Fig. 2

JIP1 is a substrate of Cdk5. A Bacterially expressed GST-tagged JIP1 was subjected to in vitro kinase assay by Cdk5/p25 and 1 μCi of [γ-³²P] ATP with or without Cdk5 inhibitor Roscovitine (Rosco) at 30°C for 30 min. The proteins were separated by SDS-PAGE for autoradiography. B, C Recombinant His-JIP1 was incubated with Cdk5/p25 with or without 20 μM of Roscovitine for the indicated duration. The proteins were subjected to western blot with anti-phospho-Serine (p-Ser) (B) or anti-phospho-Threonine (p-Thr). * and ** indicate full length of JIP1 and truncated form of JIP1 from C-terminus, respectively (C). Data representative of three independent experiments. D Schematic of domains of JIP1. Alignment of JIP1 protein sequence from different species analyzed by ClustaIW2 to show three conserved sites optimal for Cdk5 phosphorylation. E–G Identification of Cdk5 phosphorylation sites in JIP1. Ser197, Thr 205, and Ser 235 were all replaced with Ala. An in vitro kinase assay was carried out with purified active Cdk5/p35 incubated with WT-JIP1 (JIP1) or its Ala mutants, S197A (E), T205A (F), and S235A (G) and examined by western blot analysis using anti-phospho-Serine (p-Ser) or anti-phospho-Threonine (p-Thr). Data representative of three independent experiments. H Total cell lysate from p35 WT and KO neurons at DIV 3 was subjected to western blot analysis with custom pJIP1@T205 antibody (bands at ~100 and ~120 kDa, pJIP1@T205). Both bands from p35 WT or KO neurons were used for the measurement of JIP1 phosphorylation by Cdk5. I Quantification of phospho-JIP1 T205 levels relative to p35 WT neurons. WT, n=8 and KO, n=5, “n” equals the number of animals. Student’s t-test was used for statistical analysis and the data are presented as mean ± SEM. **** p < 0.0001