Cyclin-dependent kinase 5 phosphorylation of human septin SEPT5 (hCDCrel-1) modulates exocytosis.
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.
Amin
Niranjana D
ND
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Zheng
Ya-Li
YL
Kesavapany
Sashi
S
Kanungo
Jyotshnabala
J
Guszczynski
Tad
T
Sihag
Ram K
RK
Rudrabhatla
Parvathi
P
Albers
Wayne
W
Grant
Philip
P
Pant
Harish C
HC
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000166:cytology / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D051360:Cyclin-Dependent Kinase 5 / Q000502:physiology*; D004622:Embryo, Mammalian; D005089:Exocytosis / Q000502:physiology*; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D009154:Mutation / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism; D058112:Septins; D012694:Serine / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism; D050827:Syntaxin 1 / Q000378:metabolism; D014162:Transfection / Q000379:methods
D018797:Cell Cycle Proteins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D050600:SNARE Proteins; D050827:Syntaxin 1; C089495:neuronal Cdk5 activator (p25-p35); D012694:Serine; D013006:Growth Hormone; D051360:Cyclin-Dependent Kinase 5; C109366:SEPTIN5 protein, human; D058112:Septins