title: Binding of Fyn to MAP-2c through an SH3 binding domain. Regulation of the interaction by ERK2.
abstract: Microtubule-associated protein 2 (MAP-2) isoforms are developmentally expressed in the nervous system and contain a number of functional domains. Adjacent to the first repeat of the microtubule-binding domain is an RTPPKSP motif for binding SH3 domains. To identify SH3-containing proteins that interact with MAP-2, transfections, filter overlay assays, glutathione S-transferase (GST)-mediated binding assays, co-immunoprecipitations and enzyme-linked immunosorbent assays were performed. Transfections of MAP-2a, MAP-2b, and MAP-2c constructs into COS7 cells, followed by incubation of the cell lysates with SH3-GST fusion proteins, determined that the strongest interaction was between MAP-2c and the non-receptor tyrosine kinase Fyn; however, MAP-2b and MAP-2c also bound to Grb2. Co-immunoprecipitation of Fyn and MAP-2c from human fetal homogenates confirmed the interaction in vivo. MAP-2 synthetic peptides spanning the RTPPKSP motif bound to Fyn, and the interaction was regulated by phosphorylation. Co-transfections with MAP-2c and the extracellular signal-regulated kinase 2 (ERK2) demonstrated that MAP-2c is threonine/serine-phosphorylated on its RTPPKSP motif and that threonine phosphorylation abolished the MAP-2c/Fyn binding. Kinase assays and co-transfection of MAP-2c and Fyn confirmed that Fyn tyrosine kinase phosphorylates MAP-2c. Thus, the activation of signaling pathways may regulate cytoskeletal dynamics by altering the state of phosphorylation of MAP-2 by both ERK2 and Fyn kinase.
authors.affiliation: Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Zamora-Leon
authors.forename: G
authors.initials: G
authors.lastname: Lee
authors.forename: P
authors.initials: P
authors.lastname: Davies
authors.forename: B
authors.initials: B
authors.lastname: Shafit-Zagardo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001665:Binding Sites; D001921:Brain / Q000196:embryology / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011994:Recombinant Proteins / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D013912:Threonine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D019950:Mitogen-Activated Protein Kinase 1; C049301:microtubule-associated protein-2 phosphatase; D010749:Phosphoprotein Phosphatases