Doublecortin Association with Actin Filaments Is Regulated by Neurabin II

Abstract

Mutations in the human Doublecortin (DCX) gene cause X-linked lissencephaly, a neuronal migration disorder affecting the neocortex and characterized by mental retardation and epilepsy. Because dynamic cellular asymmetries such as those seen in cell migration critically depend on a cooperation between the microtubule and actin cytoskeletal filament systems, we investigated whether Dcx, a microtubule-associated protein, is engaged in cytoskeletal cross-talk. We now demonstrate that Dcx co-sediments with actin filaments (F-actin), and using light and electron microscopy and spin down assays, we show that Dcx induces bundling and crosslinking of microtubules and F-actin in vitro. It has recently been shown that binding of Dcx to microtubules is negatively regulated by phosphorylation of the Dcx at Ser-47 or Ser-297. Although the phosphomimetic green fluorescent protein (GFP)-Dcx S47E transfected into COS-7 cells had a reduced affinity for microtubules, we found that pseudophosphorylation was not sufficient to cause Dcx to bind to F-actin. When cells were co-transfected with neurabin II, a protein that binds F-actin as well as Dcx, GFP-Dcx and to an even greater extent GFP-Dcx S47E became predominantly associated with filamentous actin. Thus Dcx phosphorylation and neurabin II combinatorially enhance Dcx binding to F-actin. Our findings raise the possibility that Dcx acts as a molecular link between microtubule and actin cytoskeletal filaments that is regulated by phosphorylation and neurabin II.

The DCX Superfamily 1: Common and Divergent Roles for Members of the Mouse DCX Superfamily

“…Nevertheless, some results implicated an interaction of DCX with the actin cytoskeleton, directly or via Neurabin 2. 19,20 Interestingly, BAC26042 and, to some extent the DCX domains of DCLK2 localized to the cell periphery, at sites reminiscent of lamellipodia (Fig. 5E-H and Fig.…”

“…However, although neurabin 2 regulates the interaction of DCX with actin, DCX has the capability of directly interacting with actin and affecting its polymerization. 19,20 It is possible that additional DCX proteins interact directly with actin, alternatively additional mediators and regulators may exist.…”

“…Neurabin 2 connects DCX to actin microfilaments. 19,20 In addition, neurabin 2 binds to protein phosphates 1 (PP1), thereby regulating site-specific dephosphorylation of DCX (Shmueli et al, in press). JIP1/2 (JNK Interacting Protein 1 or 2) acts as a scaffold for both DCX and one of the kinases that phosphorylates it: JNK (cJun-N-terminal Kinase).…”

Phosphorylation of Doublecortin by Protein Kinase A Orchestrates Microtubule and Actin Dynamics to Promote Neuronal Progenitor Cell Migration

“…Interestingly, mutation of Ser-47 to Arg was found in a lissencephaly patient (15). In addition, the mutation of Ser-47 to Glu, which acts as a phospho-mimic mutation, promotes the interaction of DCX with an actin-binding protein, neurabin2/ spinophilin (25). These results highlighted DCX as a candidate downstream molecule in the G protein-signaling pathways that may act as a key regulator in orchestrating microtubule and actin dynamics.…”

The tumour suppressor function of the scaffolding protein spinophilin

“…In vitro assays showed that DCX also binds to and bundles F-actin, suggesting that the protein crosslinks microtubules and F-actin. The distribution of DCX between the two cytoskeletons can be regulated by Spn and by phosphorylation of DCX and it was proposed that Spn could localize and enhance the binding of phosphorylated DCX to F-actin (Tsukada et al, 2005). Several studies have shown that Spn preferentially binds to PP1γ1 and PP1α isoforms in brain extracts (MacMillan et al, 1999;Terry-Lorenzo et al, 2002;Carmody et al, 2004).…”