MKK4 and MKK7, and each preferentially phosphorylating a distinct site, tyrosine (MKK4) and theronine (MKK7). It remains unclear whether arrestin‐3 can promote the activation of JNK3 by MKK7. Using phospho‐Tyr and phospho‐Thr antibodies we found that arrestin‐3 promotes the phosphorylation of both Tyr and Thr within the T‐P‐Y motif in COS‐7 cells. We also found that arrestin‐3 directly binds MKK7 comparably to MKK4. The binding of JNK3 enhanced the association of arrestin‐3 with MKK4, while reduced the binding of MKK7. With pure proteins we show that arrestin‐3 scaffolds MKK7‐JNK3 module at higher concentration than MKK4‐JNK3 module. Arresint‐3 also facilitates JNK3 activation in COS‐7 cells over‐expressing either MKK4 or MKK7. This is the first evidence that arrestin‐3 promotes JNK3 phosphorylation by MKK7. The data suggest that JNK3 binding has opposite effect on arrestin‐3 interactions with MKK4 and MKK7. Support: NIH GM081756, GM077561 and EY011500 (VVG) and GM059802 and Welch Foundation Grant F‐1390 (KND).

Eukaryotic elongation factor 2 kinase (eEF-2K), through its phosphorylation of elongation factor 2 (eEF2), provides a mechanism by which cells can control the rate of the elongation phase of protein synthesis. The activity of eEF-2K is increased in rapidly proliferating malignant cells, is inhibited during mitosis, and may contribute to the promotion of autophagy in response to anti-cancer therapies. The purpose of this study was to examine the therapeutic potential of targeting eEF-2K in breast cancer tumors. Through the systemic administration of liposomal eEF-2K siRNA (twice a week, i.v. 150 µg/kg), the expression of eEF-2K was down-regulated in vivo in an orthotopic xenograft mouse model of a highly aggressive triple negative MDA-MB-231 tumor. This targeting resulted in a substantial decrease in eEF2 phosphorylation in the tumors, and led to the inhibition of tumor growth, the induction of apoptosis and the sensitization of tumors to the chemotherapy agent doxorubicin. eEF-2K down-modulation in vitro resulted in a decrease in the expression of c-Myc and cyclin D1 with a concomitant increase in the expression of p27Kip1. A decrease in the basal activity of c-Src (phospho-Tyr-416), focal adhesion kinase (phospho-Tyr-397), and Akt (phospho-Ser-473) was also detected following eEF-2K down-regulation in MDA-MB-231 cells, as determined by Western blotting. Where tested, similar results were seen in ER-positive MCF-7 cells. These effects were also accompanied by a decrease in the observed invasive phenotype of the MDA-MB-231 cells. These data support the notion that the disruption of eEF-2K expression in breast cancer cells results in the down-regulation of signaling pathways affecting growth, survival and resistance and has potential as a therapeutic approach for the treatment of breast cancer.

Arrestins are a small family of proteins with four mammalian members that play key roles in the regulation of multiple GPCR-dependent and -independent signaling pathways. Although arrestins were reported to serve as scaffolds for MAP kinase cascades, promoting the activation of JNK3, ERK1/2, and p38, the molecular mechanisms involved were not elucidated and even the direct binding of arrestins with MAP kinases were never demonstrated. Here using purified proteins we show that both non-visual arrestins directly bind JNK3α2 and its upstream activator MKK4, and that the affinity of arrestin-3 for these kinases is higher than that of arrestin-2. Reconstitution of the MKK4-JNK3α2 signaling module from pure proteins in the presence of different arrestin-3 concentrations showed that arrestin-3 acts as a “true” scaffold, facilitating JNK3α2 phosphorylation by bringing the two kinases together. Both JNK3α2 phosphorylation by MKK4 and JNK3α2 activity towards its substrate ATF2 increase at low and then decrease at high arrestin-3 levels, yielding bell-shaped concentration dependence expected with true scaffolds that do not activate the upstream kinase or its substrate. Thus, direct binding of both kinases and true scaffolding is the molecular mechanism of arrestin-3 action on the MKK4-JNK3α2 signaling module.

The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS 1 and MS 2 data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples, respectively, with 49 and 50 unique proteins identified in contrast to the more conventional MS/MS methods. The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public. The accuracy and specificity of the MassMatrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap. Analysis of a known mixture of three mitogen-activated kinases yielded similar sequence coverage percentages for UVPD of peptide anions versus conventional collisioninduced dissociation of peptide cations, and when these methods were combined into a single search, an increase of up to 13% sequence coverage was observed for the kinases. The ability to sequence peptide anions and cations in alternating scans in the same chromatographic run was also demonstrated. Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species. In this case, tryptic peptides from the cytosolic section of HeLa cells were analyzed by polarity switching nanoLC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing. Relative to searching using ETD alone, positive/negative polarity switching significantly improved sequence coverages across identified proteins, resulting in a 33% increase in unique peptide identifications and more than twice the number of peptide spectral matches. Molecular & Cellular Proteomics 12: 10.1074/mcp.O113.028258, 2604-2614, 2013.The advent of new high-performance tandem mas...