ABSTRACT
Chemotherapy remains a primary treatment for younger AML patients, though many relapse. Data from our group have shown that highly phosphorylated S6 in blasts may predict response to sirolimus given with chemotherapy. We report the results of a phase I study of this combination in newly diagnosed AML and the pharmacodynamic analysis of pS6 before and after treatment. Subjects received sirolimus (12 mg on day 1, 4 mg daily, days 2-10), then idarubicin and cytarabine (days 4-10). Response was assessed at hematologic recovery or by day 42 using a modified IWG criteria. Fifty-five patients received sirolimus. Toxicity was similar to published 7 + 3 data, and 53% had high-, 27% intermediate-, and 20% favorable-risk disease. Forty-four percent of the high-risk patients entered into CR/CRp. Seventy-nine percent of the intermediate-risk subjects had a CR/CRp. All favorable-risk patients had a CR by day 42; 9/11 remained alive and in remission with a median follow-up of 660 days. Additionally, 41/55 patients had adequate samples for pharmacodynamic analysis. All patients demonstrated activation of S6 prior to therapy, in contrast to 67% seen in previous studies of relapsed AML. mTORC1 inhibition was observed in 66% of patients without enrichment among patients who achieved remission. We conclude that sirolimus and 7 + 3 is a well-tolerated and safe regimen. mTORC1 appears to be activated in almost all patients at diagnosis of AML. Inhibition of mTORC1 did not differ based on response, suggesting that AML cells may have redundant signaling pathways that regulate chemosensitivity in the presence of mTORC1 inhibition.
ABSTRACT
Circulating IL-6, an activator of JAK/STAT signaling, is associated with poor prognosis and aromatase inhibitor (AI) resistance in hormone-receptor positive (HR+) breast cancer. Here we report the results of a phase 2 single-arm Simon 2-stage trial combining Ruxolitinib, an oral selective inhibitor of JAK1/2, with exemestane, a steroidal AI, in patients with HR+ metastatic breast cancer (MBC) after progression on non-steroidal AI (NSAI). Safety and efficacy were primary objectives, and analysis of inflammatory markers as predictors of response was a key secondary objective. Twenty-five subjects enrolled. The combination of ruxolitinib and exemestane was safe, though anemia requiring transfusion in 5/15 (33%) at the 25 mg dose in stage 1 led to a reduction to 15 mg twice daily in stage 2 (with no additional transfusions). Clinical benefit rate (CBR) in the overall study population was 24% (95% CI 9.4-45.1); 6/25 patients demonstrated stable disease for ≥6 months. Median progression-free survival was 2.8 months (95% CI 2.6-3.9). Exploratory biomarkers revealed high levels of systemic inflammation and 60% harbored a high-risk IL-6 genotype. Pharmacodynamics demonstrated modest on-target inhibition of phosphorylated-STAT3 by ruxolitinib at a tolerable dose. Thus, ruxolitinib combined with exemestane at a tolerable dose was safe but minimally active in AI-resistant tumors of patients with high levels of systemic inflammation. These findings highlight the need for more potent and specific therapies targeting inflammation in MBC.
ABSTRACT
Here, we present the largest, global dataset of Lepidopteran traits, focusing initially on butterflies (ca. 12,500 species records). These traits are derived from field guides, taxonomic treatments, and other literature resources. We present traits on wing size, phenology,voltinism, diapause/overwintering stage, hostplant associations, and habitat affinities (canopy, edge, moisture, and disturbance). This dataset will facilitate comparative research on butterfly ecology and evolution and our goal is to inspire future research collaboration and the continued development of this dataset.
Subject(s)
Butterflies , Animals , Butterflies/genetics , Ecology , PhenotypeABSTRACT
Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Imidazoles/pharmacology , Indazoles/pharmacology , Melanoma/metabolism , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Oximes/pharmacology , Piperazines/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyridones/pharmacology , Pyrimidinones/pharmacology , Skin Neoplasms/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , MAP Kinase Signaling System/drug effects , Melanoma/pathology , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphorylation/drug effects , Proteome , Proteomics/methods , Proto-Oncogene Proteins B-raf/metabolism , Skin Neoplasms/pathologyABSTRACT
Multisite phosphorylation of proteins is a common mechanism for signal integration and amplification in eukaryotic signaling networks. Proteins are commonly phosphorylated at multiple sites in an ordered manner, whereby phosphorylation by one kinase primes the substrate by generating a recognition motif for a second kinase. Here we show that substrate priming promotes phosphorylation by Saccharomyces cerevisiae Kin1 and Kin2, kinases that regulate cell polarity, exocytosis, and the endoplasmic reticulum (ER) stress response. Kin1/Kin2 phosphorylated substrates within the context of a sequence motif distinct from those of their most closely related kinases. In particular, the rate of phosphorylation of a peptide substrate by Kin1/Kin2 increased >30-fold with incorporation of a phosphoserine residue two residues downstream of the phosphorylation site. Recognition of phosphorylated substrates by Kin1/Kin2 was mediated by a patch of basic residues located in the region of the kinase αC helix. We identified a set of candidate Kin1/Kin2 substrates reported to be dually phosphorylated at sites conforming to the Kin1/Kin2 consensus sequence. One of these proteins, the t-SNARE protein Sec9, was confirmed to be a Kin1/Kin2 substrate both in vitro and in vivo Sec9 phosphorylation by Kin1 in vitro was enhanced by prior phosphorylation at the +2 position. Recognition of primed substrates was not required for the ability of Kin2 to suppress the growth defect of secretory pathway mutants but was necessary for optimal growth under conditions of ER stress. These results suggest that at least some endogenous protein substrates of Kin1/Kin2 are phosphorylated in a priming-dependent manner.
Subject(s)
Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Motifs , Membrane Proteins/chemistry , Membrane Proteins/genetics , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Qc-SNARE Proteins/genetics , Qc-SNARE Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Substrate SpecificityABSTRACT
Background Mammalian Target of Rapamycin Complex 1 (mTORC1) inhibitors enhance chemotherapy response in acute myelogenous leukemia (AML) cells in vitro. However whether inhibiting mTORC1 enhances clinical response to AML chemotherapy remains controversial. We previously optimized measurement of mTORC1's kinase activity in AML blasts during clinical trials using serial phospho-specific flow cytometry of formaldehyde-fixed whole blood or marrow specimens. To validate mTORC1 as a therapeutic target in AML, we performed two clinical trials combining an mTORC1 inhibitor (sirolimus) and MEC (mitoxantrone, etoposide, cytarabine) in patients with relapsed, refractory, or untreated high-risk AML. Methods Flow cytometric measurements of ribosomal protein S6 phosphorylation (pS6) were performed before and during sirolimus treatment to determine whether mTORC1 inhibition enriched for chemotherapy response. Results In 51 evaluable subjects, the overall response rate (ORR) to the combination regimen was 47% (95% confidence interval 33-61%, 33% CR, 2% CRi, 12% PR) and similar toxicity to historic experience with MEC alone. 37 subjects had baseline pS6 measured pre-sirolimus, of whom 27 (73%) exhibited mTORC1 activity. ORR was not significantly different between subjects with and without baseline mTORC1 activity (52% vs 40%, respectively, p = 0.20). The ORR among subjects with baseline target activation and mTORC1 inhibition during therapy was 71% (12/17) compared to 20% (2/10) in subjects without target inhibition. Conclusions Fixed, whole blood pS6 by flow cytometry may be a predictive biomarker for clinical response to mTORC1 inhibitor-based regimens. These data provide clinical confirmation that mTORC1 activation mediates chemotherapy resistance in patients with AML.
Subject(s)
Leukemia, Myeloid, Acute/drug therapy , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Sirolimus/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cytarabine/therapeutic use , Etoposide/therapeutic use , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Male , Middle Aged , Mitoxantrone/therapeutic use , Phosphorylation/drug effects , Pilot Projects , Remission Induction/methods , Signal Transduction/drug effectsABSTRACT
Janus kinase 2 (JAK2) is a central kinase in hematopoietic stem/progenitor cells (HSPCs), and its uncontrolled activation is a prominent oncogenic driver of hematopoietic neoplasms. However, molecular mechanisms underlying the regulation of JAK2 have remained elusive. Here we report that the Casitas B-cell lymphoma (CBL) family E3 ubiquitin ligases down-regulate JAK2 stability and signaling via the adaptor protein LNK/SH2B3. We demonstrated that depletion of CBL/CBL-B or LNK abrogated JAK2 ubiquitination, extended JAK2 half-life, and enhanced JAK2 signaling and cell growth in human cell lines as well as primary murine HSPCs. Built on these findings, we showed that JAK inhibitor (JAKi) significantly reduced aberrant HSPCs and mitigated leukemia development in a mouse model of aggressive myeloid leukemia driven by loss of Cbl and Cbl-b Importantly, primary human CBL mutated (CBLmut ) leukemias exhibited increased JAK2 protein levels and signaling and were hypersensitive to JAKi. Loss-of-function mutations in CBL E3 ubiquitin ligases are found in a wide range of myeloid malignancies, which are diseases without effective treatment options. Hence, our studies reveal a novel signaling axis that regulates JAK2 in normal and malignant HSPCs and suggest new therapeutic strategies for treating CBLmut myeloid malignancies.
Subject(s)
Janus Kinase 2/metabolism , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/physiopathology , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing , Animals , Cell Line, Tumor , Cell Proliferation/genetics , Cytokines/metabolism , Enzyme Stability , Hematopoietic Stem Cells/enzymology , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Janus Kinase 2/genetics , Leukemia, Myeloid, Acute/genetics , Membrane Proteins , Mice , Mutation , Proteolysis , Proto-Oncogene Proteins c-cbl/genetics , Signal Transduction/genetics , UbiquitinationABSTRACT
Genomic studies have revealed significant branching heterogeneity in cancer. Studies of resistance to tyrosine kinase inhibitor therapy have not fully reflected this heterogeneity because resistance in individual patients has been ascribed to largely mutually exclusive on-target or off-target mechanisms in which tumors either retain dependency on the target oncogene or subvert it through a parallel pathway. Using targeted sequencing from single cells and colonies from patient samples, we demonstrate tremendous clonal diversity in the majority of acute myeloid leukemia (AML) patients with activating FLT3 internal tandem duplication mutations at the time of acquired resistance to the FLT3 inhibitor quizartinib. These findings establish that clinical resistance to quizartinib is highly complex and reflects the underlying clonal heterogeneity of AML.
Subject(s)
Benzothiazoles/administration & dosage , Drug Resistance, Neoplasm , High-Throughput Nucleotide Sequencing , INDEL Mutation , Leukemia, Myeloid, Acute , Phenylurea Compounds/administration & dosage , fms-Like Tyrosine Kinase 3/genetics , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , MaleABSTRACT
Limited cell numbers in umbilical cord blood (UCB) grafts present a major impediment to favorable outcomes in adult transplantation, largely related to delayed or failed engraftment. The advent of UCB transplantation (UCBT) using two grafts successfully circumvents this obstacle, despite the engraftment of only one unit. Preclinical models suggested that the addition of UCB T cells at the time of transplant can enhance engraftment. We tested whether ex vivo activation by CD3/CD28 costimulation and expansion of T cells from a single UCB graft would be safe and feasible in adults with advanced hematologic malignancies, with an overall objective of optimizing engraftment in single unit UCBT. In this phase 1 study, recipients of single UCB units were eligible if the unit was stored in two adequate fractions. Dose limiting toxicity was defined as grade 3 or grade 4 GVHD within 90 days of UCBT. Four patients underwent UCBT; all were treated at the first dose level (10(5) cells/kg). At the 10(5) cells/kg dose level two subjects experienced grade 3 intestinal GVHD, thus meeting stopping criteria. For three subjects, neutrophil engraftment was early (12, 17, and 20 days), while one subject experienced primary graft failure. We observed early donor T cell trafficking and found that expanded T cells produced supraphysiologic levels of cytokines relevant to engraftment and to lymphoid differentiation and function. Taken together, these preliminary data suggest rapid engraftment in recipients of a single UCBT combined with relatively low doses of activated T cells, though potentially complicated by severe GVHD.
Subject(s)
Cord Blood Stem Cell Transplantation/methods , Lymphocyte Transfusion , T-Lymphocyte Subsets/transplantation , Adult , Anemia, Refractory, with Excess of Blasts/therapy , B-Cell Activating Factor/biosynthesis , Blood Preservation , CD28 Antigens/immunology , CD3 Complex/immunology , Cells, Cultured/transplantation , Cord Blood Stem Cell Transplantation/adverse effects , Cord Blood Stem Cell Transplantation/mortality , Cryopreservation , Cytokines/analysis , Dose-Response Relationship, Immunologic , Feasibility Studies , Female , Graft Survival , Graft vs Host Disease/etiology , Histocompatibility , Humans , Infant, Newborn , Leukemia, Myeloid, Acute/therapy , Lymphocyte Transfusion/adverse effects , Male , Maximum Tolerated Dose , Membrane Proteins/biosynthesis , Middle Aged , Neutrophils/transplantation , T-Cell Antigen Receptor Specificity , Transplantation Conditioning/methods , Treatment OutcomeABSTRACT
The normal expression of ß-globin protein in mature erythrocytes is critically dependent on post-transcriptional events in erythroid progenitors that ensure the high stability of ß-globin mRNA. Previous work has revealed that these regulatory processes require AUF-1 and YB-1, two RNA-binding proteins that assemble an mRNP ß-complex on the ß-globin 3'UTR. Here, we demonstrate that the ß-complex organizes during the erythropoietic interval when both ß-globin mRNA and protein accumulate rapidly, implicating the importance of this regulatory mRNP to normal erythroid differentiation. Subsequent functional analyses link ß-complex assembly to the half-life of ß-globin mRNA in vivo, providing a mechanistic basis for this regulatory activity. AUF-1 and YB-1 appear to serve a redundant post-transcriptional function, as both ß-complex assembly and ß-globin mRNA levels are reduced by coordinate depletion of the two factors, and can be restored by independent rescue with either factor alone. Additional studies demonstrate that the ß-complex assembles more efficiently on polyadenylated transcripts, implicating a model in which the ß-complex enhances the binding of PABPC1 to the poly(A) tail, inhibiting mRNA deadenylation and consequently effecting the high half-life of ß-globin transcripts in erythroid progenitors. These data specify a post-transcriptional mechanism through which AUF1 and YB1 contribute to the normal development of erythropoietic cells, as well as to non-hematopoietic tissues in which AUF1- and YB1-based regulatory mRNPs have been observed to assemble on heterologous mRNAs.
Subject(s)
Erythroid Cells/metabolism , Heterogeneous-Nuclear Ribonucleoprotein D/metabolism , Poly(A)-Binding Protein II/metabolism , Y-Box-Binding Protein 1/metabolism , beta-Globins/metabolism , 3' Untranslated Regions , Cell Differentiation , Cell Line , Heterogeneous Nuclear Ribonucleoprotein D0 , Humans , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , beta-Globins/geneticsABSTRACT
We performed a multicenter, investigator initiated, phase I dose escalation study of the oral multi-kinase inhibitor lestaurtinib in patients with JAK2V617F positive myelofibrosis, irrespective of baseline platelet count. A total of 34 patients were enrolled. Dose-limiting toxicities were observed in three patients overall, at the 100 mg (n = 1) and 160 mg (n = 2) twice-daily dose levels. The maximum tolerated dose was 140 mg twice daily. Gastrointestinal toxicity was the most common adverse event. Sixteen patients were evaluable for response at 12 weeks. Seven patients had clinical improvement by International Working Group - Myeloproliferative Neoplasms Research and Treatment criteria. Meaningful reductions in JAK2V617F allele burden were not observed. To measure JAK2 inhibition in vivo, plasma from treated patients was assayed for its ability to inhibit phosphorylation of signal transducer and activator of transcription 5 (STAT5): doses lower than 140 mg had variable and incomplete inhibition. In this phase I study, although gastrointestinal adverse events were common, significant clinical activity with lestaurtinib was observed (ClinicalTrials.gov identifier: NCT00668421).
Subject(s)
Carbazoles/therapeutic use , Primary Myelofibrosis/drug therapy , Adult , Aged , Aged, 80 and over , Carbazoles/adverse effects , Carbazoles/pharmacokinetics , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Furans , Gastrointestinal Diseases/chemically induced , Humans , Janus Kinase 2/genetics , Leukopenia/chemically induced , Male , Middle Aged , Mutation, Missense , Primary Myelofibrosis/genetics , Treatment Outcome , Young AdultABSTRACT
The Ras/MAPK signaling cascade regulates various biological functions, including cell growth and proliferation. As such, this pathway is frequently deregulated in several types of cancer, including most cases of melanoma. RSK (p90 ribosomal S6 kinase) is a MAPK-activated protein kinase required for melanoma growth and proliferation, but relatively little is known about its exact function and the nature of its substrates. Herein, we used a quantitative phosphoproteomics approach to define the signaling networks regulated by RSK in melanoma. To more accurately predict direct phosphorylation substrates, we defined the RSK consensus phosphorylation motif and found significant overlap with the binding consensus of 14-3-3 proteins. We thus characterized the phospho-dependent 14-3-3 interactome in melanoma cells and found that a large proportion of 14-3-3 binding proteins are also potential RSK substrates. Our results show that RSK phosphorylates the tumor suppressor PDCD4 (programmed cell death protein 4) on two serine residues (Ser76 and Ser457) that regulate its subcellular localization and interaction with 14-3-3 proteins. We found that 14-3-3 binding promotes PDCD4 degradation, suggesting an important role for RSK in the inactivation of PDCD4 in melanoma. In addition to this tumor suppressor, our results suggest the involvement of RSK in a vast array of unexplored biological functions with relevance in oncogenesis.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Phosphoproteins/metabolism , Proteomics/methods , RNA-Binding Proteins/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Tumor Suppressor Proteins/metabolism , 14-3-3 Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cell Line , Cell Nucleus/metabolism , Consensus Sequence , Humans , Melanoma/metabolism , Melanoma/pathology , Models, Biological , Molecular Sequence Data , Peptide Library , Phosphorylation , Phosphoserine/metabolism , Protein Binding , Protein Transport , Proteolysis , Proteome/metabolism , Substrate SpecificityABSTRACT
Tyrosine kinase inhibitors (TKIs) represent transformative therapies for several malignancies. Two critical features necessary for maximizing TKI tolerability and response duration are kinase selectivity and invulnerability to resistance-conferring kinase domain (KD) mutations in the intended target. No prior TKI has demonstrated both of these properties. Aiming to maximize selectivity, medicinal chemists have largely sought to create TKIs that bind to an inactive (type II) kinase conformation. Here we demonstrate that the investigational type I TKI crenolanib is a potent inhibitor of Fms tyrosine kinase-3 (FLT3) internal tandem duplication, a validated therapeutic target in human acute myeloid leukemia (AML), as well as all secondary KD mutants previously shown to confer resistance to the first highly active FLT3 TKI quizartinib. Moreover, crenolanib is highly selective for FLT3 relative to the closely related protein tyrosine kinase KIT, demonstrating that simultaneous FLT3/KIT inhibition, a prominent feature of other clinically active FLT3 TKIs, is not required for AML cell cytotoxicity in vitro and may contribute to undesirable toxicity in patients. A saturation mutagenesis screen of FLT3-internal tandem duplication failed to recover any resistant colonies in the presence of a crenolanib concentration well below what has been safely achieved in humans, suggesting that crenolanib has the potential to suppress KD mutation-mediated clinical resistance. Crenolanib represents the first TKI to exhibit both kinase selectivity and invulnerability to resistance-conferring KD mutations, which is unexpected of a type I inhibitor. Crenolanib has significant promise for achieving deep and durable responses in FLT3-mutant AML, and may have a profound impact upon future medicinal chemistry efforts in oncology.
Subject(s)
Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , Piperidines/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Antineoplastic Agents/chemistry , Benzimidazoles/chemistry , Cell Line, Tumor , Drug Resistance, Neoplasm , Humans , Molecular Docking Simulation , Mutation , Piperidines/chemistry , fms-Like Tyrosine Kinase 3/chemistry , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
Apicomplexan parasites rely on calcium as a second messenger to regulate a variety of essential cellular processes. Calcium-dependent protein kinases (CDPK), which transduce these signals, are conserved among apicomplexans but absent from mammalian hosts, making them attractive targets for therapeutic intervention. Despite their importance, the signaling pathways CDPK regulate remain poorly characterized, and their protein substrates are completely unknown. In Toxoplasma gondii, CDPK1 is required for calcium-regulated secretion from micronemes, thereby controlling motility, invasion, and egress from host cells. CDPK1 is unique among parasite and mammalian kinases in containing glycine at the key "gatekeeper" residue, which results in an expanded ATP-binding pocket. In the present study, we use a synthetic ATPγS analogue that displays steric complementarity to the ATP-binding pocket and hence allows identification of protein substrates based on selective thiophosphorylation. The specificity of this approach was validated by the concordance between the identified phosphorylation sites and the in vitro substrate preference of CDPK1. We further demonstrate that the phosphorylation of predicted substrates is dependent on CDPK1 both in vivo and in vitro. This combined strategy for identifying the targets of specific protein kinases provides a platform for defining the roles of CDPKs in apicomplexans.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Protein Kinases/chemistry , Protein Kinases/metabolism , Toxoplasma/enzymology , Amino Acid Sequence , Binding Sites , Humans , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Phosphates/chemistry , Phosphates/metabolism , Phosphorylation , Substrate Specificity , Toxoplasma/chemistry , Toxoplasma/metabolism , Toxoplasmosis/parasitologyABSTRACT
PURPOSE: Recent results have shown that myeloproliferative neoplasms (MPN) are strongly associated with constitutive activation of the Janus-activated kinase (JAK)2 tyrosine kinase. However, JAK2 inhibitors currently approved or under development for treating myeloproliferative neoplasms do not selectively deplete the malignant clone, and the inhibition of activity of the drug target (JAK2) has not been rigorously evaluated in the clinical studies. Therefore, in this study we developed an in vitro assay to gain insight into how effectively JAK2 activity is inhibited in the samples of patients. EXPERIMENTAL DESIGN: We treated primary cells from normal donors and patients with MPN with JAK2 inhibitors and measured phosphorylation of downstream targets STAT5 and STAT3 by flow cytometry. Obtained results were next correlated with JAK2 V617F allele burden and plasma cytokine level. RESULTS: We observed a dose-dependent decrease in pSTAT5 and pSTAT3 in ex vivo treated granulocytes. However, phosphorylation of STAT3 and STAT5 in cells from patients with myelofibrosis was significantly less inhibited when compared with cells from patients with polycythemia vera, essential thrombocythemia, and normal donors. Sensitivity to inhibition did not correlate with JAK2 V617F clonal burden. Mixing studies using plasma from patients with myelofibrosis did not transfer resistance to sensitive cells. Likewise, no single cytokine measured seemed to account for the observed pattern of resistance. CONCLUSIONS: Taken together, these observations suggest that there are cell intrinsic mechanisms that define a priori resistance to JAK2 inhibition in myelofibrosis, and the lesion is localized upstream of STAT3 and STAT5.
Subject(s)
Drug Resistance , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Primary Myelofibrosis/metabolism , Protein Kinase Inhibitors/pharmacology , Adult , Aged , Aged, 80 and over , Alleles , Carbazoles/pharmacology , Cytokines/pharmacology , Female , Furans , Gene Frequency , Humans , Janus Kinase 2/genetics , Male , Middle Aged , Mutation , Neutrophils/drug effects , Neutrophils/metabolism , Phosphorylation , Primary Myelofibrosis/genetics , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolismABSTRACT
Eukaryotic cells have evolved mechanisms for ensuring growth and survival in the face of stress caused by a fluctuating environment. Saccharomyces cerevisiae has two homologous glycerol-3-phosphate dehydrogenases, Gpd1 and Gpd2, that are required to endure various stresses, including hyperosmotic shock and hypoxia. These enzymes are only partially redundant, and their unique functions were attributed previously to differential transcriptional regulation and localization. We find that Gpd1 and Gpd2 are negatively regulated through phosphorylation by distinct kinases under reciprocal conditions. Gpd2 is phosphorylated by the AMP-activated protein kinase Snf1 to curtail glycerol production when nutrients are limiting. Gpd1, in contrast, is a target of TORC2-dependent kinases Ypk1 and Ypk2. Inactivation of Ypk1 by hyperosmotic shock results in dephosphorylation and activation of Gpd1, accelerating recovery through increased glycerol production. Gpd1 dephosphorylation acts synergistically with its transcriptional upregulation, enabling long-term growth at high osmolarity. Phosphorylation of Gpd1 and Gpd2 by distinct kinases thereby enables rapid adaptation to specific stress conditions. Introduction of phosphorylation motifs targeted by distinct kinases provides a general mechanism for functional specialization of duplicated genes during evolution.
Subject(s)
Adaptation, Physiological/genetics , Gene Expression Regulation, Fungal , Glycerol-3-Phosphate Dehydrogenase (NAD+)/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Motifs , Biological Evolution , Cell Hypoxia/genetics , Gene Duplication , Glycerol/metabolism , Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Osmolar Concentration , Osmotic Pressure , Phosphorylation , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics , Signal TransductionABSTRACT
The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. It is the primary target for antidepressants and stimulants, including "ecstasy" (3,4-methylenedioxymethamphetamine). SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and cGMP-dependent protein kinase (PKG). Here, we show that SERT was phosphorylated in a PKG Iα-dependent manner in vitro, but that SERT was not a direct substrate of PKG. We generated an analog-sensitive gatekeeper residue mutant of PKG Iα (M438G) that efficiently used the ATP analog N(6)-benzyl-ATP. This mutant, but not the wild type (WT) kinase, used the ATP analog to phosphorylate both a model peptide substrate as well as an established protein substrate of PKG (vasodilator-stimulated phosphoprotein). PKG Iα M438G effectively substituted for the WT kinase in stimulating SERT-mediated 5-hydroxytryptamine transport in cultured cells. Addition of either WT or mutant PKG Iα M438G to membranes containing SERT in vitro led to radiolabel incorporation from [γ-(33)P]ATP but not from similarly labeled N(6)-benzyl-ATP, indicating that SERT was phosphorylated by another kinase that could not utilize the ATP analog. These results are consistent with the proposed SERT phosphorylation site, Thr-276, being highly divergent from the consensus PKG phosphorylation site sequence, which we verified through peptide library screening. Another proposed SERT kinase, the p38 mitogen-activated protein kinase, could not substitute for PKG in this assay, and p38 inhibitors did not block PKG-dependent phosphorylation of SERT. The results suggest that PKG initiates a kinase cascade that leads to phosphorylation of SERT by an as yet unidentified protein kinase.
Subject(s)
Cyclic GMP-Dependent Protein Kinase Type I/metabolism , Cyclic GMP/metabolism , Serotonin Plasma Membrane Transport Proteins/metabolism , Serotonin/metabolism , Amino Acid Substitution , Biological Transport, Active/physiology , Cyclic GMP/genetics , Cyclic GMP-Dependent Protein Kinase Type I/genetics , HEK293 Cells , Humans , Mutation, Missense , Phosphorylation/physiology , Serotonin/genetics , Serotonin Plasma Membrane Transport Proteins/genetics , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The normal accumulation of ß-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate ß-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of ß-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted ß-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34(+) cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the ß-globin 3'untranslated region that has recently been implicated as a determinant of ß-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of ß-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of ß-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of ß-globin gene expression.
Subject(s)
Erythroid Cells/metabolism , Gene Expression Regulation , Heterogeneous-Nuclear Ribonucleoprotein D/metabolism , RNA Stability , RNA, Messenger/metabolism , Y-Box-Binding Protein 1/metabolism , beta-Globins/biosynthesis , 3' Untranslated Regions , Antigens, CD34/metabolism , Cells, Cultured , Electrophoretic Mobility Shift Assay , Fetal Blood/cytology , Gene Silencing , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein D0 , Heterogeneous-Nuclear Ribonucleoprotein D/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoprotein D/genetics , Humans , K562 Cells , Mutation , RNA, Small Interfering , Recombinant Proteins/metabolism , Ribonucleoproteins/metabolism , Y-Box-Binding Protein 1/antagonists & inhibitors , Y-Box-Binding Protein 1/genetics , beta-Globins/geneticsABSTRACT
C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed ß-synuclein as a potential CHK substrate, and Y127 in ß-synuclein as the preferential phosphorylation site. Using peptides derived from ß-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides ß-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.
Subject(s)
Protein-Tyrosine Kinases/chemistry , Structural Homology, Protein , beta-Synuclein/chemistry , src Homology Domains , Amino Acid Motifs , Amino Acid Sequence , Animals , CSK Tyrosine-Protein Kinase , Catalytic Domain , Cytosol/enzymology , Cytosol/metabolism , HEK293 Cells , Humans , Molecular Sequence Data , Peptide Library , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Rats , Substrate Specificity , beta-Synuclein/metabolism , src-Family KinasesABSTRACT
Phosphorylation is a universal mechanism for regulating cell behavior in eukaryotes. Although protein kinases target short linear sequence motifs on their substrates, the rules for kinase substrate recognition are not completely understood. We used a rapid peptide screening approach to determine consensus phosphorylation site motifs targeted by 61 of the 122 kinases in Saccharomyces cerevisiae. By correlating these motifs with kinase primary sequence, we uncovered previously unappreciated rules for determining specificity within the kinase family, including a residue determining P-3 arginine specificity among members of the CMGC [CDK (cyclin-dependent kinase), MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), and CDK-like] group of kinases. Furthermore, computational scanning of the yeast proteome enabled the prediction of thousands of new kinase-substrate relationships. We experimentally verified several candidate substrates of the Prk1 family of kinases in vitro and in vivo and identified a protein substrate of the kinase Vhs1. Together, these results elucidate how kinase catalytic domains recognize their phosphorylation targets and suggest general avenues for the identification of previously unknown kinase substrates across eukaryotes.