Regulation of Exocytosis by Cyclin-dependent Kinase 5 via Phosphorylation of Munc18

Abstract

Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p protein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is derived from SNAP receptor (the soluble Nethylmaleimide-sensitive fusion protein)). However, this interaction inhibits vesicle SNARE interactions with syntaxin that are required for secretory vesicles to achieve competency for membrane fusion. As such, regulation of the interaction between Munc18a and syntaxin 1a may provide an important mechanism controlling secretory responsiveness. Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts directly with Munc18a in vitro, and utilizes Munc18a as a substrate for phosphorylation. We have now demonstrated that Cdk5 is capable of phosphorylating Munc18a in vitro within a preformed Munc18a⅐syntaxin 1a heterodimer complex and that this results in the disassembly of the complex. Using sitedirected mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr 574 . Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fraction and an increase of Cdk5 kinase activity. Inhibition of Cdk5 with olomoucine decreased evoked norepinephrine secretion from chromaffin cells, an effect not observed with the inactive analogue iso-olomoucine. The effects of olomoucine were independent of calcium influx as evidenced by secretory inhibition in permeabilized chromaffin cells and in cells under whole-cell voltage clamp. Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotinic agonist-induced secretory responses. Our data suggest a model whereby Cdk5 acts to regulate Munc18a interaction with syntaxin 1a and thereby modulates the level of vesicle SNARE interaction with syntaxin 1a and secretory responsiveness.

Cocaine-induced proliferation of dendritic spines in nucleus accumbens is dependent on the activity of cyclin-dependent kinase-5

“…There were no significant differences in mean dendritic length in any of the experimental conditions; this indicates that the observed changes in spine density do not result from dendritic lengthening or shortening. Together, these data demonstrate that the ability of chronic cocaine to increase NAc spine density is dependent on the activity of Cdk5.Previous studies have demonstrated that Cdk5 co-localizes with presynaptic proteins involved in exocytosis and synaptic transmission (Shuang et al, 1998;Fletcher et al, 1999) as well as post-synaptic D1 receptor-mediated second messenger cascades (Bibb et al, 1999a). Cdk5 co-localizes with DARPP-32 (Dopamine and cyclic AMP [cAMP] Regulated Phosphoprotein, Mr 32 kDa) in cell bodies and dendritic shafts in NAc (Bibb et al, 2001).…”

“…Previous studies have demonstrated that Cdk5 co-localizes with presynaptic proteins involved in exocytosis and synaptic transmission (Shuang et al, 1998;Fletcher et al, 1999) as well as post-synaptic D1 receptor-mediated second messenger cascades (Bibb et al, 1999a). Cdk5 co-localizes with DARPP-32 (Dopamine and cyclic AMP [cAMP] Regulated Phosphoprotein, Mr 32 kDa) in cell bodies and dendritic shafts in NAc (Bibb et al, 2001).…”

“…It is possible that Cdk5 is locally involved in spine outgrowth through an interaction with actin and its associated cytoskeletal proteins as in Cdk5-mediated axonal migration and dendritic morphogenesis (Humbert et al, 2000;Smith et al, 2001;Dhavan and Tsai, 2001). Presynaptically, Cdk5 binds to or phosphorylates several proteins that mediate vesicular exocytosis and synaptic transmission such as Munc-18, syntaxin 1A, and synapsin I (Matsubara et al, 1996;Fletcher et al, 1999). Thus, changes in Cdk5 activity within nerve terminals of NAc could alter neurotransmitter release that, in turn, could change spine density.…”

Cyclin-dependent Kinase-5 Is Involved in Neuregulin-dependent Activation of Phosphatidylinositol 3-Kinase and Akt Activity Mediating Neuronal Survival

“…Recently, Cdk5 has been suggested as contributing to the control of neuronal positioning in Reelin signaling during neural development (5) and to mediate neuronal guidance by regulating semaphorin-3A with Fyn kinase (6). Cdk5 and p35 have recently also been shown to regulate presynaptic and postsynaptic activity by phosphorylating Munc-18, amphiphysin, and the NR2A subunit of the N-methyl-D-aspartate receptor (7)(8)(9)(10)(11). Cdk5 activity also regulates dopamine signaling by phosphorylating DARPP-32 protein (12) and has been shown to up-regulate the expression of acetylcholine receptor at the neuromuscular junction (13).…”

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

“…Phosphorylated Munc18 have shown the reduced affinity for syntaxin1A but with some difference where phosphorylation by Cdk5 resulted in a greater reduction in syntaxin1A binding affinity (Shuang et al, 1996;Fletcher et al, 1999). The phosphorylation of SV proteins may alter physiological function.…”