pY Sites Research Articles

The fitness cost of spurious phosphorylation.

The fidelity of signal transduction requires the binding of regulatory molecules to their cognate targets. However, the crowded cell interior risks off-target interactions between proteins that are functionally unrelated. How such off-target interactions impact fitness is not generally known. Here, we use Saccharomyces cerevisiae to inducibly express tyrosine kinases. Because yeast lacks bona fide tyrosine kinases, the resulting tyrosine phosphorylation is biologically spurious. We engineered 44 yeast strains each expressing a tyrosine kinase, and quantitatively analysed their phosphoproteomes. This analysis resulted in ~30,000 phosphosites mapping to ~3500 proteins. The number of spurious pY sites generated correlates strongly with decreased growth, and we predict over 1000 pY events to be deleterious. However, we also find that many of the spurious pY sites have a negligible effect on fitness, possibly because of their low stoichiometry. This result is consistent with our evolutionary analyses demonstrating a lack of phosphotyrosine counter-selection in species with tyrosine kinases. Our results suggest that, alongside the risk for toxicity, the cell can tolerate a large degree of non-functional crosstalk as interaction networks evolve.

Deep phosphotyrosine characterisation of primary murine T cells using broad spectrum optimisation of selective triggering.

Sequencing the tyrosine phosphoproteome using MS-based proteomics is challenging due to the low abundance of tyrosine phosphorylation in cells, a challenge compounded in scarce samples like primary cells or clinical samples. The broad-spectrum optimisation of selective triggering (BOOST) method was recently developed to increase phosphotyrosine sequencing in low protein input samples by leveraging tandem mass tags (TMT), phosphotyrosine enrichment, and a phosphotyrosine-loaded carrier channel. Here, we demonstrate the viability of BOOST in T cell receptor (TCR)-stimulated primary murine T cells by benchmarking the accuracy and precision of the BOOST method and discerning significant alterations in the phosphoproteome associated with receptor stimulation. Using 1mg of protein input (about 20 million cells) and BOOST, we identify and precisely quantify more than 2000 unique pY sites compared to about 300 unique pY sites in non-BOOST control samples. We show that although replicate variation increases when using the BOOST method, BOOST does not jeopardise quantitative precision or the ability to determine statistical significance for peptides measured in triplicate. Many pY previously uncharacterised sites on important T cell signalling proteins are quantified using BOOST, and we identify new TCR responsive pY sites observable only with BOOST. Finally, we determine that the phase-spectrum deconvolution method on Orbitrap instruments can impair pY quantitation in BOOST experiments.

Dissecting phospho-motif-dependent Shc1 interactome using long synthetic protein fragments.

Activated receptor tyrosine kinases (RTKs) rely on the assembly of signaling proteins into high-dimensional protein complexes for signal transduction. Shc1, a prototypical scaffold protein, plays a pivotal role in directing phosphotyrosine (pY)-dependent protein complex formation for numerous RTKs typically through its two pY-binding domains. The three conserved pY sites within its CH1 region (Shc1CH1) hold particular significance due to their substantial contribution to its functions. However, how Shc1 differentially utilizes these sites to precisely coordinate protein complex assembly remains unclear. Here, we employed multiple peptide ligation techniques to synthesize an array of long protein fragments (107 amino acids) covering a significant portion of the Shc1CH1 region with varying phosphorylation states at residues Y239, 240, 313, and S335. By combining these phospho-Shc1CH1 fragments with integrated proteomics sample preparation and quantitative proteomic analysis, we were able to comprehensively resolve the site-specific interactomes of Shc1 with single amino acid resolution. By applying this approach to different cancer cell lines, we demonstrated that these phospho-Shc1CH1 fragments can be effectively used as a diagnostic tool to assess cell type-specific RTK signaling networks. Collectively, these biochemical conclusions help to better understand the sophisticated organization of pY-dependent Shc1 adaptor protein complexes and their functional roles in cancer.

BRK confers tamoxifen-resistance in breast cancer via regulation of tyrosine phosphorylation of CDK1

Tamoxifen (Tam) has been the first-line therapy for estrogen receptor-positive breast cancer since its FDA-approval in 1998. Tam-resistance, however, presents a challenge and the mechanisms that drive it have yet to be fully elucidated. The non-receptor tyrosine kinase BRK/PTK6 is a promising candidate as previous research has shown that BRK knockdown resensitizes Tam-resistant breast cancer cells to the drug. However, the specific mechanisms that drive its importance to resistance remain to be investigated. Here, we investigate the role and mechanism of action of BRK in Tam-resistant (TamR), ER+, and T47D breast cancer cells using phosphopeptide enrichment and high throughput phopshoproteomics analysis. We conducted BRK-specific shRNA knockdown in TamR T47D cells and compared phosphopeptides identified in these cells with their Tam-resistant counterpart and parental, Tam-sensitive cells (Par). A total of 6492 STY phosphosites were identified. Of these sites, 3739 high-confidence pST sites and 118 high-confidence pY sites were analyzed for significant changes in phosphorylation levels to identify pathways that were differentially regulated in TamR versus Par and to investigate changes in these pathways when BRK is knocked down in TamR. We observed and validated increased CDK1 phosphorylation at Y15 in TamR cells compared to BRK-depleted TamR cells. Our data suggest that BRK is a potential Y15-directed CDK1 regulatory kinase in Tam-resistant breast cancer.

Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY can handle up to 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic, and tyrosine-specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.

Accurately predicting microbial phosphorylation sites using evolutionary and structural features

Post-translational modification (PTM) is a biological process involving a protein’s enzymatic changes after its translation by the ribosome. Phosphorylation is one of the most critical PTMs that occurs when a phosphate group interacts with an amino acid residue along protein sequence. It contributes to cell communication, DNA repair, and gene regulation. Predicting microbial phosphorylation sites can provide better understanding of host-pathogen interaction and the development of anti-microbial agents. Experimental methods such as mass spectrometry are time-consuming, laborious, and expensive. This paper proposes a new approach, called RotPhoPred, for predicting phospho-serine (pS), phospho-threonine (pT), and phospho-tyrosine (pY) sites in the microbial organism by integrating evolutionary bigram profile with structural information and using Rotation Forest as the classification technique. To the best of our knowledge, our extracted features and employed classifier have never been utilized for this task. Comparative results demonstrate that the RotPhoPred surpasses its peers in terms of different metrics such as sensitivity (90.0%, 75.4% and 78.2%), specificity (92.1%, 97.2% and 94.7%), accuracy (91.0%, 86.3%, 86.4%), and MCC (0.82, 0.74 and 0.74) for pS, pT, and pY sites predictions, respectively. RotPhoPred as a standalone predictor and all its source codes are publicly available at: https://github.com/faisalahm3d/RotPredPho.

Aberrant function of pathogenic STAT3 mutant proteins is linked to altered stability of monomers and homodimers

AbstractSTAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide–binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

Motif-dependent immune co-receptor interactome profiling by photoaffinity chemical proteomics

Identification of the tyrosine phosphorylation (pY)-dependent interactome of immune co-receptors is crucial for understanding signal pathways involved in immunotherapy. However, identifying the motif-specific interactome for each pY commonly found on these multi-phosphorylated membrane proteins remains challenging. Here, we describe a photoaffinity-based chemical proteomic approach to dissect the motif-specific cytoplasmic interactomes of the critical immune co-receptor CD28. Various full-length CD28 cytoplasmic tails (CD28cyto) with defined pY and selectively replaced photo-methionine were synthesized and applied to explore three pY-motif-dependent CD28cyto interactomes. We identified a stand-alone interaction of phospholipase PLCG1 with the Y191 motif with enhanced affinity for the sequence neighboring the transmembrane domain. Importantly, taking advantage of native top-down mass spectrometry with a 193-nm laser, we discovered the direct association of a previously undefined pY218 motif with the kinase PKCθ through its C2 domain. This synthetic CD28cyto-based photoaffinity proteomic approach is generically applicable to the study of other immune co-receptors with multiple pY sites on their linear cytoplasmic tail.

Phospho-proteomic discovery of novel signal transducers including thioredoxin-interacting protein as mediators of erythropoietin-dependent human erythropoiesis

Erythroid cell formation critically depends on signals transduced via erythropoietin (EPO)/EPO receptor (EPOR)/JAK2 complexes. This includes not only core response modules (e.g., JAK2/STAT5, RAS/MEK/ERK), but also specialized effectors (e.g., erythroferrone, ASCT2 glutamine transport, Spi2A). By using phospho-proteomics and a human erythroblastic cell model, we identify 121 new EPO target proteins, together with their EPO-modulated domains and phosphosites. Gene ontology (GO) enrichment for "Molecular Function" identified adaptor proteins as one top EPO target category. This includes a novel EPOR/JAK2-coupled network of actin assemblage modifiers, with adaptors DLG-1, DLG-3, WAS, WASL, and CD2AP as prime components. "Cellular Component" GO analysis further identified 19 new EPO-modulated cytoskeletal targets including the erythroid cytoskeletal targets spectrin A, spectrin B, adducin 2, and glycophorin C. In each, EPO-induced phosphorylation occurred at pY sites and subdomains, which suggests coordinated regulation by EPO of the erythroid cytoskeleton. GO analysis of "Biological Processes" further revealed metabolic regulators as a likewise unexpected EPO target set. Targets included aldolase A, pyruvate dehydrogenase α1, and thioredoxin-interacting protein (TXNIP), with EPO-modulated p-Y sites in each occurring within functional subdomains. In TXNIP, EPO-induced phosphorylation occurred at novel p-T349 and p-S358 sites, and was paralleled by rapid increases in TXNIP levels. In UT7epo-E and primary human stem cell (HSC)-derived erythroid progenitor cells, lentivirus-mediated short hairpin RNA knockdown studies revealed novel pro-erythropoietic roles for TXNIP. Specifically, TXNIP's knockdown sharply inhibited c-KIT expression; compromised EPO dose-dependent erythroblast proliferation and survival; and delayed late-stage erythroblast formation. Overall, new insight is provided into EPO's diverse action mechanisms and TXNIP's contributions to EPO-dependent human erythropoiesis.

The Effects of Pertuzumab and Its Combination with Trastuzumab on HER2 Homodimerization and Phosphorylation.

Pertuzumab (Perjeta) is an anti-HER2 monoclonal antibody that is used for treatment of HER2-positive breast cancers in combination with trastuzumab (Herceptin) and docetaxel and showed promising clinical outcomes. Pertuzumab is suggested to block heterodimerization of HER2 with EGFR and HER3 that abolishes canonical function of HER2. However, evidence on the exact mode of action of pertuzumab in homodimerization of HER2 are limited. In this study, we investigated the effect of pertuzumab and its combination with trastuzumab on HER2 homodimerization, phosphorylation and whole gene expression profile in Chinese hamster ovary (CHO) cells stably overexpressing human HER2 (CHO-K6). CHO-K6 cells were treated with pertuzumab, trastuzumab, and their combination, and then HER2 homodimerization and phosphorylation at seven pY sites were investigated. The effects of the monoclonal antibodies on whole gene expression and the expression of cell cycle stages, apoptosis, autophagy, and necrosis were studied by cDNA microarray. Results showed that pertuzumab had no significant effect on HER2 homodimerization, however, trastuzumab increased HER2 homodimerization. Interestingly, pertuzumab increased HER2 phosphorylation at Y1127, Y1139, and Y1196 residues, while trastuzumab increased HER2 phosphorylation at Y1196. More surprisingly, combination of pertuzumab and trastuzumab blocked the phosphorylation of Y1005 and Y1127 of HER2. Our results also showed that pertuzumab, but not trastuzumab, abrogated the effect of HER2 overexpression on cell cycle in particular G1/S transition, G2/M transition, and M phase, whereas trastuzumab abolished the inhibitory effect of HER2 on apoptosis. Our findings confirm that pertuzumab is unable to inhibit HER2 homodimerization but induces HER2 phosphorylation at some pY sites that abolishes HER2 effects on cell cycle progress. These data suggest that the clinical effects of pertuzumab may mostly through the inhibition of HER2 heterodimers, rather than HER2 homodimers and that pertuzumab binding to HER2 may inhibit non-canonical HER2 activation and function in non-HER-mediated and dimerization-independent pathway(s).

Combinatorial Peptide Ligand Library-Based Photoaffinity Probe for the Identification of Phosphotyrosine-Binding Domain Proteins.

Phosphotyrosine (pY) serves as a docking site for the recognition proteins containing pY-binding (pYB) modules, such as the SH2 domain, to mediate cell signal transduction. Thus, it is vital to profile these binding proteins for understanding of signal regulation. However, identification of pYB proteins remains a significant challenge due to their low abundance and typically weak and transient interactions with pY sites. Herein, we designed and prepared a pY-peptide photoaffinity probe for the robust and specific enrichment and identification of its binding proteins. Using SHC1-pY317 as a paradigm, we showed that the developed probe enables to capture target protein with high selectivity and remarkable specificity even in a complex context. Notably, we expanded the strategy to a combinatorial pY-peptide-based photoaffinity probe by using combinatorial peptide ligand library (CPLL) technique and identified 24 SH2 domain proteins, which presents a deeper profiling of pYB proteins than previous reports using affinity probes. Moreover, the method can be used to mine putative pYB proteins and confirmed PKN2 as a selective binder to pY, expanding the repertoire of known domain proteins. Our approach provides a general strategy for rapid and robust interrogating pYB proteins and will promote the understanding of the signal transduction mechanism.

Conformational Clusters of Phosphorylated Tyrosine.

Tyrosine phosphorylation plays an important role in many cellular and intercellular processes including signal transduction, subcellular localization, and regulation of enzymatic activity. In 1999, Blom et al., using the limited number of protein data bank (PDB) structures available at that time, reported that the side chain structures of phosphorylated tyrosine (pY) are partitioned into two conserved conformational clusters ( Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999 , 294 , 1351 - 1362 ). We have used the spectral clustering algorithm to cluster the increasingly growing number of protein structures with pY sites, and have found that the pY residues cluster into three distinct side chain conformations. Two of these pY conformational clusters associate strongly with a narrow range of tyrosine backbone conformation. The novel cluster also highly correlates with the identity of the n + 1 residue, and is strongly associated with a sequential pYpY conformation which places two adjacent pY side chains in a specific relative orientation. Further analysis shows that the three pY clusters are associated with distinct distributions of cognate protein kinases.

A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription.

We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.

Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate.

Systematic assessment of tyrosine kinase-substrate relationships is fundamental to a better understanding of cellular signaling and its profound alterations in human diseases such as cancer. In human cells, such assessments are confounded by complex signaling networks, feedback loops, conditional activity, and intra-kinase redundancy. Here we address this challenge by exploiting the yeast proteome as an in vivo model substrate. We individually expressed 16 human non-receptor tyrosine kinases (NRTKs) in Saccharomyces cerevisiae and identified 3,279 kinase-substrate relationships involving 1,351 yeast phosphotyrosine (pY) sites. Based on the yeast data without prior information, we generated a set of linear kinase motifs and assigned ∼1,300 known human pY sites to specific NRTKs. Furthermore, experimentally defined pY sites for each individual kinase were shown to cluster within the yeast interactome network irrespective of linear motif information. We therefore applied a network inference approach to predict kinase-substrate relationships for more than 3,500 human proteins, providing a resource to advance our understanding of kinase biology.

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity.

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long-term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multisite phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity, and reduced GEF activity.

Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.

SH2 Binding Site Protection Assay: A Method for Identification of SH2 Domain Interaction Partners by Exploiting SH2 Mediated Phosphosite Protection.

Over the last two decades there has been a significant effort in the field to characterize the phosphosite binding specificities of SH2 domains with the goal of deciphering the pY signaling code. Although high throughput studies in various formats using most SH2 domains have collectively provided a rich resource of in vitro SH2-pTyr site specificity maps, this data can only be used approximate what is happening in the cell where protein concentrations and localization are not homogenous, as they are for in vitro experiments. Here we describe an in vivo approach, SH2 site protection assay, which can capture the pTyr binding specificity of SH2 domains in the cell. The basis of this approach is SH2-pY site protection, the ability of SH2 domains to prevent the PTP-dependent dephosphorylation of their pY site binding partners. We overexpress a tracer SH2 domain in cells and quantify the change in abundance of tyrosine phosphorylated sites using MS. Since the method is performed in vivo, it has the advantage of identifying SH2-pY interactions as they occur within in the cell.

Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters.

Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets.

Homo‐ and Heterodinuclear Rh and Ir Complexes Supported by SNn Mixed‐Donor Ligands (n = 2–4): Stereochemistry and Coordination‐Site‐Exchange Reactions of Cp*M (M = Rh, Ir) Units

A series of SNn mixed‐donor ligands [n = 2: H2NC2H4SCH2‐2‐pyridyl (2‐NSpy) (1a), H2NC2H4SCH2‐4‐pyridyl (4‐NSpy) (1b), n = 3: 2‐pyridylCH2NHC2H4SCH2‐2‐pyridyl (2‐pyNSpy) (2), n = 4: (2‐pyridylCH2)2NC2H4SCH2‐2‐pyridyl (2‐py2NSpy) (3)] was utilized to support homo‐ and heterodinuclear complexes including Cp*MIII units (M = Rh, Ir; Cp* = pentamethylcyclopentadienyl). Reactions of [Cp*MCl2]2 with 2‐pyNSpy (2), 2‐py2NSpy (3), and 4‐NSpy (1b) afforded homodinulear complexes, [(Cp*MCl)(2‐pyNSpy)(Cp*MCl)](PF6)2 [M = Rh (5a), Ir (5b)], [(Cp*M)(2‐py2NSpy)(Cp*MCl)](PF6)3 [M = Rh (6a), Ir (6b)], [(Cp*MCl)(4‐NSpy)(Cp*MCl2)]Cl [M = Rh (8a), Ir (8b)]. Heterodinuclear complexes [(Cp*MCl)(4‐NSpy)(Cp*M′Cl2)]Cl [M, M′ = Rh, Ir (8c), Ir, Rh (8d)] were prepared using mononuclear complexes [(Cp*MCl)(4‐NSpy)]Cl [M = Rh (7a), Ir (7b)] reacted with [Cp*MCl2]2 (M = Ir, Rh), respectively. Complexes 5–8 were characterized by X‐ray crystallography to determine the configurations around the M, M′, S, and N centers. The solid‐state structures of 6 are retained in acetonitrile solution whereas four diastereomers are generated in the case of 5 due to low stereoselectivity around the coordinated amine nitrogen atom, in contrast to the sulfur atom. Heterodinuclear complexes 8c,d are unstable in solution at 55 °C, readily affording mixtures of 8a–d via intra‐ and intermolecular coordination‐site‐exchange reactions of Cp*M fragments between the SN moiety and the py site. In order to evaluate the selectivity of Cp*M fragments for the SN and py coordination sites, several competitive reactions of [Cp*MCl2]2 (M = Rh, Ir) with H2NC2H4SCH2C6H5 (NSph) (4) and/or 4‐methylpyridine (4‐Mepy) were carried out to demonstrate predominant formation of iridium complexes 9b and 10b among [(Cp*MCl)(NSph)]Cl [M = Rh (9a), Ir (9b)] and [(Cp*MCl)(4‐Mepy)]Cl [M = Rh (10a), Ir (10b)]. These reactions indicated higher affinity of the Cp*Ir fragment to both the NS and py sites relative to the rhodium analogue.

Abstract 4119: Tyrosine phosphorylation of MACC1 is essential and druggable for colorectal cancer metastasis restriction

Abstract We identified the previously undescribed gene Metastasis Associated in Colon Cancer 1 (MACC1). MACC1 is a metastasis inducer and prognostic biomarker for colorectal cancer (CRC). MACC1 has meanwhile been confirmed as decisive driver for tumorigenesis and metastasis for a broad variety of solid cancers. Here we aim to identify the impact of defined tyrosine phosphorylations on the function of the metastasis inducer MACC1. In silico identified MACC1 tyrosine phosphorylation (pY) sites were ranked and most promising positions were site-directed mutated in wildtype (wt) MACC1 constructs. Human CRC cells were transduced with these constructs. Tyrosine phosphorylation was quantified by selected reaction monitoring (SRM)-mass spectrometry. Effects of pY-MACC1 mutants on cell migration, proliferation, dissemination and colony formation were assessed in cell culture as well as on liver metastasis formation in human CRC-xenografted mice. To identify MACC1-phosphorylating kinases the MACC1 interactome was identified using mass spectrometry. Most promising binding partners were validated by immunoprecipitation. Impact of inhibitors targeting kinases phosphorylating MACC1 was assessed on MACC1-induced tumor growth and liver metastasis in human CRC-xenografted mice by in vivo imaging. We report that tyrosine phosphorylation of MACC1 is hierarchical and essential for motility and proliferation in cell culture, as well as for tumor growth and metastasis in mice. We identified kinases for MACC1 phosphorylation. Targeting these kinases using inhibitors employed in clinical trials restricts MACC1-induced tumor growth and metastasis in mice. MACC1 tyrosine phosphorylation is crucial for metastasis and is druggable by small molecule inhibitors. This fundamental finding opens new therapeutic options for inhibition of MACC1-induced cancer metastasis. Citation Format: Dennis Kobelt, Gunnar Dittmar, Claudia Fleuter, Janice Smith, Mathias Dahlmann, Rebekka Migotti, Peter M. Schlag, Ulrike S. Stein. Tyrosine phosphorylation of MACC1 is essential and druggable for colorectal cancer metastasis restriction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4119. doi:10.1158/1538-7445.AM2015-4119