Figure 1.

CDK5 directly phosphorylates Cort in vitro and this phosphorylation alters the interaction between Cort and F-actin. (A) Dyn 1 or Cort was incubated with or without 1 µg/ml CDK5 in the presence of [γ³²P]-ATP. ³²P-Dyn1 and ³²P-Cort was then analyzed by SDS-PAGE and visualized by autoradiography (upper panels). Total protein was stained with SYPRO Orange (bottom panels). Proteins were incubated without CDK5 as a control. (B) Sites phosphorylated by CDK5 in Cort (T145 and T219) identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. These residues are located in the second and fourth Cort R units. (C) Verification of the identified phosphorylation sites in Cort. WT Cort (left) and a Cort mutant in which the T145/T219 phosphorylation sites were substituted with alanine (right) at 3.2 µg were incubated with CDK5/p35 and [γ³²P]-ATP for 1 h. Total Cort stained with coomassie brilliant blue is presented (bottom). ³²P-Cort was detected by autoradiography. (D) In vitro actin polymerization in the presence of WT Cort or the T145D, T219D, or T145DT219D mutant. Actin polymerization was initiated by adding K⁺ and Mg²⁺. Actin filament formation was measured by determining the change in the fluorescence intensity of pyrene-actin. (E) F-actin formation at 1 h following initiation of actin polymerization with or without WT Cort or the T145D, T219D or T145DT219D mutant was measured by analyzing pyrene-actin fluorescence. Actin alone was used as a control. The mean ± standard error of the mean of 3-6 independent experiments is plotted. ^*P<0.05. (F) Phosphorylation of Cort by CDK5 decreases its actin-crosslinking capability. (a) Actin alone appeared as individual filaments. (b) Tightly cross-linked F-actin in the presence of WT Cort (+Cort WT). Partially assembled filaments were observed in the presence of the (c) T145D, (d) T219D or (e) T145DT219D mutants. Preformed F-actin (3.3 µM) was incubated with or without the indicated proteins (5 µM each; b-e). F-actin was stained with Alexa Fluor 488-conjugated phalloidin. Scale bar, 30 µm. CDK5, cyclin-dependent kinase 5; Dyn 1, dynamin 1; Cort, cortactin; ³²P, phosphorylated; WT, wild-type; NTA, N-terminal acidic region; R, repeat; PR, a proline-rich region; SH3, Src homology 3.