LMTK1, a Novel Modulator of Endosomal Trafficking in Neurons

Abstract

Neurons extend long processes known as axons and dendrites, through which they communicate with each other. The neuronal circuits formed by the axons and dendrites are the structural basis of higher brain functions. The formation and maintenance of these processes are essential for physiological brain activities. Membrane components, both lipids, and proteins, that are required for process formation are supplied by vesicle transport. Intracellular membrane trafficking is regulated by a family of Rab small GTPases. A group of Rabs regulating endosomal trafficking has been studied mainly in nonpolarized culture cell lines, and little is known about their regulation in polarized neurons with long processes. As shown in our recent study, lemur tail (former tyrosine) kinase 1 (LMTK1), an as yet uncharacterized Ser/Thr kinase associated with Rab11-positive recycling endosomes, modulates the formation of axons, dendrites, and spines in cultured primary neurons. LMTK1 knockdown or knockout (KO) or the expression of a kinase-negative mutant stimulates the transport of endosomal vesicles in neurons, leading to the overgrowth of axons, dendrites, and spines. More recently, we found that LMTK1 regulates TBC1D9B Rab11 GAP and proposed the Cdk5/p35-LMTK1-TBC1D9B-Rab11 pathway as a signaling cascade that regulates endosomal trafficking. Here, we summarize the biochemical, cell biological, and physiological properties of LMTK1.

Keywords: Cdk5; LMTK1; Rab GAP; Rab11; endosome; neurite outgrowth; spine; vesicle transport.

Figure 1

Domain structure of the two isoforms of mouse lemur tail (former tyrosine) kinase 1 (LMTK1) and human LMTK1-3. Mouse LMTK1A and LMTK1B are alternatively spliced isoforms of LMTK1 that consist of 1,317 and 1,374 amino acids, respectively. The kinase domain (Kinase) is present in the N-terminal region with the long C-terminal tail. The amino acids Lys (K) and Asp (D) that are essential for the kinase activity are conserved. LMTK1A is a cytosolic protein that binds to the cytoplasmic surface of endosomal membranes via palmitoylation at N-terminal three cysteines (ccc). LMTK1B is a transmembrane protein with two transmembrane sequences at the N-terminus, which is also suggested to be palmitoylated at cysteines (ccc). The Cdk5 phosphorylation site is Ser34 of LMTK1A. Human LMTK1-3 is shown below the mouse LMTK1. Human LMTK1 is translated from the methionine in the first transmembrane sequence according to the amino acid sequences (NCBI accession number: NP_001073864.2). While the identity of the kinase domain is 92%, the identity of the full-length protein is 73% between mouse and human LMTK1, which is a relatively low value. Human LMTK2 (NCBI accession number: NP_055731.2) and LMTK3 (NCBI accession number: NP_001073903.1) are also shown below with % homology to human LMTK1.

Figure 2

Subcellular localization of LMTK1A and LMTK1B and the regulation of vesicle trafficking at the perinuclear endosomal recycling compartment (ERC) or ERGIC. (A) Immunostaining of wild type (WT) and kinase negative (KN) mutants of LMTK1A (1A) and LMTK1B (1B) expressed in CHO-K1 cells. SIM superresolution microscopic images in the right show the localization of LMTK1B KN, but not LMTK1A KN, on ER stained with a KDEL ER marker. Bars represent 20 and 5 μm, respectively (modified from Wei et al., 2020). (B) Proteins regulating endosomal trafficking bind to endosomal membranes through different binding modes. Cdk5 associated with the membranes through the myristoylation of the N-terminal glycine of its activator protein p35. LMTK1A accumulates on endosomes through the palmitoylation of cysteine residues in the N-terminal region. Active Rab11 binds to endosomes via the geranylgeranylation of the C-terminal cysteine. GDI is a GDP dissociation inhibitor, which binds to the GDP-bound form of Rabs in the cytosol. (C) LMTK1A and LMTK1B bind to recycling endosomes (RE) at the perinuclear ERC and regulate Rab11-dependent endosomal transport through TBC1D9B Rab11 GAP. LMTK1B is a transmembrane protein that would be synthesized on the ER and transported to the perinuclear ERC via vesicle transport. Because LMTK1B KN is localized to the elongated tubular ER, LMTK1B likely regulates vesicle transport at the ERGIC. The mechanism might be similar to transport at the perinuclear ERC; TBC1D20 and Rab1 are described as possible downstream candidates. EE represents the early endosome and Golgi represents the Golgi apparatus.

Figure 3

LMTK1 regulates axon outgrowth, dendrite arborization, and spine density through TBC1D9B Rab11 GAP. (A) The proper regulation of Rab11A is important for controlling the morphogenesis of neurons. The Cdk5-LMTK1A-TBC1D9B pathway is the upstream cascade of Rab11A endosomal regulation. Cdk5/p35 regulates LMTK1A activity by phosphorylation. LMTK1 activates TBC1D9B, leading to the inactivation of Rab11A activity. This regulation cascade operates on the endosome. (B) Downregulation of LMTK1 results in overactivation of Rab11A, leading to: (1) overgrowth of axons; (2) higher dendrite arborization; and (3) excessive dendritic spine formation.