Synapsins as major neuronal Ca2+/S100A1-interacting proteins.
The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca(2+)-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca(2+)/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca(2+)-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca(2+)-dependent and Zn(2+)-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.
Heierhorst
J
J
St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy, VIC 3065, Australia. heier@ariel.its.unimelb.edu.au
Mitchelhill
K I
KI
Mann
R J
RJ
Tiganis
T
T
Czernik
A J
AJ
Greengard
P
P
Kemp
B E
BE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002451:Cell Compartmentation; D002846:Chromatography, Affinity; D008969:Molecular Sequence Data; D016501:Neurites / Q000737:chemistry; D009474:Neurons / Q000737:chemistry* / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D009418:S100 Proteins; D020539:Sequence Analysis, Protein; D016704:Synapsins / Q000302:isolation & purification / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D020033:Protein Isoforms; D009418:S100 Proteins; C110294:S100A1 protein; D016704:Synapsins; D000255:Adenosine Triphosphate; D002118:Calcium