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

Abstract

Doublecortin (DCX) is expressed in young neurons and functions as a microtubule-associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migration. Previous in vitro phosphorylation experiments revealed that cyclin-dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5-/- mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP-DCX-WT, or GFP-DCX-S332A and analyzed neurite extension and migration. Introduction of GFP-DCX-WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP-DCX-S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5-/- brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function.

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…S1C). These cytoskeletal JNK substrate proteins include those associating with microtubules (DCX [260][261][262], MAP1B [180,263], MAP2 [264], Tau [265], and WDR62 [213]), the actin cytoskeleton (MARCKSL1 [266] and SMTL2 [267]), or focal adhesions (paxillin [268,269] and ␤-catenin [270][271][272]). In addition, JNK-mediated phosphorylation of additional proteins may also direct vesicular transport (through phosphorylation of JIP1 and ␤-APP [104,217]), as well as the exocytosis of specialized, Glut4-containing vesicles (through insulin-stimulated phosphorylation of IRS1 and IRS2 [273,274]).…”

“…DCX is a critical regulator of neurogenesis and postmitotic neuroblast migration, capable of binding to JNK directly and also containing numerous JNK phosphorylation sites (261,262). Interestingly, most of the sites located N-terminally and Cterminally from the microtubule-binding tandem doublecortin domains are also conserved in the closely related doublecortinlike kinases 1 and 2 (DCLK1 and DCLK2), but not in the moredivergent DCLK3.…”

Dyrk kinases regulate phosphorylation of doublecortin, cytoskeletal organization, and neuronal morphology

“…As a MT-associated protein, DCX is involved in MT polymerization, bundling and stabilization (Kim et al, 2003; Schaar et al, 2004; Bielas et al, 2007). DCX can be phosphorylated by at least 4 different kinases including Cdk5, PKA, Jnk, and GSK3β (Gdalyahu et al, 2004; Graham et al, 2004; Schaar et al, 2004; Tanaka et al, 2004; Bilimoria et al, 2010; Jin et al, 2010); our results add Dyrks to this list. Phosphorylation of DCX at specific sites can either increase or decrease its affinity to MTs and result in distinct functional effects (Reiner et al, 2004).…”

“…Both the N- and C- termini of DCX can interact with MTs (Kim et al, 2003) and this interaction is regulated by DCX phosphorylation at multiple sites. Several kinases, including Cyclin-dependent kinase 5 (Cdk5), c-Jun N-terminal kinase (Jnk), protein kinase A (PKA) and Glycogen synthase kinase 3β (Gsk3β) have been shown to phosphorylate DCX (Graham et al, 2004; Reiner et al, 2004; Tanaka et al, 2004; Bilimoria et al, 2010; Jin et al, 2010). …”

A critical and previously unsuspected role for doublecortin at the neuromuscular junction in mouse and human

“…In support of this hypothesis, Dcx has been involved in vesicle transport and sorting based on specific partners. These include the µ subunits of clathrin adaptor complexes [45] and two kinases, Cdk5 and Jnk [37,46]. Dcx has also been shown to interact with and regulate endocytosis and surface distribution of the phosphorylated neurofascin adhesion molecule [47,48].…”