MYO18A is found as a novel PAK2 binding partner via βPIX/GIT1. MYO18A-depleted cells showed dramatic changes in shape, actin stress fiber and membrane ruffle formation, and displayed increases in the number and size of focal adhesions and a decrease in cell migration, suggesting an important role of MYO18A in regulating epithelial cell migration.

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.

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BackgroundGolgin-97 is a tethering factor in the trans-Golgi network (TGN) and is crucial for vesicular trafficking and maintaining cell polarity. However, the significance of golgin-97 in human diseases such as cancer remains unclear.MethodsWe searched for a potential role of golgin-97 in cancers using Kaplan-Meier Plotter (http://kmplot.com) and Oncomine (www.oncomine.org) datasets. Specific functions of golgin-97 in migration and invasion were examined in golgin-97-knockdown and golgin-97-overexpressing cells. cDNA microarray, pathway analysis and qPCR were used to identify gene profiles regulated by golgin-97. The role of golgin-97 in NF-κB signaling pathway was examined by using subcellular fractionation, luciferase reporter assay, western blot analysis and immunofluorescence assay (IFA).ResultsWe found that low expression of golgin-97 correlated with poor overall survival of cancer patients and was associated with invasiveness in breast cancer cells. Golgin-97 knockdown promoted cell migration and invasion, whereas re-expression of golgin-97 restored the above phenotypes in breast cancer cells. Microarray and pathway analyses revealed that golgin-97 knockdown induced the expression of several invasion-promoting genes that were transcriptionally regulated by NF-κB p65. Mechanistically, golgin-97 knockdown significantly reduced IκBα protein levels and activated NF-κB, whereas neither IκBα levels nor NF-κB activity was changed in TGN46- or GCC185-knockdown cells. Conversely, golgin-97 overexpression suppressed NF-κB activity and restored the levels of IκBα in golgin-97-knockdown cells. Interestingly, the results of Golgi-disturbing agent treatment revealed that the loss of Golgi integrity was not involved in the NF-κB activation induced by golgin-97 knockdown. Moreover, both TGN-bound and cytosolic golgin-97 inhibited NF-κB activation, indicating that golgin-97 functions as an NF-κB suppressor regardless of its subcellular localization.ConclusionOur results collectively demonstrate a novel and suppressive role of golgin-97 in cancer invasiveness. We also provide a new avenue for exploring the relationship between the TGN, golgin-97 and NF-κB signaling in tumor progression.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0230-5) contains supplementary material, which is available to authorized users.