Ca2+-dependent phosphorylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18.
Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that binds with VAMP/synaptobrevin and SNAP-25 to form the SNARE complex. Modulation of syntaxin binding properties by protein kinases could be critical to control of neurotransmitter release. Using yeast two-hybrid selection with syntaxin-1A as bait, we have isolated a cDNA encoding the C-terminal domain of death-associated protein (DAP) kinase, a calcium/calmodulin-dependent serine/threonine protein kinase. Expression of DAP kinase in adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies demonstrate that DAP kinase binds to and phosphorylates syntaxin-1 at serine 188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A phosphorylation by DAP kinase or its S188D mutant, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation and is required for synaptic vesicle docking. Our results suggest that syntaxin is a DAP kinase substrate and provide a novel signal transduction pathway by which syntaxin function could be regulated in response to intracellular [Ca2+] and synaptic activity.
Synaptic Function Unit, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4154, USA.
Jin-Hua
JH
Tian
Sunit
S
Das
Zu-Hang
ZH
Sheng
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins; D001224:Aspartic Acid / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D051938:Munc18 Proteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000378:metabolism; D050827:Syntaxin 1; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D051017:Apoptosis Regulatory Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D012333:RNA, Messenger; C494208:SNAP25 protein, human; C494212:STX1A protein, human; C497937:STXBP1 protein, human; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; C497939:Stxbp1 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D001224:Aspartic Acid; D012694:Serine; D005982:Glutathione Transferase; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium