Chemical Rescue of Protein Kinases for Cell Signaling Applications
用于细胞信号传导应用的蛋白激酶的化学救援
基本信息
- 批准号:10331818
- 负责人:
- 金额:$ 6.67万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-02-01 至 2022-12-31
- 项目状态:已结题
- 来源:
- 关键词:Active SitesAddressAdoptedAreaBackBiologyBiomedical ResearchCRISPR/Cas technologyCatalytic DomainCell LineCell modelCell physiologyCellsCellular biologyChemicalsComplementCyclic AMP-Dependent Protein KinasesDevelopmentDiseaseEngineeringEnzymatic BiochemistryEnzymesFunctional disorderGenesGoalsHealthHumanImidazoleImpairmentIn VitroJurkat CellsKineticsLaboratoriesLaboratory ChemicalsLifeLigandsMalignant NeoplasmsMammalian CellMass Spectrum AnalysisMethodsModelingModernizationMutateMutationNeuroblastomaOncogenicPathway interactionsPhenotypePhosphorylationPhosphotransferasesPhysiologicalPhysiologyPlayPoint MutationPositioning AttributeProtein AnalysisProtein KinaseProteinsProteomicsPublishingReceptor Protein-Tyrosine KinasesRegulationResearchRoleSignal PathwaySignal TransductionSignaling ProteinSpecificityStructureSystemT-Cell ActivationT-LymphocyteTechniquesTechnologyTrainingWorkZAP-70 Geneanaplastic lymphoma kinasebasecareercellular targetingcomparativeexperimental studyextracellularhuman diseaseinterestmutantphosphoproteomicspreservationprotein-tyrosine kinase c-srcrapid techniquerational designreceptorskillssmall moleculesmall molecule librariessuccessvirtual
项目摘要
ABSTRACT
This is a new F32 application to develop and apply chemical rescue technology to the functional analysis of
protein kinases. Protein kinases regulate virtually every aspect of cellular physiology in health and disease
through phosphorylation of their substrates, typically on Tyr and Ser/Thr residues. Despite intense efforts even
the most “well-studied” protein kinases remain incompletely understood. Methods to study protein kinase
signaling in cellular contexts are urgently needed, but unfortunately, few methods exist for activating specific
kinases in living cells. The techniques that are available for this purpose require extensive engineering, which
raises concerns about preservation of natural regulatory mechanisms and has likely limited their widespread
application. Previously, our lab developed chemical rescue as a method for rapidly activating protein Tyr kinases
in living cells. A single point mutant is generated within the catalytic site, which renders the kinase catalytically
inactive without disturbing its structure or regulation. A small molecule is then added which complements this
mutation and rapidly switches the kinase back on in a reversible manner. This technique is conceptually simple
and has been proven useful in dissecting cell signaling of the protein Tyr kinases Csk, Src, and Abl. However,
due to the historical difficulty with generating point mutants in mammalian cells, chemical rescue has not yet
been widely applied. Additionally, a chemical rescue technique has not been developed for Ser/Thr kinases,
which make up approximately 80% of the human kinome. This proposal seeks to overcome both of these
limitations. By using CRISPR/Cas9 gene editing to introduce the required point mutations in model cell lines, we
will use chemical rescue to identify substrates and cell signaling pathways of two enigmatic Tyr kinases. In Aim
1, ALK, a receptor Tyr kinase commonly mutated in cancer but with unclear physiological function will be
analyzed in its wild-type and oncogenic forms to identify its cellular targets. In Aim 2, ZAP70, a nonreceptor Tyr
kinase essential for T-cell activation but with very few known substrates will be investigated. For these two aims,
we will use an unbiased mass spectrometry based phosphoproteomic approach to identify substrate proteins
and activated signaling pathways. In Aim 3, we will develop proof-of-concept for chemical rescue of Ser/Thr
kinases using the model protein Akt1. Collectively, this work will open new doors to investigating kinase signaling
and advance our understanding of three key enzymes in biomedical research. In addition, this proposal builds
on the chemical biology background of the PI and promises to greatly broaden the PI's skills in enzymology,
mass spectrometry, and cell biology. It is anticipated that the training plan outlined here will position the PI for
success in an independent academic research career.
摘要
项目成果
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