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dc.contributor.authorZamora-Leon, Sonia Pilar
dc.date.accessioned2018-07-12T17:32:55Z
dc.date.available2018-07-12T17:32:55Z
dc.date.issued2004
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 64-12, Section: B, page: 5945.;Advisors: Bridget Shafit-Zagardo.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3117012
dc.identifier.urihttps://hdl.handle.net/20.500.12202/677
dc.description.abstractMAP-2 belongs to the family of structural MAPs that binds microtubules and regulates cytoskeletal dynamics. All MAP-2 isoforms are developmentally regulated in the central nervous system, and contain functional domains that include the regulatory subunit of PKA and the repeats of the microtubule-binding-domain. Adjacent to the first repeat is the RTPPKSP motif for binding of SH3 domains. Transfections of MAP-2 cDNAs into COS7 cells, and incubation of cell lysates with SH3-GST fusion proteins determined that the strongest interaction was between MAP-2c and SH3-Fyn. ELISAs confirmed the binding of the SH3-domain of Fyn on the RTPPKSP motif on MAP-2c. A peptide containing the arginine to alanine mutation within the RTPPKSP motif abolished the interaction, confirming MAP-2 as a class I ligand for the SH3-domain of Fyn. A peptide containing a phosphorylated-threonine abolished the MAP-2/Fyn binding, indicating a phosphorylated-dependent regulation of the interaction. MAP-2c and ERK2 co-transfections demonstrated that ERK2 phosphorylated MAP-2c within the RT&barbelow;PPKS&barbelow;P motif on threonine/serine residues. Pull-downs with human fetal brain homogenates and Fyn protein showed that threonine-phosphorylated MAP-2c did not bind to Fyn.;Co-transfection of MAP-2c and Fyn followed by immunoprecipitation with a phosphotyrosine-antibody confirmed MAP-2c tyrosine-phosphorylation by Fyn. In vitro kinase reactions performed with wild-type and mutant MAP-2c, determined that Tyr-67 was phosphorylated by Fyn, generating a pYSN motif for the binding of the SH2-domain of Grb2. Filter-overlay assays and ELISAs confirmed the SH2-Grb2/MAP-2c interaction. GST-mediated binding assays with human fetal brain homogenates showed that MAP-2c bound to SH2-Grb2, indicating tyrosine-phosphorylation of MAP-2c in vivo.;Single transfections of COS7 cells with Fyn or MAP-2c resulted in a decrease in flat-rounded cells and an increase in the number of cells with processes. When Fyn and MAP-2c were co-transfected, the number of double-labeled transfected cells having short-spike processes decreased, and the number of cells with >2 processes increased. Immunostaining indicated that MAP-2c and Fyn partially co-localized in the cell body and processes.;The results support a role for MAP-2c as a scaffold protein recruiting Fyn, Grb2 and other signaling proteins. We postulate that the MAP-2c/Fyn interaction activates a novel intracellular signaling pathway that regulates cytoskeletal dynamics.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleInteraction of the microtubule-associated protein MAP -2c with Fyn, Grb2, and ERK2: Implications for a signaling cascade
dc.typeDissertation


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