GTP binding and hydrolysis kinetics of human septin 2.
Septins are a family of conserved proteins that are essential for cytokinesis in a wide range of organisms including fungi, Drosophila and mammals. In budding yeast, where they were first discovered, they are thought to form a filamentous ring at the bridge between the mother and bud cells. What regulates the assembly and function of septins, however, has remained obscure. All septins share a highly conserved domain related to those found in small GTPases, and septins have been shown to bind and hydrolyze GTP, although the properties of this domain and the relationship between polymerization and GTP binding/hydrolysis is unclear. Here we show that human septin 2 is phosphorylated in vivo at Ser218 by casein kinase II. In addition, we show that recombinant septin 2 binds guanine nucleotides with a Kd of 0.28 microm for GTPgammaS and 1.75 microm for GDP. It has a slow exchange rate of 7 x 10(-5) s(-1) for GTPgammaS and 5 x 10(-4) s(-1) for GDP, and an apparent kcat value of 2.7 x 10(-4) s(-1), similar to those of the Ras superfamily of GTPases. Interestingly, the nucleotide binding affinity appears to be altered by phosphorylation at Ser218. Finally, we show that a single septin protein can form homotypic filaments in vitro, whether bound to GDP or GTP.
Huang
Yi-Wei
YW
Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada.
Surka
Mark C
MC
Reynaud
Denis
D
Pace-Asciak
Cecil
C
Trimble
William S
WS
D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D012694:Serine; D006160:Guanosine Triphosphate; D047390:Casein Kinase II; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium