LMTK1/AATYK1 Is a Novel Regulator of Axonal Outgrowth That Acts via Rab11 in a Cdk5-Dependent Manner

Abstract

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/ threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.

Cdk5 Regulation of the GRAB-Mediated Rab8-Rab11 Cascade in Axon Outgrowth

“…wt Rab11A or ca Rab11A overexpression induced approximately a twofold increase in axonal length, whereas ci Rab11A did not (Fig. 2 F, G), consistent with our previous report (Takano et al, 2012).…”

“…Membrane trafficking is a dynamic process involving vesicle formation in donor compartments for transport and their delivery, tethering, and docking to their acceptor membranes (Takai et al, 2001;Zerial and McBride, 2001;Stenmark, 2009). The small GTPases of the Rab family regulate these processes.…”

“…A deficiency in Rab8 activity results in decreased neurite outgrowth in embryonic hippocampal neurons (Huber et al, 1995). In the endocytic pathway, which requires several Rabs (e.g., Rab5 and Rab11), plasma membranes are endocytosed and then processed as recycling vesicles, which are delivered back to the cell surface (Takai et al, 2001;Zerial and McBride, 2001). Rab11 regulates endosome transport to axon tips (Ascano et al, 2009;Eva et al, 2010;Takano et al, 2012).…”

“…In the endocytic pathway, which requires several Rabs (e.g., Rab5 and Rab11), plasma membranes are endocytosed and then processed as recycling vesicles, which are delivered back to the cell surface (Takai et al, 2001;Zerial and McBride, 2001). Rab11 regulates endosome transport to axon tips (Ascano et al, 2009;Eva et al, 2010;Takano et al, 2012). However, whether these endocytic and exocytic Rabs act independently or cooperatively during axon outgrowth is not known.…”

“…Cdk5 is active mainly in postmitotic neurons and is involved in a variety of neuronal functions (e.g., neuronal migration and axonal outgrowth) (Smith and Tsai, 2002;Kawauchi, 2014). Recently, we have shown that Cdk5-p35 regulates trafficking of Rab11A-positive recycling endosomes via lemur kinase 1A (LMTK1A) during axon outgrowth (Takano et al, 2012(Takano et al, , 2014. However, other molecular mechanisms regulated by Cdk5-p35 and involving membrane delivery to supoport axonal outgrowth are currently unknown.…”