Binding of Fyn to MAP-2c through an SH3 binding domain: Regulation of the interaction by ERK2

S. Pilar Zamora-Leon, Gloria Lee, Peter Davies, Bridget Shafit-Zagardo

Research output: Contribution to journalArticle

41 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)39950-39958
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number43
DOIs
StatePublished - Oct 26 2001

Fingerprint

Microtubule-Associated Proteins
src Homology Domains
Mitogen-Activated Protein Kinase 1
Phosphorylation
Transfection
Assays
Proto-Oncogene Proteins c-fyn
Threonine
Glutathione Transferase
Immunoprecipitation
Phosphotransferases
Immunosorbents
Neurology
Microtubules
Serine
Nervous System
Protein Isoforms
Proteins
Fusion reactions
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Binding of Fyn to MAP-2c through an SH3 binding domain : Regulation of the interaction by ERK2. / Zamora-Leon, S. Pilar; Lee, Gloria; Davies, Peter; Shafit-Zagardo, Bridget.

In: Journal of Biological Chemistry, Vol. 276, No. 43, 26.10.2001, p. 39950-39958.

Research output: Contribution to journalArticle

Zamora-Leon, S. Pilar ; Lee, Gloria ; Davies, Peter ; Shafit-Zagardo, Bridget. / Binding of Fyn to MAP-2c through an SH3 binding domain : Regulation of the interaction by ERK2. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 43. pp. 39950-39958.
@article{2c646843fc2a487c92c23689505adfc0,
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.",
author = "Zamora-Leon, {S. Pilar} and Gloria Lee and Peter Davies and Bridget Shafit-Zagardo",
year = "2001",
month = "10",
day = "26",
doi = "10.1074/jbc.M107807200",
language = "English (US)",
volume = "276",
pages = "39950--39958",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "43",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=0035955702&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035955702&partnerID=8YFLogxK

U2 - 10.1074/jbc.M107807200

DO - 10.1074/jbc.M107807200

M3 - Article

C2 - 11546790

AN - SCOPUS:0035955702

VL - 276

SP - 39950

EP - 39958

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 43

ER -