Development and Characterization of Selective FAK Inhibitors and PROTACs with In Vivo Activity.

Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.

A multi-purpose, regenerable, proteome-scale, human phosphoserine resource for phosphoproteomics.

Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation. We demonstrate possible uses of iSPI, including use as a phosphopeptide standard, a tool to evaluate and optimize phosphorylation-site localization algorithms, and a benchmark to compare performance across data analysis pipelines. We also present AScorePro, an updated version of the AScore algorithm specifically optimized for phosphorylation-site localization in higher energy fragmentation spectra, and the FLR viewer, a web tool for phosphorylation-site localization, to enable community use of the iSPI resource. iSPI and its associated data constitute a useful, multi-purpose resource for the phosphoproteomics community.

Mutant and Wild-Type Isocitrate Dehydrogenase 1 Share Enhancing Mechanisms Involving Distinct Tyrosine Kinase Cascades in Cancer.

Isocitrate dehydrogenase 1 (IDH1) is important for reductive carboxylation in cancer cells, and the IDH1 R132H mutation plays a pathogenic role in cancers including acute myeloid leukemia (AML). However, the regulatory mechanisms modulating mutant and/or wild-type (WT) IDH1 function remain unknown. Here, we show that two groups of tyrosine kinases (TK) enhance the activation of mutant and WT IDH1 through preferential Y42 or Y391 phosphorylation. Mechanistically, Y42 phosphorylation occurs in IDH1 monomers, which promotes dimer formation with enhanced substrate (isocitrate or α-ketoglutarate) binding, whereas Y42-phosphorylated dimers show attenuated disruption to monomers. Y391 phosphorylation occurs in both monomeric and dimeric IDH1, which enhances cofactor (NADP+ or NADPH) binding. Diverse oncogenic TKs phosphorylate IDH1 WT at Y42 and activate Src to phosphorylate IDH1 at Y391, which contributes to reductive carboxylation and tumor growth, whereas FLT3 or the FLT3-ITD mutation activates JAK2 to enhance mutant IDH1 activity through phosphorylation of Y391 and Y42, respectively, in AML cells. SIGNIFICANCE: We demonstrated an intrinsic connection between oncogenic TKs and activation of WT and mutant IDH1, which involves distinct TK cascades in related cancers. In particular, these results provide an additional rationale supporting the combination of FLT3 and mutant IDH1 inhibitors as a promising clinical treatment of mutant IDH1-positive AML.See related commentary by Horton and Huntly, p. 699.This article is highlighted in the In This Issue feature, p. 681.

The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3.

The sorting of signaling receptors into and out of cilia relies on the BBSome, a complex of Bardet-Biedl syndrome (BBS) proteins, and on the intraflagellar transport (IFT) machinery. GTP loading onto the Arf-like GTPase ARL6/BBS3 drives assembly of a membrane-apposed BBSome coat that promotes cargo entry into cilia, yet how and where ARL6 is activated remains elusive. Here, we show that the Rab-like GTPase IFT27/RABL4, a known component of IFT complex B, promotes the exit of BBSome and associated cargoes from cilia. Unbiased proteomics and biochemical reconstitution assays show that, upon disengagement from the rest of IFT-B, IFT27 directly interacts with the nucleotide-free form of ARL6. Furthermore, IFT27 prevents aggregation of nucleotide-free ARL6 in solution. Thus, we propose that IFT27 separates from IFT-B inside cilia to promote ARL6 activation, BBSome coat assembly, and subsequent ciliary exit, mirroring the process by which BBSome mediates cargo entry into cilia.

Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding and promotes the Warburg effect.

The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

Regulation of microtubule-based transport by MAP4.

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2-dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2-based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2-dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.

Tyr-94 phosphorylation inhibits pyruvate dehydrogenase phosphatase 1 and promotes tumor growth.

Many cancer cells rely more on aerobic glycolysis (the Warburg effect) than mitochondrial oxidative phosphorylation and catabolize glucose at a high rate. Such a metabolic switch is suggested to be due in part to functional attenuation of mitochondria in cancer cells. However, how oncogenic signals attenuate mitochondrial function and promote the switch to glycolysis remains unclear. We previously reported that tyrosine phosphorylation activates and inhibits mitochondrial pyruvate dehydrogenase kinase (PDK) and phosphatase (PDP), respectively, leading to enhanced inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) and consequently inhibition of pyruvate dehydrogenase complex (PDC) in cancer cells. In particular, Tyr-381 phosphorylation of PDP1 dissociates deacetylase SIRT3 and recruits acetyltransferase ACAT1 to PDC, resulting in increased inhibitory lysine acetylation of PDHA1 and PDP1. Here we report that phosphorylation at another tyrosine residue, Tyr-94, inhibits PDP1 by reducing the binding ability of PDP1 to lipoic acid, which is covalently attached to the L2 domain of dihydrolipoyl acetyltransferase (E2) to recruit PDP1 to PDC. We found that multiple oncogenic tyrosine kinases directly phosphorylated PDP1 at Tyr-94, and Tyr-94 phosphorylation of PDP1 was common in diverse human cancer cells and primary leukemia cells from patients. Moreover, expression of a phosphorylation-deficient PDP1 Y94F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at different tyrosine residues inhibits PDP1 through independent mechanisms, which act in concert to regulate PDC activity and promote the Warburg effect.

Tyr phosphorylation of PDP1 toggles recruitment between ACAT1 and SIRT3 to regulate the pyruvate dehydrogenase complex.

Mitochondrial pyruvate dehydrogenase complex (PDC) is crucial for glucose homeostasis in mammalian cells. The current understanding of PDC regulation involves inhibitory serine phosphorylation of pyruvate dehydrogenase (PDH) by PDH kinase (PDK), whereas dephosphorylation of PDH by PDH phosphatase (PDP) activates PDC. Here, we report that lysine acetylation of PDHA1 and PDP1 is common in epidermal growth factor (EGF)-stimulated cells and diverse human cancer cells. K321 acetylation inhibits PDHA1 by recruiting PDK1, and K202 acetylation inhibits PDP1 by dissociating its substrate PDHA1, both of which are important in promoting glycolysis in cancer cells and consequent tumor growth. Moreover, we identified mitochondrial ACAT1 and SIRT3 as the upstream acetyltransferase and deacetylase, respectively, of PDHA1 and PDP1, while knockdown of ACAT1 attenuates tumor growth. Furthermore, Y381 phosphorylation of PDP1 dissociates SIRT3 and recruits ACAT1 to PDC. Together, hierarchical, distinct posttranslational modifications act in concert to control molecular composition of PDC and contribute to the Warburg effect.

Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.

Alternative RISC assembly: binding and repression of microRNA-mRNA duplexes by human Ago proteins.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate protein output from the majority of human mRNAs. In contrast to the consensus view that all miRNAs are associated with Argonaute (Ago) proteins, we determine that miRNAs are expressed in a 13-fold excess relative to Agos in HeLa cells and that miRNAs are bound to mRNAs in a sevenfold excess relative to Agos, implying the existence of miRNA-mRNA duplexes not stoichiometrically bound by Agos. We show that all four human Agos can repress miRNA-mRNA duplexes, but only Ago2 can cleave small interfering RNA-mRNA duplexes in vitro. We visualize direct Ago binding to miRNA-mRNA duplexes in live cells using fluorescence lifetime imaging microscopy. In contrast to the consensus view that Agos bind miRNA duplexes, these data demonstrate that Agos can bind and repress miRNA-mRNA duplexes and support a model of catalytic Ago function in translational repression.

Activated inositol 1,4,5-trisphosphate receptors are modified by homogeneous Lys-48- and Lys-63-linked ubiquitin chains, but only Lys-48-linked chains are required for degradation.

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP(3) and Ca(2+)-gated Ca(2+) channels. Endogenous IP(3)Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP(3)R1 in αT3-1 pituitary gonadotrophs and found that IP(3)R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D. A., Kubota, K., Kirkpatrick, D. S., Alzayady, K. J., Gygi, S. P., and Wojcikiewicz, R. J. H. (2008) J. Biol. Chem. 283, 35319-35328). Here, we have extended these studies to determine whether IP(3)R2 and IP(3)R3 are similarly modified and if ubiquitination is cell type-dependent. Using mass spectrometry and linkage-specific ubiquitin antibodies, we found that all IP(3)R types are subject to ubiquitination at approximately the same locations and that, independent of cell type, IP(3)Rs are modified by monoubiquitin and Lys-48- and Lys-63-linked ubiquitin chains, although in differing proportions. Remarkably, the attached Lys-48- and Lys-63-linked ubiquitin chains are homogeneous and are segregated to separate IP(3)R subunits, and Lys-48-linked ubiquitin chains, but not Lys-63-linked chains, are required for IP(3)R degradation. Together, these data provide unique insight into the complexities of ubiquitination of an endogenous ubiquitin-proteasome pathway substrate in unperturbed mammalian cells. Importantly, although Lys-48-linked ubiquitin chains appear to trigger proteasomal degradation, the presence of Lys-63-linked ubiquitin chains suggests that ubiquitination of IP(3)Rs may have physiological consequences beyond signaling for degradation.

The conserved Bardet-Biedl syndrome proteins assemble a coat that traffics membrane proteins to cilia.

The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII, and clathrin coats. Here, we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. The BBSome is the major effector of the Arf-like GTPase Arl6/BBS3, and the BBSome and GTP-bound Arl6 colocalize at ciliary punctae in an interdependent manner. Strikingly, Arl6(GTP)-mediated recruitment of the BBSome to synthetic liposomes produces distinct patches of polymerized coat apposed onto the lipid bilayer. Finally, the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome in order to mediate targeting of membrane proteins to cilia. Thus, we propose that trafficking of BBSome cargoes to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals by the BBSome.

Gas-phase rearrangements do not affect site localization reliability in phosphoproteomics data sets.

Intramolecular transfer of phosphate during collision-induced dissociation (CID) in ion-trap mass spectrometers has recently been described. Because phosphorylation events are assigned to discrete serine, threonine, and tyrosine residues based on the presence of site-determining ions in MS/MS spectra, phosphate transfer may invalidate or confound site localization in published large-scale phosphorylation data sets. Here, we present evidence for the occurrence of this phenomenon using synthetic phosphopeptide libraries, specifically for doubly charged species. We found, however, that the extent of the transfer reaction was insufficient to cause localization of phosphorylation sites to incorrect residues. We further compared CID to electron-transfer dissociation (ETD) for site localization using synthetic libraries and a large-scale yeast phosphoproteome experiment. The agreement in site localization was >99.5 and 93%, respectively, suggesting that ETD-based site localization is no more reliable than CID. We conclude that intramolecular phosphate transfer does not affect the reliability of current or past phosphorylation data sets.

Single copies of Sec61 and TRAP associate with a nontranslating mammalian ribosome.

During cotranslational protein translocation, the ribosome associates with a membrane channel, formed by the Sec61 complex, and recruits the translocon-associated protein complex (TRAP). Here we report the structure of a ribosome-channel complex from mammalian endoplasmic reticulum in which the channel has been visualized at 11 A resolution. In this complex, single copies of Sec61 and TRAP associate with a nontranslating ribosome and this stoichiometry was verified by quantitative mass spectrometry. A bilayer-like density surrounds the channel and can be attributed to lipid and detergent. The crystal structure of an archaeal homolog of the Sec61 complex was then docked into the map. In this model, two cytoplasmic loops of Sec61 may interact with RNA helices H6, H7, and H50, while the central pore is located below the ribosome tunnel exit. Hence, this copy of Sec61 is positioned to capture and translocate the nascent chain. Finally, we show that mammalian and bacterial ribosome-channel complexes have similar architectures.

Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides.

Given the extensive efforts applied toward proteomics and research in biomarkers, methods for the simultaneous measurement of proteins, peptides, metabolic intermediates, hormones, etc. in a complex sample may be required in the foreseeable future. Assays based on mass spectrometric detection may be suitable for meeting the demands of such complex samples with sensitivity and specificity. An analytical method for the quantitation of C-reactive protein (CRP), a well-known marker of inflammation, is described. Exact quantities of two synthetic (13)C-labeled CRP tryptic peptides were added as internal standards directly to the sample prior to chemical treatment, trypsinization, and liquid chromatography/mass spectrometry quantitation. C-reactive protein levels based on isotopic response ratios were measured. Intact C-reactive protein was spiked into blank rat urine for chemical and enzymatic treatment, producing linear response ratios of labeled to unlabeled peptides. For rigorous quantitation, standard curves, and quality control samples were prepared in rat urine with highly purified labeled and unlabeled peptides over the 50 pg-5 ng/muL concentration range. Using the same chemical and enzymatic treatment used for digestion of intact CRP, data from these samples demonstrated excellent analytical performance. The method was successfully applied toward the quantitation of urinary C-reactive protein from a study of drug-induced nephrotoxicity.

Regulation of cytochrome P450 by posttranslational modification.

Cytochrome P450s are a family of enzymes represented in all kingdoms with expression in many species. Over 3,000 enzymes have been identified in nature. Humans express 57 putatively functional enzymes with a variety of critical physiological roles. They are involved in the metabolic oxidation, peroxidation, and reduction of many endogenous and exogenous compounds including xenobiotics, steroids, bile acids, fatty acids, eicosanoids, environmental pollutants, and carcinogens [Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P., Estabrook, R. W., Feyereisen, R., Gonzalez, F. J., Coon, M. J., Gunsalus, I. C., Gotoh, O. (1993) The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 12(1):1-51.] The development of numerous diseases and disorders including cancer and cardiovascular and endocrine dysfunction has been linked to P450s. Several levels of regulation, including transcription, translation, and posttranslational modification, participate in maintaining the proper function of P450s. Modifications including phosphorylation, glycosylation, nitration, and ubiquitination have been described for P450s. Their physiological significance includes modulation of enzyme activity, targeting to specific cellular compartments, and tagging for proteasomal degradation. Knowledge of P450 posttranslational regulation is derived from studies with relatively few enzymes. In many cases, there is only enough evidence to suggest the occurrence and a possible role for the modification. Thus, many P450 enzymes have not been fully characterized. With the introduction of current proteomics tools, we are primed to answer many important questions regarding regulation of P450 in response to a posttranslational modification. This review considers regulation of P450 in a context that describes the potential role and physiological significance of each modification.

Sensitive capillary chromatography mass spectrometric methods for the determination of salcatonin in human biological matrices.

New methods employing capillary liquid chromatography in combination with time-of-flight mass spectrometry (microLC-TOF/MS) were developed for the rapid determination of salcatonin in human urine and plasma. The present approaches utilize (13)C(6)-leucine (19)-labeled salcatonin as internal standard, small matrix volumes and simple sample preparation procedures. They allow TOF/MS to be used as a highly selective detector for providing accurate quantitation of salcatonin. Data acquisition was performed in enhanced mode optimizing the signal for the triply charged species of salcatonin and its internal standard. We demonstrate that the determination of salcatonin is straightforward and reliable and can be performed with excellent linearity (R(2)>0.999), precision and accuracy over the concentration ranges of 2.9-290 pmol/mL in human urine, and 7.3-730 pmol/mL in human plasma.