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 (cyclindependent 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 32 Plabelled, cdk5-phosphorylated DCX using a combination of offline 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 32 P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/prolinerich 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. Sitedirected 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.

JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration

“…Ser297 of DCX was shown to be phosphorylated by Cdk5 in vivo (Tanaka et al, 2004). A previous in vitro study demonstrated that Cdk5 phosphorylates multiple sites within the Cterminus of DCX, including Ser332 (Graham et al, 2004). Unexpectedly, we found elevated phosphorylation levels of DCX at Ser332 in the Cdk5À/À mouse brain.…”

“…Experiments by Graham and colleagues demonstrated that DCX was phosphorylated by Cdk5 in vitro and identified multiple phosphorylation sites, including S332, by mass spectrometry (Graham et al, 2004). To date, Ser297 (S297) is the only reported in vivo Cdk5 phosphorylation site of DCX, and phosphorylation levels of DCXS297 are decreased in the Cdk5À/À mouse brain (Tanaka et al, 2004;T.O., unpublished data).…”

“…1) in vitro and in mouse brain (Gdalyahu et al, 2004). Graham et al (2004) reported that Cdk5 phosphorylates multiple sites within the DCX C-terminal region in vitro, including Ser332 (S332). However, among them, only Ser297 was reported to be phosphorylated in the mouse brain (Tanaka et al, 2004).…”

Missense mutations resulting in type 1 lissencephaly

“…JNK phosphorylates DCX on at least three different sites, T321, T331 and S334 [93]. Cdk5 phosphorylates DCX on S297 [115] or, as other results indicate, S28, and S339 as the main phosphorylated sites [118], and PKA and/or the MARK kinase phosphorylates DCX on several sites, with the most significant one being S47 [116]. In vitro analysis indicated that DCXs phosphorylation by Cdk5, PKA and MARK reduced the affinity of DCX to MTs [115,116].…”

The in Vivo Phosphorylation and Glycosylation of Human Insulin-like Growth Factor-binding Protein-5

“…Tryptic Digestion and HPLC-Approximately 15 g of purified 32 Plabeled IGFBP-5 from T47D cells was prepared and purified by HPLC as described previously (19). The protein was dissolved in Laemmli sample buffer and heated to near boiling for 5 min.…”