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.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Microtubule-Associated Proteins/metabolism , Neuropeptides/metabolism , Amino Acid Substitution/physiology , Animals , Cloning, Molecular/methods , Cyclin-Dependent Kinase 5 , Doublecortin Domain Proteins , Doublecortin Protein , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Mice , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Mutation/physiology , Neuropeptides/chemistry , Neuropeptides/genetics , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
A rapid method to detect extracellular proteolytic activity around colonies of Cryptococcus neoformans was developed with tannic acid used to complex with residual protein in a solid medium. A survey was conducted with 32 isolates of C. neoformans var. gattii and 31 isolates of C. neoformans var. neoformans which were cultured on medium containing gelatin as the sole nitrogen source. The annulus of clearing around fungal colonies was > 1.2 mm in 24 (77%) isolates of C. neoformans var. neoformans compared with only 7 (22%) isolates of C. neoformans var. gattii. There was no difference in proteolytic activity between environmental and human clinical isolates of C. neoformans. However, there was a difference between the size of the annulus around animal isolates of C. neoformans var. neoformans and isolates of the same variety from other sources. The annuli around the 14 animal isolates were all >1.2 mm, while 7 (70%) of 10 human clinical isolates and only 3 (43%) of 7 environmental isolates were scored in the high proteinase range. A difference between the genetic types (as characterised by RAPD typing) of C. neoformans var. gattii was also evident with 17 (77%) of 22 VG-I isolates having a small annulus compared with only 1 (17%) of 6 VG-II and VG-III isolates with annuli of similar size. Relatively low proteinase production by C. neoformans var. gattii may reduce local and systemic spread of infection in mammalian hosts.
