TY - JOUR
T1 - Binding of Fyn to MAP-2c through an SH3 binding domain
T2 - Regulation of the interaction by ERK2
AU - Zamora-Leon, S. Pilar
AU - Lee, Gloria
AU - Davies, Peter
AU - Shafit-Zagardo, Bridget
PY - 2001/10/26
Y1 - 2001/10/26
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M107807200
DO - 10.1074/jbc.M107807200
M3 - Article
C2 - 11546790
AN - SCOPUS:0035955702
SN - 0021-9258
VL - 276
SP - 39950
EP - 39958
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -