Trk Activation of the ERK1/2 Kinase Pathway Stimulates Intermediate Chain Phosphorylation and Recruits Cytoplasmic Dynein to Signaling Endosomes for Retrograde Axonal Transport

Abstract

The retrograde transport of Trk containing endosomes from the axon to the cell body by cytoplasmic dynein is necessary for axonal and neuronal survival. We investigated the recruitment of dynein to signaling endosomes in rat embryonic neurons and PC12 cells. We identified a novel phospho-serine on the dynein intermediate chains (IC) and we observed a time-dependent neurotrophin-stimulated increase in intermediate chain phosphorylation on this site in both cell types. Pharmacological studies, over-expression of constitutively active MEK, and an in vitro assay with recombinant proteins demonstrated that the intermediate chains are phosphorylated by the MAP kinase ERK1/2, extracellular-signal-regulated kinase, a major downstream effector of Trk. Live cell imaging with fluorescently-tagged IC mutants demonstrated that the dephospho-mimic mutants had significantly reduced co-localization with Trk and Rab7, but not a mitochondrial marker. The phosphorylated intermediate chains were enriched on immuno-affinity purified Trk containing organelles. Inhibition of ERK reduced the amount of phospho-IC and the total amount of dynein that co-purified with the signaling endosomes. In addition, inhibition of ERK1/2 reduced the motility of Rab7 and TrkB containing endosomes and the extent of their co-localization with dynein in axons. NGF-dependent survival of sympathetic neurons was significantly reduced by the over-expression of the dephospho-mimic mutant IC-1B-S80A, but not WT IC-1B, further demonstrating the functional significance of phosphorylation on this site. These results demonstrate that neurotrophin binding to Trk initiates the recruitment of cytoplasmic dynein to signaling endosomes through ERK1/2 phosphorylation of intermediate chains for their subsequent retrograde transport in axons.

Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

“…Dynein IC1 can also interact with the G␤␥ subunit, which plays a central role in G-protein signaling and is involved in cell division, vesicle trafficking, and signal integration (74). The Trk-ERK1/2-stimulated phosphorylation of dynein IC is required for regulation of dynein activity and affects certain cellular events, such as microtubule-dependent sliding and mitosis (75)(76)(77). The middle part of IC1 consists of repetitive sequences homologous to the sequence of the nucleoside diphosphate kinase (NDPK), indicating that IC1 may function as an NDPK that regulates RNA, DNA, and polysaccharide synthesis (78)(79)(80).…”

Interaction of SQSTM1 with the motor protein dynein: SQSTM1 is required for normal dynein function and trafficking

“…Phosphorylation of dynein subunits has also been implicated in dynein regulation (Dillman and Pfister, 1994;Lin et al, 1994;Vaughan et al, 2001), with some examples showing that phosphorylation can inhibit or enhance dynein binding to organelles or some cargoes (Mitchell et al, 2012). Atypical PKC (aPKC) can phosphorylate DICs, regulating their binding with paxillin, thus controlling the focal adhesion disassembly (Rosse et al, 2012).…”

“…Two dynein subunits, intermediate chains 1 and 2 (DIC1 and DIC2, also known as DYNC1I1 and DYNC1I2) have been implicated in dynein binding to both aggregated proteins (Xu et al, 2013) and cellular organelles (Pfister et al, 2006;Mitchell et al, 2012) for retrograde transport to aggresomes. The adaptor protein SQSTM1 (Okatsu et al, 2010) has also been implicated in aggresome formation by contributing to the transport of both ubiquitylated (Behl, 2011) and nonubiquitylated proteins (Watanabe and Tanaka, 2011).…”

“…DICs have been implicated in the retrograde transport of Trkcontaining signaling endosomes (Mitchell et al, 2012). The basic endosomal maturation program begins with endocytosis of cargo from the plasma membrane into endocytic vesicles, which then fuse into early endosomes (Huotari and Helenius, 2011).…”

Microtubule-based transport – basic mechanisms, traffic rules and role in neurological pathogenesis

“…Many other lines of research support the view that kinesin-and dynein-mediated transport is modulated by phosphorylation. For example, ERK1/2 phosphorylation of dynein intermediate chain (DIC) recruits cytoplasmic dynein to signaling endosomes for retrograde transport in axons (Mitchell et al, 2012). Moreover, recent studies in Caenorhabditis elegans have shown that two cyclin-dependent kinases, PCT-1 and CDK-5, directly polarize trafficking of presynaptic components by inhibiting dyneinmediated retrograde transport and setting the balance between anterograde and retrograde motors (Ou et al, 2010).…”