{"@context":"http://iiif.io/api/presentation/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/manifest.json","@type":"sc:Manifest","label":"Study of STAT3 Protein Interactions Using an In Vitro Binding Assay","metadata":[{"label":"dc.description.sponsorship","value":"This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree."},{"label":"dc.format","value":"Monograph"},{"label":"dc.format.medium","value":"Electronic Resource"},{"label":"dc.identifier.uri","value":"http://hdl.handle.net/11401/76514"},{"label":"dc.language.iso","value":"en_US"},{"label":"dc.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.abstract","value":"Transcription factors function as regulators of gene expression by controlling the activation of target genes.  As a member of the STAT (Signal Transducer and Activators of Transcription) family, STAT3 regulates gene expression responsible for controlling differentiation, cell survival, proliferation, migration, and apoptosis.  Activation of STAT3 conventionally occurs through tyrosine phosphorylation by JAK kinases stimulated following cytokine binding receptors.  Cytokines like IL-6 bind to cell surface receptors, leading to the activation of associated JAK kinases. JAKs phosphorylate the cytoplasmic domains of the receptors, and STAT3 is recruited through its SH2 domain resulting in its phosphorylation by JAKs.  It has been determined that the phosphorylation state of STAT3 does not impact its ability to enter the nucleus, but its precise mechanism of nuclear import remains to be completely understood. The importin \u00ce\u00b1 family is known to bind conventional nuclear localization signals (NLS) in proteins destined for the nucleus.  Importin \u00ce\u00b1 associates with importin \u00ce\u00b2, which mediates interaction with the nuclear pore complexes to transport the cargo.  The importin \u00ce\u00b1 family contains several members, and the purpose of this study is to determine if there is an interaction present between STAT3 and members of the importin \u00ce\u00b1 family, and to identify the region of STAT3 recognized.  The approach taken involved preparing tyrosine phosphorylated STAT3 (pTyr-STAT3) or unphosphorylated STAT3 (USTAT3)  from bacteria.  Recombinant clones were generated and STAT3 protein was expressed containing a Histidine and Maltose Binding Protein tag.  Different members of the importin alpha family including importin \u00ce\u00b11, \u00ce\u00b13, and \u00ce\u00b15 tagged with glutathione-S-transferase (GST) were expressed in bacteria cells and purified using glutathione beads.  The STAT3 proteins (USTAT3 and pTyr-STAT3) and purified GST-Importins were used in an in vitro binding assay. STAT3 was pulled down with amylose beads and analyzed for associated importins with GST antibodies. Multiple importin \u00ce\u00b1 proteins were able to interact with both USTAT3 and pTyr STAT3.  The phosphorylation of STAT3 impacts which importin \u00ce\u00b1 will recognize the protein.  I also cloned STAT3 mutants which contain N-terminal deletions. The STAT3 mutants include amino acids encoding 127-770, 320-770, 495-770, and 585-770.  These mutants were used to determine which region of the STAT3 is needed for binding to the importin \u00ce\u00b1 protein.  The DNA binding region of recombinant STAT3 appeared necessary for pTyr STAT3 and importin \u00ce\u00b11 and \u00ce\u00b15 interaction.   STAT3 is also suspected of playing a role in the ability of gammaherpesvirus to establish latency.  Following lytic replication, gammaherpesviruses are maintained in host B cells as viral DNA episomes. Latent viral gene expression is known to be able to promote the development of various cancers. The latent virus can subsequently reactivate, and this process requires the viral protein, replication and transcription activator (RTA). I tested the murine gammaherpesvirus 68 (MHV68) RTA protein for its ability to bind recombinant STAT3 protein in an in vitro binding assay. My results indicate that RTA binds STAT3 with a preference for tyrosine phosphorylated STAT3. Binding to tyrosine phosphorylated STAT3 N-terminal deletions demonstrated RTA association requires the DNA binding domain of STAT3.  My results demonstrate STAT3 interaction with the transport protein, importin \u00ce\u00b1, and a viral protein, RTA.  Understanding the interface of STAT3 with these proteins could lead to the development of potential therapeutics to inhibit the role of STAT3 in proliferative or viral diseases."},{"label":"dcterms.available","value":"2017-09-20T16:50:31Z"},{"label":"dcterms.contributor","value":"Reich, Nancy C"},{"label":"dcterms.creator","value":"Armstrong, Julie"},{"label":"dcterms.dateAccepted","value":"2017-09-20T16:50:31Z"},{"label":"dcterms.dateSubmitted","value":"2017-09-20T16:50:31Z"},{"label":"dcterms.description","value":"Department of Molecular and Cellular Pharmacology."},{"label":"dcterms.extent","value":"55 pg."},{"label":"dcterms.format","value":"Application/PDF"},{"label":"dcterms.identifier","value":"http://hdl.handle.net/11401/76514"},{"label":"dcterms.issued","value":"2015-12-01"},{"label":"dcterms.language","value":"en_US"},{"label":"dcterms.provenance","value":"Made available in DSpace on 2017-09-20T16:50:31Z (GMT). No. of bitstreams: 1\nArmstrong_grad.sunysb_0771M_12277.pdf: 1113425 bytes, checksum: 9eddabb0d8512ee5681de61ded9b4317 (MD5)\n  Previous issue date:    1"},{"label":"dcterms.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.subject","value":"Immunology"},{"label":"dcterms.title","value":"Study of STAT3 Protein Interactions Using an In Vitro Binding Assay"},{"label":"dcterms.type","value":"Thesis"},{"label":"dc.type","value":"Thesis"}],"description":"This manifest was generated dynamically","viewingDirection":"left-to-right","sequences":[{"@type":"sc:Sequence","canvases":[{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json","@type":"sc:Canvas","label":"Page 1","height":1650,"width":1275,"images":[{"@type":"oa:Annotation","motivation":"sc:painting","resource":{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/74%2F46%2F71%2F74467167247957475814490270412588922080/full/full/0/default.jpg","@type":"dctypes:Image","format":"image/jpeg","height":1650,"width":1275,"service":{"@context":"http://iiif.io/api/image/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/74%2F46%2F71%2F74467167247957475814490270412588922080","profile":"http://iiif.io/api/image/2/level2.json"}},"on":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json"}]}]}]}