CAPS activity in priming vesicle exocytosis requires CK2 phosphorylation.
CAPS (Ca(2+)-dependent activator protein for secretion) functions in priming Ca(2+)-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca(2+)-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca(2+) in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.
Nojiri
Mari
M
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Loyet
Kelly M
KM
Klenchin
Vadim A
VA
Kabachinski
Gregory
G
Martin
Thomas F J
TF
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D047390:Casein Kinase II / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D005089:Exocytosis; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D014230:Triazoles / Q000494:pharmacology
C485812:Cadps protein, rat; D002135:Calcium-Binding Proteins; D014230:Triazoles; D012694:Serine; C012771:benzotriazole; D047390:Casein Kinase II; D002118:Calcium