Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals.
The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.
Xue
Jing
J
Cell Signalling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia.
Milburn
Peter J
PJ
Hanna
Bernadette T
BT
Graham
Mark E
ME
Rostas
John A P
JA
Robinson
Phillip J
PJ
D000818:Animals; D001665:Binding Sites; D001921:Brain; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D017729:Presynaptic Terminals / Q000201:enzymology*; D051381:Rats; D058112:Septins; D012694:Serine / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D010455:Peptides; D010768:Phosphoserine; D012694:Serine; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D020558:GTP Phosphohydrolases; D058112:Septins; C407343:septin 3