Septin 2 phosphorylation: theoretical and mass spectrometric evidence for the existence of a single phosphorylation site in vivo.
Septins constitute a family of conserved proteins that are required for cytokinesis in a wide range of organisms. Most cells express a set of septin proteins and these are found to assemble into hetero-oligomeric septin complexes that appear filamentous. However, the mechanisms controlling the function and polymerization of septins are not known. We therefore examined the possibility that septins could be post-translationally modified by phosphorylation. We present herein a combined theoretical and experimental approach for the analysis of Septin 2 (Sept2) monophosphorylation in vivo. We purified and characterized the human recombinant Sept2, a 45-kDa protein, expressed from Sf21 insect cells. Analysis by matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry on the full-length protein sequence of wild-type Sept2 revealed a unique phosphorylation site at residue Ser248 in vivo, which is consistent with one of the twelve phosphorylation sites in the protein sequence theoretically predicted by the Netphos program. Additional predictions with the motif scan programs Scansite and Prosite suggest that the phosphorylation of wild-type Sept2 might be a potential substrate for casein kinase 2. Site-directed mutagenesis of residue 248 from serine to alanine abrogated this phosphorylation. The location of phosphorylation in Sept2 differs from the sites predicted for cGMP-dependent protein kinase (PKG) phosphorylation in Septin 3, raising the possibility that different septins may undergo distinct phosphorylation events that could control their functions in important cellular processes such as neurotransmission or cytokinesis.
She
Yi-Min
YM
Program in Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. yimin.she@utoronto.ca
Huang
Yi-Wei
YW
Zhang
Li
L
Trimble
William S
WS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D012694:Serine; D010744:Phosphoric Monoester Hydrolases