Cyclin-Dependent Kinase 5 Phosphorylation of Human Septin SEPT5 (hCDCrel-1) Modulates Exocytosis

Abstract

Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in the nervous system, where it is involved in neuronal migration, synaptic transmission, and survival. The role of Cdk5 in synaptic transmission is mediated by regulating the cellular functions of presynaptic proteins such as synapsin, Munc18, and dynamin 1. Its multifunctional role at the synapse is complex and probably involves other novel substrates. To explore this possibility, we used a yeast two-hybrid screen of a human cDNA library with p35 as bait and isolated human septin 5 (SEPT5), known also as hCDCrel-1, as an interacting clone. Here we report that p35 associates with SEPT5 in GST (glutathione S-transferase)-pull-down and coimmunoprecipitation assays. We confirmed that Cdk5/p35 phosphorylates SEPT5 in vitro and in vivo and identified S327 of SEPT5 as a major phosphorylation site. A serine (S)-to-alanine (A) 327 mutant of SEPT5 bound syntaxin more efficiently than SEPT5 wild type. Additionally, coimmunoprecipitation from synaptic vesicle fractions and Cdk5 wild-type and knock-out lysates showed that phosphorylation of septin 5 by Cdk5/p35 decreases its binding to syntaxin-1. Moreover, mutant nonphosphorylated SEPT5 potentiated regulated exocytosis more than the wild type when each was expressed in PC12 cells. These data suggest that Cdk5 phosphorylation of human septin SEPT5 at S327 plays a role in modulating exocytotic secretion.

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator, Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

“…Kinase assays were performed in the same buffer containing 1 mM DTT, 0.1 mM ATP, and 0.185 MBq of [␥- 32 P]ATP with 20 g of histone H1 as the substrate. Phosphorylation was performed in a final volume of 50 l, incubated at 30°C for 60 min, and stopped by the addition of 10% SDS sample buffer and heating at 95°C for 5 min.…”

“…It reduces Tau hyperphosphorylation and protects transfected HEK cells and neurons from apoptosis induced by Cdk5-p25 (23,27,(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). To produce a smaller and more effective inhibitory peptide of Cdk5-p25 for potential therapeutic use, we designed and constructed five short peptides in GST vectors, as shown in Fig.…”

“…p5 Inhibits Both Cdk5-p25 and Cdk5-p35 Activities in Vitro; a Dose-dependent Study-In a dose-dependent kinase assay, we compared the effective inhibitory concentration of p5 for both the Cdk5-p25 and Cdk5-p35 complexes. His-p5 peptide was added at different concentrations to the in vitro assay containing 100 M [␥ 32 -P]ATP and histone H1. The results are shown in Fig.…”

Mitotic Regulation of SEPT9 Protein by Cyclin-dependent Kinase 1 (Cdk1) and Pin1 Protein Is Important for the Completion of Cytokinesis

“…Based on the location of the Pin1-binding site near the extreme N terminus of SEPT9, it is tempting to speculate that the N-terminal region of septins may serve as an important regulatory domain. Consistent with this, phosphorylation near the extreme N terminus of mouse SEPT5 by Cdk5 regulates association with syntaxin-1 in the brain (39,40). In addition, phosphorylation near the extreme C terminus of the yeast septin Cdc3p by Cdc28p is required for septin ring disassembly (41), suggesting that the C-terminal domain of septins may also serve a regulatory role.…”

Septin 8 is an interaction partner and in vitro substrate of MK5

“…In previous studies, several septins have been shown to be phosphorylated in vitro and/or in vivo, including SEPT1, SEPT2, SEPT3 and SEPT5 [40][41][42][43][44] . After describing the interaction between two proteins, we wanted to know whether MK5 possesses catalytic activity towards SEPT8.…”