Báo cáo khoa học: Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase

Roles of the SH2 and SH3 domains in the regulation of

neuronal Src kinase functions

Bradley R. Groveman1

, Sheng Xue2

, Vedrana Marin1

, Jindong Xu2

, Mohammad K. Ali1

,

Ewa A. Bienkiewicz1 and Xian-Min Yu1,2

1 Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, USA

2 Faculty of Dentistry, University of Toronto, Ontario, Canada

Introduction

Src family kinases (SFKs) are critically involved in the

regulation of many biological functions mediated

through growth factors, G-protein-coupled receptors

or ligand-gated ion channels. As such, SFKs have

become important targets for therapeutic treatments

[1,2]. Based on crystallographic studies of inactive and

active Src, the SH2 and SH3 domains are believed to

form a ‘regulatory apparatus’. Binding of the phos￾phorylated C-terminus to the SH2 domain and ⁄ or

binding of the SH2-kinase linker to the SH3 domain

inactivates SFKs [3–6]. It has been shown that

mutating Tyr527 to phenylalanine (Y527F) in the

Keywords

NMDA receptor regulation; phosphorylation;

Src; the SH2 domain; the SH3 domain

Correspondence

X.-M. Yu, 1115 West Call Street,

Tallahassee, FL 32306-4300, USA

Fax: +1 850 644 5781

Tel: +1 850 645 2718

E-mail: [email protected]

(Received 10 September 2010, revised

3 November 2010, accepted 6 December

2010)

doi:10.1111/j.1742-4658.2010.07985.x

Previous studies demonstrated that intra-domain interactions between

Src family kinases (SFKs), stabilized by binding of the phosphorylated

C-terminus to the SH2 domain and ⁄ or binding of the SH2 kinase linker to

the SH3 domain, lock the molecules in a closed conformation, disrupt the

kinase active site, and inactivate SFKs. Here we report that the up-regula￾tion of N-methyl-D-aspartate receptors (NMDARs) induced by expression

of constitutively active neuronal Src (n-Src), in which the C-terminus tyro￾sine is mutated to phenylalanine (n-Src ⁄Y535F), is significantly reduced by

dysfunctions of the SH2 and ⁄ or SH3 domains of the protein. Furthermore,

we found that dysfunctions of SH2 and ⁄ or SH3 domains reduce auto￾phosphorylation of the kinase activation loop, depress kinase activity, and

decrease NMDAR phosphorylation. The SH2 domain plays a greater regu￾latory role than the SH3 domain. Our data also show that n-Src binds

directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a

KD of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent,

and dysfunctions of the SH2 and ⁄ or SH3 domains do not significantly

affect the binding. These data indicate that the SH2 and SH3 domains may

function to promote the catalytic activity of active n-Src, which is impor￾tant in the regulation of NMDAR functions.

Structured digital abstract

l MINT-8074560: NR2A (uniprotkb:Q00959) binds (MI:0407) to n-Src (uniprotkb:P05480) by

surface plasmon resonance (MI:0107)

l MINT-8074641, MINT-8074668, MINT-8074679, MINT-8074693, MINT-8074813: n-Src

(uniprotkb:P05480) and n-Src (uniprotkb:P05480) phosphorylate (MI:0217) by protein kinase

assay (MI:0424)

l MINT-8074576, MINT-8074726, MINT-8074741, MINT-8074777: n-Src (uniprotkb:P05480)

phosphorylates (MI:0217) NR2A (uniprotkb:Q00959) by protein kinase assay (MI:0424)

Abbreviations

c-Src, cellular Src; NMDAR, N-methyl-D-aspartate receptor; n-Src, neuronal Src; SFK, Src family kinase; v-Src, viral Src.

FEBS Journal 278 (2011) 643–653 ª 2010 The Authors Journal compilation ª 2010 FEBS 643