The Neural Cell Adhesion Molecule (NCAM) Associates with and Signals through p21-Activated Kinase 1 (Pak1)

Abstract

The Neural cell adhesion molecule (NCAM) plays an important role in regulation of nervous system development. To expand our understanding of the molecular mechanisms via which NCAM influences differentiation of neurons, we used a yeast two-hybrid screening to search for new binding partners of NCAM and identified p21-activated kinase 1 (Pak1). We show that NCAM interacts with Pak1 in growth cones of neurons. The autophosphorylation and activity of Pak1 were enhanced when isolated growth cones were incubated with NCAM function triggering antibodies, which mimic the interaction between NCAM and its extracellular ligands. The association of Pak1 with cell membranes, the efficiency of Pak1 binding to its activators, and Pak1 activity were inhibited in brains of NCAM-deficient mice. NCAM-dependent Pak1 activation was abolished after lipid raft disruption, suggesting that NCAM promotes Pak1 activation in the lipid raft environment. Phosphorylation of the downstream Pak1 effectors LIMK1 and cofilin was reduced in growth cones from NCAM-deficient neurons, which was accompanied by decreased levels of filamentous actin and inhibited filopodium mobility in the growth cones. Dominant-negative Pak1 inhibited and constitutively active Pak1 enhanced the ability of neurons to increase neurite outgrowth in response to the extracellular ligands of NCAM. Our combined observations thus indicate that NCAM activates Pak1 to drive actin polymerization to promote neuronal differentiation.

Kinesin-1 promotes post-Golgi trafficking of NCAM140 and NCAM180 to the cell surface

“…We thus propose that this domain competes with KLC1 for binding to NCAM and contributes to the detachment of NCAM from KLC1 upon delivery of NCAM to the cell surface. In agreement with this idea, activation of NCAM functions induces activation of PAK1 (Li et al, 2013) and exocytosis in growth cones (Chernyshova et al, 2011). Interestingly, the proposed model also suggests that activation of NCAM by homophilic and heterophilic interactions at sites of contacts between cells can promote exocytosis of NCAMcontaining vesicles to stabilize these contacts.…”

“…PAK1 interacts with NCAM at the cell surface of growing neurites and growth cones (Li et al, 2013). It binds to the amino acid sequence in the intracellular domain of NCAM, which we now show also contains the binding site for KLC1 (Li et al, 2013).…”

“…Interestingly, the amino acid sequence in NCAM-ID that is required for its binding to KLC1 overlaps with a recently identified binding site for p21-activated protein kinase (PAK1), which interacts with NCAM at the cell surface (Li et al, 2013). This observation prompted us to analyze whether PAK1 and KLC1 compete with each other for binding to NCAM-ID.…”

“…For transfection of cells, cDNAs coding for full length non-mutated hNCAM180 (Diestel et al, 2004); hNCAMΔCT (Boutin et al, 2009);rat NCAM140-ID (Leshchyns'ka et al, 2003); rat NCAM-ID-729-750, rat NCAM-ID-777-810, and Sec8Y401F,Y616F (Chernyshova et al, 2011); rat NCAM-ID-748-777 (Li et al, 2013); GFP-KLC1 and GFP-KHC1 (generous gifts of Toshiharu Suzuki, Hokkaido University, Sapporo, Japan) (Araki et al, 2007;Kawano et al, 2012), and GFP (Clontech) were used. To construct cDNA coding for hNCAM180-Δ747-754, site-specific mutagenesis was carried out using the overlap extension PCR method (Ho et al, 1989) with hNCAM180 as a template.…”

“…In the adult NCAM is involved in regulating synaptic plasticity (Chazal et al, 2000;Chipman et al, 2014;Cremer et al, 1994Cremer et al, , 1997Doherty et al, 1990;Hinsby et al, 2004;Kiryushko et al, 2004;Maness and Schachner, 2007;Muller et al, 1996;Panicker et al, 2003;Sytnyk et al, 2004). Being expressed at the cell surface of neurons and glial cells, NCAM induces intracellular signal transduction pathways and cytoskeleton rearrangements in response to interactions with its extracellular domain (Büttner et al, 2003;Ditlevsen et al, 2008;Leshchyns'ka et al, 2003;Li et al, 2013;Nielsen et al, 2010;Pollerberg et al, 1987;Walmod et al, 2004).…”