Binding of the extreme carboxyl-terminus of PAK-interacting exchange factor β (βPIX) to myosin 18A (MYO18A) is required for epithelial cell migration

Abstract

The PAK2/βPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-β/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/βPIX/GIT1 complex through direct binding to βPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα-βPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of βPIX and inhibited cell migration. βPIX interacts through its most carboxyl-terminus, PAWDETNL (639-646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas βPIX(1-638), a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in βPIX(1-638)-expressing cells were greater than those in cells expressing wild-type βPIX(FL). Further experiments using deletion mutants of MYO18A and βPIX showed that disruption of MYO18A-βPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and βPIX and provide evidence for the critical role of this interaction in regulating cellular localization of βPIX, Rac1 activity, and adhesion and migration in epithelial cells.

Myosin 18A Coassembles with Nonmuscle Myosin 2 to Form Mixed Bipolar Filaments

“…These sites, which include a PDZ domain, PDZ domain ligand, and potential SH3 domain ligands, could recruit specific molecules to filaments ( Figure 4B). M18A has been shown to interact with the Rac GEF b-PIX in a complex with PAK2 and GIT1 [14,18], with a complex containing LRAP35A and the NM2 kinase MRCK [15], and with the Golgiresident protein GOLPH3 [2,19]. M18A could also link hybrid filaments to firmly anchored structures against which hybrid filaments could generate force even when actin filaments are not present on both sides of the bipolar myosin filament (Figure 4C).…”

Identification of CD245 as myosin 18A, a receptor for surfactant A: A novel pathway for activating human NK lymphocytes

“…Myosin 2A is required for cytolytic granule exocytosis in human NK cells. 36 Class 18 myosins have been involved in fundamental tissular processes in mammalians, including epithelial cell migration, 37 stromal cell differentiation 38 and tumor suppression. 39,40 In humans and mice, myosin 18A is expressed in hematopoietic cells as two splice variants, referred to as a (230 kDa) and b (190 kDa).…”

Myosin 18Aα targets guanine nucleotide exchange factor β-Pix to dendritic spines of cerebellar Purkinje neurons to promote spine maturation

“…As discussed in the Introduction, myosin 18A isoforms probably contribute to cell function primarily by co-assembling with myosin 2, where they then recruit proteins that regulate the myosin filament, influence the local environment around the filament, and/or attach the filament to cellular structures. One protein known to interact with myosin 18Aα is the Rac/Cdc42 GEF β-Pix, which binds to the myosin's C-terminal non-helical (Hsu et al, 2010(Hsu et al, , 2014. This interaction could be particularly relevant here, as β-Pix has been implicated in promoting spine maturation via several distinct pathways involving actin assembly and myosin assembly and contractility (see Introduction).…”

“…Similar results were obtained using GFP-tagged β-Pix, which is also no longer concentrated in spines relative to dendrites following myosin 18Aα knockdown ( Figure 6, Panels E1 through E3, and Panel I). Finally, a GFP-tagged version of β-Pix lacking C-terminal residues 639-647 (β-Pix-ΔCT), which is no longer able to bind to myosin 18Aα (Hsu et al, 2010(Hsu et al, , 2014, does not exhibit significant spine enrichment ( Figure 6, Panels F1 through F3, and Panel I). Together, these results argue that the recruitment of β-Pix to Purkinje neuron spines depends on myosin 18Aα.…”

“…One bona fide myosin 18A binding partner is β-Pix, which binds via 12 residues near its Cterminus to myosin 18A's ~80-residue non-helical tailpiece (Hsu et al, 2010(Hsu et al, , 2014. This interaction has been shown to regulate cell migration and focal adhesion dynamics in mesenchymal cells (Hsu et al, 2010(Hsu et al, , 2014. A second binding partner is the myosin 2 regulatory light chain kinase MRCK, which binds to myosin 18A's N-terminal PDZ domain through the adaptor protein LRAP (Tan et al, 2008).…”