SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

A Scalable Platform for Producing Recombinant Nucleosomes with Codified Histone Methyltransferase Substrate Preferences.

Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1; also known as NSD2) is a SET domain-containing histone lysine methyltransferase. A chromosomal translocation occurs in 15-20% of multiple myeloma patients and is associated with increased production of WHSC1 and poor clinical prognosis. To define the substrate requirements of NSD2, we established a platform for the large-scale production of recombinant polynucleosomes, based on authentic human histone proteins, expressed in E. coli, and complexed with linearized DNA. A brief survey of methyltransferases whose substrate requirements are recorded in the literature yielded expected results, lending credence to the fitness of our approach. This platform was readily 'codified' with respect to both position and extent of methylation at histone 3 lysines 18 and 36 and led to the conclusion that the most readily discernible activity of NSD2 in contact with a nucleosome substrate is dimethylation of histone 3 lysine 36. We further explored reaction mechanism, and conclude a processive, rather than distributive mechanism best describes the interaction of NSD2 with intact nucleosome substrates. The methods developed feature scale and flexibility and are suited to thorough pharmaceutical-scale drug discovery campaigns.

Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36.

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-(14)C and (36)S and secondary Me-(3)H3, Me-(2)H3, 5'-(14)C, and 5'-(3)H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

An optimized platform for hydrophilic interaction chromatography-immobilized metal affinity chromatography enables deep coverage of the rat liver phosphoproteome.

While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe(3+) immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectivity, and increase phosphopeptide identifications. Here we present refinements to our overall protocol that make it simpler and more efficient, while they provide greater coverage of the phosphoproteome. We introduce filter-aided sample prep (FASP) for cell lysis and trypsin digestion. Following HILIC separation, fractions are IMAC enriched using a 96-well filter plate. Finally, enriched samples are analyzed using an LC-MS strategy optimized for the fractionation scheme. The optimized protocol improves protein recovery, simplifies phosphopeptide enrichment, and optimizes instrument time, while it maintains deep coverage of the phosphoproteome. By using the refined protocol, we identified more than 16,000 unique phosphosites from rat liver in a single experiment, which used approximately 1 day of instrument time. All together, we present evidence for 24,485 rat liver phosphosites that represents the deepest coverage of a tissue phosphoproteome to date.

Mapping posttranslational modifications of proteins by MS-based selective detection: application to phosphoproteomics.

This chapter outlines general principals that apply to the analysis of posttranslational modifications of proteins, with an emphasis on phosphoproteins. Mass spectrometry (MS)-based approaches for selective detection and site-specific analysis of posttranslationally modified peptides are described, and an MS-based method that relies on production and detection of fragment ions specific for the modification(s) of interest and that was developed in the authors' laboratory is described in detail. The method is applicable to selective detection of N- and O-linked carbohydrates in glycoproteins, O-linked sulfate, and N- and O-linked lipids. Detailed procedures for application of this strategy to phosphorylation-site mapping are presented here.

Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex.

BACKGROUND: In S. cerevisiae, the mitotic exit network (MEN) proteins, including the Polo-like protein kinase Cdc5 and the protein phosphatase Cdc14, are required for exit from mitosis. In pre-anaphase cells, Cdc14 is sequestered to the nucleolus by Net1 as a part of the RENT complex. When cells are primed to exit mitosis, the RENT complex is disassembled and Cdc14 is released from the nucleolus. RESULTS: Here, we show that Cdc5 is necessary to free nucleolar Cdc14 in late mitosis, that elevated Cdc5 activity provokes ectopic release of Cdc14 in pre-anaphase cells, and that the phosphorylation state of Net1 is regulated by Cdc5 during anaphase. Furthermore, recombinant Cdc5 and Xenopus Polo-like kinase can disassemble the RENT complex in vitro by phosphorylating Net1 and thereby reducing its affinity for Cdc14. Surprisingly, although RENT complexes containing Net1 mutants (Net1(7m) and Net1(19m') lacking sites phosphorylated by Cdc5 in vitro are refractory to disassembly by Polo-like kinases in vitro, net1(7m) and net1(19m') cells grow normally and exhibit only minor defects in releasing Cdc14 during anaphase. However, net1(19m') cells exhibit a synergistic growth defect when combined with mutations in CDC5 or DBF2 (another MEN gene). CONCLUSIONS: We propose that although Cdc5 potentially disassembles RENT by directly phosphorylating Net1, Cdc5 mediates exit from mitosis primarily by phosphorylating other targets. Our study suggests that Cdc5/Polo is unusually promiscuous and highlights the need to validate Cdc5/Polo in vitro phosphorylation sites by direct in vivo mapping experiments.

Comparative phosphorylation site mapping from gel-derived proteins using a multidimensional ES/MS-based approach.

Understanding how phosphorylation regulates the behavior of individual proteins is critical to understanding signaling pathways. These studies usually involve knowledge of which amino acid residues are phosphorylated on a given protein and the extent of such a modification. This is often a rather difficult task in that most phosphoproteins contain multiple substoichiometric sites of phosphorylation. Here we describe the multidimensional electrospray (ES) mass spectrometry (MS)-based phosphopeptide-mapping strategy developed in our laboratory. In the first dimension of the process, phosphopeptides present in a protein digest are selectively detected and collected into fractions during on-line liquid chromatography (LC)/ES/MS, which monitors for phosphopeptide-specific marker ions. This analysis generates a phosphorylation profile that can be used to assess changes in the phosphorylation state of a protein pointing to those phosphopeptides that require further investigation. The phosphopeptide-containing fractions are then analyzed in the second dimension by nano-ES with precursor-ion scan for the marker ion m/z 79. As the final step, direct sequencing of the phosphopeptides is performed by LC/ES/MS/MS. Merits and limitations of the strategy, as well as experimental details and suggestions, are described here.

A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival.

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

Dbf2-Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis.

Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2-Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.

A quantitative results-driven approach to analyzing multisite protein phosphorylation: the phosphate-dependent phosphorylation profile of the transcription factor Pho4.

Multisite protein phosphorylation appears to be quite common. Nevertheless our understanding of how multiple phosphorylation events regulate the function of a protein is limited in many cases. The ability to measure temporal changes in the site-specific phosphorylation profile of a protein in response to a given stimulus or cellular activity would provide an immediate indication of the functional significance of any phosphorylation site to a given process. Here we describe a mass spectrometry-based method to identify functionally relevant phosphorylation sites on a protein. It combines stable isotope labeling with a highly selective mass spectrometry analysis to detect and quantitate phosphorylation sites in response to a cellular signal. This approach requires no a priori knowledge of the phosphorylation state of the protein, does not require purification of phosphopeptides, and reliably detects substoichiometric levels of phosphorylation. Following a review of the quantitative results, only those phosphorylation sites that show a change in relative abundance are selected for identification and further study. We used this results-driven approach to study phosphorylation of the budding yeast transcription factor Pho4 in response to phosphate starvation. Phosphorylation of Pho4 on five cyclin-dependent kinase (Cdk) consensus sites has been shown to regulate the transcriptional activity of Pho4 in response to changes in environmental phosphate levels. Here we show that in phosphate-rich medium Pho4 is phosphorylated on at least 15 distinct sites including the five Cdk sites described previously. In excellent agreement with the known mechanism for regulation of Pho4 we found that phosphorylation at all five of the Cdk sites was repressed in phosphate-depleted medium. In addition to these five sites, we identified four novel phosphorylation sites that were also responsive to changes in phosphate availability. Selecting a limited number of Pho4 phosphorylation sites, we performed a more detailed kinetic analysis using an isotope-free strategy. We used LC-MS with selected reaction monitoring to greatly improve the accuracy, sensitivity, and dynamic range of the subsequent experiments. A detailed analysis of the cell-based phosphorylation at the selected Pho4 sites confirmed an apparent site preference for the Pho80-Pho85 cyclin-cyclin-dependent kinase complex.

Identification of tyrosine residues on ELMO1 that are phosphorylated by the Src-family kinase Hck.

The SH3 and SH2 domains of hematopoietic cell kinase (Hck) play important roles in substrate targeting. To identify new components of Hck signaling pathways, we identified proteins that bind to the SH3 domain of Hck (Scott et al. (2002) J. Biol. Chem. 277, 28238). One such protein was ELMO1, the mammalian orthologue of the Caenorhabditis elegans gene, ced-12. ELMO1 is an approximately 80-kD protein containing a PH domain and a C-terminal Pro-rich sequence. In C. elegans, ced-12 is required for the engulfment of dying cells and for cell migration. In mammalian fibroblasts, ELMO1 binds to Dock180, and functions upstream of Rac during phagocytosis and cell migration. We previously showed that ELMO1 binds directly to the Hck SH3 domain and is phosphorylated by Hck. In this study, we used mass spectrometry to identify the following sites of ELMO1 phosphorylation: Tyr 18, Tyr 216, Tyr 511, Tyr 395, and Tyr 720. Mutant forms of ELMO1 lacking these sites were defective in their ability to promote phagocytosis and migration in fibroblasts. Single tyrosine mutations showed that Tyr 511 is particularly important in mediating these biological effects. These mutants displayed comparable binding to Dock180 and Crk as wild-type ELMO1, but gave a lowered activation of Rac. The data suggest that Src family kinase mediated tyrosine phosphorylation of ELMO1 might represent an important regulatory mechanism that controls signaling through the ELMO1/Crk/Dock180 pathway.

Improved sensitivity for phosphopeptide mapping using capillary column HPLC and microionspray mass spectrometry: comparative phosphorylation site mapping from gel-derived proteins.

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.

Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast.

Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1.Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies.

Mistranslational errors associated with the rare arginine codon CGG in Escherichia coli.

In Escherichia coli, CGG is a rare arginine codon occurring at a frequency of 0.54% in all E. coli mRNAs or 9.8% when an arginine residue is encoded for. When present in high numbers or in clusters in highly expressed recombinant mRNA, rare codons can cause expression problems compromising product yield and translational fidelity. The coding region for an N-terminally polyhistidine tagged p27 protease domain from Herpes Simplex Virus 2 (HSV-2) contains 11 of these rare arginine codons, with 3 occurring in tandem near the C-terminus of the protein. When expressed in E. coli, the majority of the recombinant material produced had an apparent molecular mass of 31 kDa by SDS-PAGE gels or 3 kDa higher than predicted. Detailed biochemical analysis was performed on chemical and enzymatic digests of the protein and peptide fragments were characterized by Edman and MS/MS sequencing approaches. Two major species were isolated comprising +1 frameshift events at both the second and third CGG codons in the triplet cluster. Translation proceeded in the missense frame to the next termination codon. In addition, significant levels of glutamine misincorporating for arginine were discovered, suggesting second base misreading of CGG as CAG. Coexpression of the argX gene, which encodes the cognate tRNA for CGG codons, largely eliminated both the frameshift and misincorporation events, and increased expression levels of authentic product by up to 7-fold. We conclude that supplementation of the rare arginyl tRNA(CGG) levels by coexpression of the argX gene can largely alleviate the CGG codon bias present in E. coli, allowing for efficient and accurate translation of heterologous gene products.