title: Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5.
abstract: Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.
authors.affiliation: Children's Medical Research Institute, Locked Bag 23, Wentworthville, NSW 2145, Australia.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Graham
authors.forename: Patricia
authors.initials: P
authors.lastname: Ruma-Haynes
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Capes-Davis
authors.forename: Joanne M
authors.initials: JM
authors.lastname: Dunn
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Tan
authors.forename: Valentina A
authors.initials: VA
authors.lastname: Valova
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
authors.forename: Peter L
authors.initials: PL
authors.lastname: Jeffrey
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: C000714328:DCX protein, human; C000714847:Dcx protein, mouse; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D008869:Microtubule-Associated Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D051360:Cyclin-Dependent Kinase 5; C495906:Cdk5 protein, mouse; D018844:Cyclin-Dependent Kinases