Tài liệu Báo cáo Y học: Identification of syntaxin-1A sites of phosphorylation by casein kinase I and

Identification of syntaxin-1A sites of phosphorylation

by casein kinase I and casein kinase II

Thierry Dubois1

, Preeti Kerai2,*, Michele Learmonth1

, Andy Cronshaw1 and Alastair Aitken1

1

The University of Edinburgh, Division of Biomedical and Clinical Laboratory Sciences, UK; 2

Division of Protein Structure,

National Institute for Medical Research, London, UK

Casein kinases I (CKI) are serine/threonine protein kinases

widely expressed in a range of eukaryotes including yeast,

mammals and plants. They have been shown to play a role in

diverse physiological events including membrane trafficking.

CKIa is associated with synaptic vesicles and phosphory￾lates some synaptic vesicle associated proteins including

SV2. In this report, we show that syntaxin-1A is phospho￾rylated in vitro by CKI on Thr21. Casein kinase II (CKII)

has been shown previously to phosphorylate syntaxin-1A

in vitro and we have identified Ser14 as the CKII phospho￾rylation site, which is known to be phosphorylated in vivo.

As syntaxin-1A plays a key role in the regulation of neuro￾transmitter release by forming part of the SNARE (soluble

N-ethylmaleimide-sensitive factor attachment protein

receptor) complex, we propose that CKI may play a role in

synaptic vesicle exocytosis.

Keywords: CKI; CKII; syntaxin-1A; trafficking.

Casein kinase I (CKI) belongs to a family of serine/

threonine protein kinases with seven isoforms identified in

mammals (CKI a, b, d, e, c1, c2, and c3; reviewed in [1]).

The kinase domain is highly conserved between members

of the CKI family but unique N- and C-terminal tails

characterize each isoform. In yeast, the functions of CKI

have been much more extensively studied compared to

their mammalian counterparts. Recently, many reports

have linked yeast CKIs to cytokinesis and vesicle traffick￾ing especially in endocytosis [2–9]. Mammalian CKIs

appear to have similar functions and also have been

involved in DNA repair, circadian rhythms, and wnt

signaling. Like their yeast counterparts, CKIcs have been

implicated in cytokinesis and in membrane trafficking [10].

CKIa interacts with and phosphorylates the clathrin

adapter AP-3 [11] that is involved in endocytosis. CKIa

has been found to colocalize in neurones with synaptic

vesicle markers and to phosphorylate some synaptic vesicle

associated proteins including SV2 [12]. More importantly,

the phosphorylation of SV2 by CKI modulates its ability

to interact with synaptotagmin [13]. SV2 plays a role in

neurotransmitter release suggesting a role for CKI in this

biological process. We have recently identified centaurin-a1,

a protein shown to associate with presynaptic vesicular

structures [14], as a novel CKI partner [15].

In this report, we have identified syntaxin-1A as a

novel substrate for CKI, which further supports a role

for CKI in membrane trafficking. Indeed, the involve￾ment of syntaxin-1A in neurotransmitter release is well

documented (reviewed in [16,17]). Regulated neurotrans￾mitter secretion is the key step in synaptic transmission

and is the basis of intercellular communication in the

nervous system. Synaptic vesicle exocytosis is regulated

by Ca2+ and by a large number of proteins (reviewed in

[17,18]). Syntaxin-1A is associated with the presynaptic

membrane and associates with the plasma membrane

protein SNAP-25 and the synaptic vesicle protein syna￾ptobrevin to form a ÔSNARE complexÕ. Assembly of this

complex is necessary and may be sufficient to trigger

membrane fusion (reviewed in [17]).

Syntaxin-1A has been previously shown to be phospho￾rylated in vitro by casein kinase II (CKII) [19–21]. Although

the site of phosphorylation was not identified, Ser14 was

speculated to be the phosphorylation site as it is present

within a CKII consensus motif. Recently, it has been shown

using phospho-specific antibodies that Ser14 is phosphory￾lated in vivo [22]. However, the kinase responsible for this

was not identified. Here, we have identified the in vitro

phosphorylation sites within syntaxin-1A that are phospho￾rylated by both CKI and CKII. CKI and CKII phospho￾rylate the N-terminal domain of syntaxin-1A on Thr21 and

on Ser14, respectively.

MATERIALS AND METHODS

Materials

[c￾32P]ATP was from Amersham. Casein and histone H1

were purchased from Sigma. Recombinant casein kinase II

and the catalytic subunit of protein kinase A (PKA) were

from Calbiochem-Novabiochem. The plasmids encoding

the cytoplasmic domains of rat syntaxin-1A-pGEX4T-1

(1–265, 1–190 and 191–265) were obtained from T. Abe,

Niigata University, Japan. The rat munc18-1-pGEX2T

Correspondence to T. Dubois, Institut Curie – Section Recherche,

CNRS UMR 144, 26 rue d’Ulm, 75 248 Paris cedex 05, France.

Fax: + 33 1 42 34 63 77, Tel.: + 33 1 42 34 63 67,

E-mail: [email protected]

Abbreviations: CKI, casein kinase I; CKII, casein kinase II; PKA,

protein kinase A; SNARE, soluble N-ethylmaleimide-sensitive factor

attachment protein receptor.

*Present address: Wolfson Institute for Biomedical Research,

University College London, Cruciform Building, Gower Street,

London WC1E 6BT.

(Received 20 September 2001, revised 3 December 2001, accepted 4

December 2001)

Eur. J. Biochem. 269, 909–914 (2002) Ó FEBS 2002