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Early detection of cancer remains a significant challenge. Existing liquid biopsy approaches to detect early-stage cancer show promise but suffer significant limitations including the abundance and stability of the analyte, large sample requirements, and insufficient specificity to distinguish biomarkers as originating from benign or cancerous cells. Extracellular vesicles (EVs) from cancer cells exhibit both stability and abundance in human biofluids which makes them an attractive target for early-cancer detection. Measurement of multiple, colocalized cancer-associated biomarkers on single EVs may improve upon existing methods for the non-invasive detection of cancer. To demonstrate this, we developed Mercy Halo™, an immunoassay designed to capture and detect cancer-specific biomarkers on the surface of EVs derived from cancer cells. Our platform shows promise for the sensitive and specific detection of early-stage ovarian tumors in human plasma, outperforming a single biomarker (CA125), for the detection of stage I and II high-grade serous ovarian cancer (HGSOC).

Introduction: Extracellular vesicles (EVs) are abundant in biological fluids and increasingly recognized for their diagnostic and therapeutic potential. Unlike circulating tumor DNA which scales in abundance with tumor size and is a product of tumor cell death; tumor EVs are secreted continuously, are remarkably stable, and used by cancer cells as messengers to benefit tumor growth and metastasis, suggesting they may be a better indicator of early-stage cancer. Tumor-derived EVs reflect their cell of origin, making them an ideal analyte for liquid biopsy diagnostic assays. We describe a platform technology, Mercy Halo™, which interrogates millions of individual EVs within a plasma or serum sample to capture and detect cancer cell derived EVs. Using panels of antibodies targeting up to three cancer-associated biomarkers simultaneously, we demonstrate Mercy Halo enables sensitive and specific detection of early-stage high-grade serous ovarian carcinoma (HGSOC) with excellent reproducibility in a proof-of-concept (PoC) study. Methods: Following purification of a sample by size-exclusion chromatography, EVs were captured using antibody-functionalized beads and interrogated using proximity ligation qPCR. EVs were characterized and biomarker expression confirmed by traditional immunoassay and Mercy Halo. The colocalization of specific biomarkers on single EVs was measured using a panel of antibodies against biomarkers upregulated in HGSOC and human cancer cell lines. Optimized antibody panels were then used in a feasibility study using EVs purified from plasma samples from early- and late-stage HGSOC, benign ovarian masses, and healthy controls. Results: Mercy Halo detects colocalized biomarkers specifically and sensitively on the surface of tumor derived EVs. Surface biomarkers expressed by cancer cells were detected in our assay and signal strength correlated with protein expression, demonstrating that EVs can be used as a surrogate to indicate the presence or absence of cancer. We demonstrate that biomarkers present on cancer cells are colocalized on EVs derived from a tumor and are readily detectable using the Mercy Halo platform. A positive signal was dependent on the presence of all biomarkers on the same EV. Importantly, there was significant improvement in both the specificity and sensitivity of the assay as the number of biomarker antibodies used to interrogate the EVs increases. Finally, in a small clinical cohort PoC study, we demonstrate improved detection of early stage HGSOC relative to CA125 ELISA. Conclusions: Tumor-derived EVs are a robust analyte for liquid-biopsy assays which can readily be detected using our platform technology. This PoC study suggests Mercy Halo can discriminate all stages of HGSOC from benign and healthy samples in human plasma with high sensitivity and specificity and is being investigated for extensibility in other cancers and diseases. Citation Format: Daniel P. Salem, Laura T. Bortolin, Anthony D. Couvillon, Dan Gusenleitner, Jonian Grosha, Sanchari Banerjee, Kelly M. Biette, Ibukunoluwapo O. Zabroski, Christopher R. Sedlak, Delaney M. Byrne, Peter A. Duff, Bilal F. Hamzeh, MacKenzie S. King, Lauren T. Cuoco, Emily S. Winn-Deen, Eric K. Huang, Joseph C. Sedlak. Colocalization of cancer-associated biomarkers on single extracellular vesicles for early-cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3058.

Introduction: Screening for lung cancer (LC), the leading cause of cancer deaths, with helical computerized tomography lowers mortality but uptake is poor. Investigations into new approaches such as using circulating tumor cells and circulating tumor DNA for LC detection have soared in the last decade. However, the low abundance of these targets has limited the performance of these approaches as screening tools. We hypothesize that co-localization of biomarkers on the surface of individual extracellular vesicles (EVs), which are shed into the circulation by cancer cells, may lead to development of a blood test for early stage LC. We evaluated the potential of our approach in detecting early stage LC in clinical samples. Methods: EVs were purified from plasma using size-exclusion chromatography and immunoaffinity capture, and biomarkers co-localized on the EV surface were detected with proximity ligation qPCR. We used antibody combinations comprising 1 capture antibody and 2 oligonucleotide-tagged detection antibodies, recognizing 1, 2 or 3 unique biomarkers. We evaluated this approach by testing plasma samples from early stage I/II lung adenocarcinoma (LUAD) patients (15 smokers, 19 non-smokers), late stage III/IV LUAD patients (16 smokers, 18 non-smokers), and healthy donors (34 smokers, 33 non-smokers). Samples were from one vendor, processed using a standardized protocol. LUAD samples were sourced from a cancer research center and healthy samples from a primary care facility. PCR cycle threshold (Ct) values were generated for each combination and data was evaluated using univariate analysis. Results: Combinations recognizing 3 biomarkers were better in detecting all stages of LUAD (AUC=0.83, 95% CI 0.77-0.90), as compared to combinations recognizing 2 biomarkers (AUC=0.71, 95% CI 0.63-0.80) or 1 biomarker (AUC=0.50, 95% CI 0.35-0.55), demonstrating greater accuracy with an increasing number of co-localized biomarkers. In detecting LUAD (all stages) at a specificity of 0.80 (95% CI 0.69-0.88), sensitivity improved as the number of co-localized biomarkers increased from 1 (0.08, 95% CI 0.03-0.18) to 2 (0.60, 95% CI 0.48-0.72) to 3 (0.76, 95% CI 0.65-0.86). In detecting early stage I/II LUAD, the most effective combination used 3 biomarkers (STn, MUC1, CEACAM6) and had a sensitivity of 0.56 (95% CI 0.38-0.73). Conclusions: These preliminary data highlight the potential of detecting biomarkers co-localized on the surface of single EVs as an effective tool for early stage LC detection, and the benefit of using 3 biomarkers simultaneously. Despite inherent challenges associated with commercial samples, our finding that detection of co-localized EV surface biomarkers distinguished LUAD is promising. Additional studies with LC cohorts beyond LUAD are underway to refine combinations and independently validate our assay for early stage LC detection. Citation Format: Daniel P. Salem, Laura T. Bortolin, Sanchari Banerjee, Kelly M. Biette, Delaney M. Byrne, Anthony D. Couvillon, Peter A. Duff, Jonian Grosha, Daniel Gusenleitner, MacKenzie Sadie King, Christopher R. Sedlak, Ibukunoluwapo O. Zabroski, Karen Copeland, Emily S. Winn-Deen, Eric K. Huang, Christine D. Berg, Joseph C. Sedlak. Preliminary results for a novel single extracellular vesicle assay for early lung cancer: The power of co-localized detection of surface biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2232.

Introduction: Ovarian cancer (OC) is one of the deadliest cancers, with 314,000 new cases and 207,000 deaths globally in 2020. Serum CA125 has been explored as an OC biomarker for the past 40 years, but lacks sensitivity for early stage OC and is not recommended for screening average-risk, asymptomatic women. We hypothesize that co-localization of biomarkers on the surface of individual extracellular vesicles (EVs), which are shed into the circulation by cancer cells, may lead to development of a blood test for early stage OC. We evaluated the potential of our approach in detecting early stage OC in clinical samples. Methods: We isolated EVs using size-exclusion chromatography and immunoaffinity capture, and detected biomarkers co-localized on the surface of individual EVs with proximity ligation qPCR. Using this approach, we evaluated 49 antibody combinations recognizing 2 or more biomarkers. Each combination consisted of 1 capture antibody and 2 oligonucleotide-tagged detection antibodies. We tested plasma samples from women with early stage I/II high-grade serous ovarian carcinoma (HGSOC)(n=18; 48-80 yr, med 57) and late stage HGSOC (n=24; 37-80 yr, med 54). HGSOC samples were sourced from 2 commercial vendors. Controls comprised samples from women with benign ovarian masses (n=26; 23-76 yr, med 39.5) sourced from a single vendor, and samples prospectively collected by Mercy from healthy women with no cancer history (n=24; 22-72 yr, med 52.5). PCR cycle threshold (Ct) values were measured for each of 49 combinations and data was evaluated using univariate analysis. Performance was compared to plasma CA125 measured at Mercy by commercial ELISA. Results: 8 of 49 combinations distinguished all stages of HGSOC relative to benign and healthy controls with AUCs ranging from 0.86 (95% CI 0.78-0.94) to 0.95 (95% CI 0.90-1.00), comparable to CA125 with an AUC of 0.87 (95% CI 0.79-0.95). One of the most effective combinations (STn, BST2, MUC1) had a sensitivity of 0.78 (95% CI 0.52-0.94) at a specificity of 0.96 (95% CI 0.87-0.99) in detecting early stage HGSOC. This combination also detected HGSOC in 6 of 11 women (3 early stage, 3 late stage) with normal CA125 (< 25 U/mL) and correctly classified 7 of 8 women with benign masses and high CA125 (> 25 U/mL). Conclusions: These preliminary data suggest that co-localization of surface biomarkers in single EVs may provide an effective means to identify women with early stage HGSOC, including those with normal CA125, while avoiding false positives in women with benign masses and high CA125. Despite the inherent challenges associated with commercial samples, our finding that several combinations detected early stage HGSOC is promising. Statistically powered studies with curated repository specimens are underway to refine combinations and independently validate our assay for early stage OC detection. Citation Format: Laura T. Bortolin, Daniel P. Salem, Sanchari Banerjee, Kelly M. Biette, Delaney M. Byrne, Anthony D. Couvillon, Peter A. Duff, Jonian Grosha, MacKenzie Sadie King, Christopher R. Sedlak, Ibukunoluwapo O. Zabroski, Claire Alexander, Karen Copeland, Daniel Gusenleitner, Emily S. Winn-Deen, Eric K. Huang, Bo R. Rueda, Joseph Charles Sedlak. Preliminary results for a novel single extracellular vesicle assay for early stage ovarian cancer: The power of co-localized detection of surface biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3390.

5542 Background: Detection of cancer with improved discrimination compared to current blood tests could be achieved using an approach that assesses extracellular vesicles (EVs). This approach should have high sensitivity (se) because of EVs abundance in blood and high specificity (sp) by assaying EVs with multiple cancer-related protein and glycosylation epitopes (PGEs) co-localized on their surfaces. We are developing a platform technology that detects multiple cancer-related PGEs co-localized on the same EV using immunoaffinity-capture and proximity-ligation qPCR. In this study, we compare the performance of this technology vs plasma CA125 for correctly categorizing early-stage high-grade serous ovarian cancer (HGSOC) vs healthy/benign ovarian tumors (OT). Methods: We evaluated our EV-based platform technology using 7 PGE combinations to discriminate HGSOC from benign adnexal masses. We first derived a prediction model on a retrospectively collected cohort of 42 HGSOC and 26 benign OT samples from 2 commercial vendors and 24 healthy controls (HC) using a machine-learning algorithm. We validated this model on an independent cohort [89 HGSOC: Stage I (17), II (35), III (37); 192 benign OT] from university-associated biobanks and 124 HC. We also assessed the assay’s performance in plasma from 87 women with off-target cancers and 42 women with inflammatory conditions from commercial vendors. For each sample, we also measured CA125 levels using a commercial ELISA. Results: The prediction model distinguishes HGSOC from benign and HC with an AUC of 0.965 (95% CI 0.93-0.99), with 89.9% (0.82-0.95) se at 98% sp. For stage I/II HGSOC, the model achieves an AUC of 0.942 (0.9-0.99), with 84.6% (0.72-0.93) se at 98% sp. By comparison, CA125 achieves an AUC of 0.875 (0.81-0.94) and 44.2% (0.3-0.59) se at 98% sp. Direct comparison of CA125 and our model shows a significant difference at 98% sp for both all and stage I/II HGSOC (McNemar p-val < 0.001). When comparing HGSOC to HC, there is no significant difference between our model and CA125 (p-val = 1.0). There is a significant difference when comparing patients with all stage and stage I/II HGSOC to patients with benign OT (p-val < 0.001). Our assay had 1 false positive and CA125 had 3 false positives out of 42 inflammatory cases. Conclusions: These preliminary data suggest our platform technology for detecting PGEs co-localized on individual EVs may detect all stages of HGSOC from plasma with high se at a very high sp. Our assay may improve on CA125 by distinguishing stage I/II HGSOC from benign OT and could have clinical utility for both early detection and surgical referral recommendation for benign and malignant OT. While the diverse cohorts in this study may present challenges in interpretation, the reproducibility in an independent cohort is encouraging and supports further investigation using cases and controls from well-defined cohort studies.

Phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP3 or Gβγ, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP3 and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.

mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.

Protein histidine phosphorylation plays a crucial role in cell signaling and central metabolism. However, its detailed functions remain elusive due to technical challenges in detecting and isolating proteins bearing phosphohistidine (pHis), a labile posttranslational modification (PTM). To address this issue, we previously developed the first pHis-specific antibodies using stable, synthetic triazole-based pHis analogs. A second-generation, pyrazole-based pHis analog that enabled the development of a pan-pHis antibody with much improved pHis specificity is now reported.

NF-kappaB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-kappaB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1alpha through the adapter protein TRAF2. ER stress-induced NF-kappaB activation is impaired in IRE1alpha knockdown cells and IRE1alpha(-/-) MEFs. We found, however, that inhibiting NF-kappaB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-alpha) was IRE1alpha and NF-kappaB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-alpha-induced activation of NF-kappaB and c-Jun N-terminal kinase and turns TNF-alpha from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-alpha induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-Ras V12 oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-Ras V12 oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.

Extracellular signal-regulated kinases (ERKs) play critical roles in numerous cellular processes, including proliferation and differentiation. ERK5 contains a kinase domain at the N-terminal, and the unique extended C-terminal includes multiple autophosphorylation sites that enhance ERK5-dependent transcription. However, the impact of phosphorylation at the various sites remain unclear. In this study, we examined the role of phosphorylation at the ERK5 C-terminal. We found that a constitutively active MEK5 mutant phosphorylated ERK5 at the TEY motif, resulting in the sequential autophosphorylation of multiple C-terminal residues, including Thr732 and Ser769/773/775. However, when ERK1/2 was selectively activated by an oncogenic RAS mutant, ERK5 phosphorylation at Thr732 was induced without affecting the phosphorylation status at TEY or Ser769/773/775. The Thr732 phosphorylation was U0126-sensitive and was observed in a kinase-dead mutant of ERK5 as well, suggesting that ERK1/2 can phosphorylate ERK5 at Thr732. This phosphorylation was also promoted by epidermal growth factor and nerve growth factor in HEK293 and PC12 cells, respectively. The ERK5–T732A mutant was localized in the cytosol under basal conditions. In contrast, ERK5 phosphorylated at Thr732 via the RAS-ERK1/2 pathway and ERK5–T732E, which mimics the phosphorylated form, were localized in both the nucleus and cytosol. Finally, ER–32A and U0126 blocked ERK5-dependent MEF2C transcriptional activity. Based on these findings, we propose a novel cross-talk mechanism in which ERK1/2, following activation by growth factor stimulation, phosphorylates ERK5 at Thr732. This phosphorylation event is responsible for ERK5 nuclear localization and ERK5-dependent transcription.

The liver plays a critical role in glucose metabolism and communicates with peripheral tissues to maintain energy homeostasis. Obesity and insulin resistance are highly associated with nonalcoholic fatty liver disease (NAFLD). However, the precise molecular details of NAFLD remain incomplete. The p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) regulate liver metabolism. However, the physiological contribution of MAPK phosphatase 1 (MKP-1) as a nuclear antagonist of both p38 MAPK and JNK in the liver is unknown. Here we show that hepatic MKP-1 becomes overexpressed following high-fat feeding. Liver-specific deletion of MKP-1 enhances gluconeogenesis and causes hepatic insulin resistance in chow-fed mice while selectively conferring protection from hepatosteatosis upon high-fat feeding. Further, hepatic MKP-1 regulates both interleukin-6 (IL-6) and fibroblast growth factor 21 (FGF21). Mice lacking hepatic MKP-1 exhibit reduced circulating IL-6 and FGF21 levels that were associated with impaired skeletal muscle mitochondrial oxidation and susceptibility to diet-induced obesity. Hence, hepatic MKP-1 serves as a selective regulator of MAPK-dependent signals that contributes to the maintenance of glucose homeostasis and peripheral tissue energy balance. These results also demonstrate that hepatic MKP-1 overexpression in obesity is causally linked to the promotion of hepatosteatosis.

The Ras-related guanosine triphosphatase RhoA mediates pathological cardiac hypertrophy, but also promotes cell survival and is cardioprotective after ischemia/reperfusion injury. To understand how RhoA mediates these opposing roles in the myocardium, we generated mice with a cardiomyocyte-specific deletion of RhoA. Under normal conditions, the hearts from these mice showed functional, structural, and growth parameters similar to control mice. Additionally, the hearts of the cardiomyocyte-specific, RhoA-deficient mice subjected to transverse aortic constriction (TAC)-a procedure that induces pressure overload and, if prolonged, heart failure-exhibited a similar amount of hypertrophy as those of the wild-type mice subjected to TAC. Thus, neither normal cardiac homeostasis nor the initiation of compensatory hypertrophy required RhoA in cardiomyocytes. However, in response to chronic TAC, hearts from mice with cardiomyocyte-specific deletion of RhoA showed greater dilation, with thinner ventricular walls and larger chamber dimensions, and more impaired contractile function than those from control mice subjected to chronic TAC. These effects were associated with aberrant calcium signaling, as well as decreased activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT. In addition, hearts from mice with cardiomyocyte-specific RhoA deficiency also showed less fibrosis in response to chronic TAC, with decreased transcriptional activation of genes involved in fibrosis, including myocardin response transcription factor (MRTF) and serum response factor (SRF), suggesting that the fibrotic response to stress in the heart depends on cardiomyocyte-specific RhoA signaling. Our data indicated that RhoA regulates multiple pathways in cardiomyocytes, mediating both cardioprotective (hypertrophy without dilation) and cardio-deleterious effects (fibrosis).

Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine (pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC–MS/MS. We show that collision-induced dissociation (CID) of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da. Using isotopic labeling studies, we also demonstrate that the 98 Da neutral loss occurs via gas-phase phosphoryl transfer from pHis to the peptide C-terminal α-carboxylate or to Glu/Asp side chain residues if present. To exploit this property, we developed a software tool that screens LC–MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern. This tool was integrated into a proteomics workflow for the identification of endogenous pHis-containing proteins in cellular lysates. As an illustration of this strategy, we analyzed pHis peptides from glycerol-fed and mannitol-fed Escherichia coli cells. We identified known and a number of previously speculative pHis sites inferred by homology, predominantly in the phosphoenolpyruvate:sugar transferase system (PTS). Furthermore, we identified two new sites of histidine phosphorylation on aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF) enzymes, previously not known to bear this modification. This study lays the groundwork for future pHis proteomics studies in bacteria and other organisms.

The phosphorylation of phosphatidylinositol (PtdIns) on the 3′ position of the inositol ring by phosphoinositide 3-kinase (PI 3-kinase) is shown to depend strongly on the curvature of liposomes containing a mixture of phosphatidylcholine (PtdCho) and PtdIns. Vesicles with an average diameter of 50 nm are phosphorylated 100 times faster than chemically identical vesicles with an average diameter greater than 300 nm. The low reactivity of large vesicles is not due to the difference in vesicle number for large and small vesicles at constant total lipid, nor to occlusion of lipid surfaces in multilammelar structures, and can be reversed by addition of low (

The assembly of neuronal circuits during development requires the precise navigation of axons, which is controlled by attractive and repulsive guidance cues. In the developing spinal cord, ephrinB3 functions as a short-range repulsive cue that prevents EphA4 receptor-expressing corticospinal tract and spinal interneuron axons from crossing the midline, ensuring proper formation of locomotor circuits. Here we report that the small GTPase RhoA, a key regulator of cytoskeletal dynamics, is also required for ephrinB3/EphA4-dependent locomotor circuit formation. Deletion of RhoA from neural progenitor cells results in mice that exhibit a rabbit-like hopping gait, which phenocopies mice lacking ephrinB3 or EphA4. Consistent with this locomotor defect, we found that corticospinal tract axons and spinal interneuron projections from RhoA-deficient mice aberrantly cross the spinal cord midline. Furthermore, we determined that loss of RhoA blocks ephrinB3-induced growth cone collapse of cortical axons and disrupts ephrinB3 expression at the spinal cord midline. Collectively, our results demonstrate that RhoA is essential for the ephrinB3/EphA4-dependent assembly of cortical and spinal motor circuits that control normal locomotor behavior.

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.

In neuronal cells, current evidence suggests that G(13)alpha and RhoA play significant roles in LPA-mediated neurite retraction; however, the contribution of other G-proteins to this process is less well-understood. We provide evidence that LPA activation of G(13), G(q) and G(i) occurs rapidly in neuroblastoma cells, but that stimulation of RhoA is transient whereas the activation of G(q)- and G(i)-mediated pathways is sustained. In addition to G(13)alpha, we demonstrate that G(q)alpha is capable of promoting neurite retraction. G(q)-mediated retraction is RhoA-independent and is likely mediated via a mechanism involving protein kinase C and calcium flux. Additionally, we provide evidence that activation of adenylyl cyclase via G(s) inhibits RhoA-mediated neurite retraction via protein kinase A-mediated inhibition of RhoA action. Taken together, we hypothesize that LPA promotes neurite retraction via RhoA-dependent and -independent pathways involving G(13) and G(q), respectively, and that agonists that activate G(s) inhibit the RhoA-dependent pathway.

Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.

Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5 (amino acids 2-17), which is distinct from that of class I or class III ARFs, is essential for binding to the C-terminus of arfophilin (amino acids 612-756). This study using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates showed that, unexpectedly, ARF6 also bound to full-length arfophilin or the C-terminus of arfophilin (amino acids 612-756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further showed that the amino acid sequence of residues 37-80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, was essential for binding to arfophilin. Both GTP-ARF5 and GTP-ARF6 bound to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 did not bind. In contrast, all three forms of ARF bound to arfaptin 2, with ARF1 showing the strongest binding. Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 was autoactivating. Our findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector.

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4, 5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.

The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology.