Identification of syntaxin‐1A sites of phosphorylation by casein kinase I and casein kinase II

Abstract

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 phosphorylates some synaptic vesicle associated proteins including SV2. In this report, we show that syntaxin-1A is phosphorylated 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 phosphorylation site, which is known to be phosphorylated in vivo. As syntaxin-1A plays a key role in the regulation of neurotransmitter 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 trafficking especially in endocytosis [2][3][4][5][6][7][8][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 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-a 1 , 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 involvement of syntaxin-1A in neurotransmitter release is well documented (reviewed in [16,17]). Regulated neurotransmitter 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 Ca 2+ 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 synaptobrevin to form a ÔSNARE complexÕ. Assembly of this complex is necessary ...

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

“…In this context it is worthwhile to notice that the CK1 family members α and δ have been localised to the synaptosome [45], [46]. They interact and phoshorylate several SNARE proteins, among them SV-2, syntaxin and snapin [47]–[49]. Phosphorylation of SNARE proteins has been suggested to influence their interaction with other proteins as well as vesicle transport and neurotransmitter release [48]–[55].…”

Analysis of Cell Type-specific Expression of CK1ε in Various Tissues of Young Adult BALB/c Mice and in Mammary Tumors of SV40 T-Ag-transgenic Mice

“…We observed strong cytoplasmic CK1ε positivity in all three parts of the pituitary gland, indicating that CK1ε might be involved in specific functions of the SNARE complex and in neurotransmitter release, like CK1a and -y (Gross et al 1995;Shimazaki et al 1996;Reisinger and Allerberger 1999;Kataoka et al 2000;Pyle et al 2000;Chheda et al 2001;Dubois et al 2002;Snyder et al 2006;Wolff et al 2006;Pozzi et al 2008). In the testis, CK1ε positivity of Leydig cells suggests regulatory functions of CK1ε in vesicle transport and hormone release (Knippschild et al 2005, and references therein).…”

A Plasma Membrane Syntaxin Is Phosphorylated in Response to the Bacterial Elicitor Flagellin

“…Although well documented in vitro (25,48), only a few reports exist on stimulated phosphorylation of a syntaxin in vivo. It is interesting that the Nterminal phosphorylation site of Syp122 (upstream of the coiled-coil H ABC domain) is similar to Ser-14 phosphorylation of human syntaxin 1A (49), but no stimulus is known yet that induces the latter phosphorylation.…”