Interaction of myosin VI and its binding partner DOCK7 plays an important role in NGF-stimulated protrusion formation in PC12 cells

Abstract

DOCK7 (dedicator of cytokinesis 7) is a guanidine nucleotide exchange factor (GEF) for Rac1 GTPase that is involved in neuronal polarity and axon generation as well in Schwann cell differentiation and myelination. Recently, we identified DOCK7 as the binding partner of unconventional myosin VI (MVI) in neuronal-lineage PC12 cells and postulated that this interaction could be important in vivo [Majewski et al. (2012) Biochem Cell Biol., 90:565-574]. Herein, we found that MVI-DOCK7 interaction takes also place in other cell lines and demonstrated that MVI cargo domain via its RRL motif binds to DOCK7 C-terminal M2 and DHR2 domains. In MVI knockdown cells, lower Rac1 activity and a decrease of DOCK7 phosphorylation on Tyr1118 were observed, indicating that MVI could contribute to DOCK7 activity. MVI and DOCK7 co-localization was maintained during NGF-stimulated PC12 cell differentiation and observed also in the outgrowths. Also, during differentiation an increase in phosphorylation of DOCK7 as well as of its downstream effector JNK kinase was detected. Interestingly, overexpression of GFP-tagged MVI cargo domain (GFP-GT) impaired protrusion formation indicating that full length protein is important for this process. Moreover, a transient increase in Rac activity observed at 5min of NGF-stimulated differentiation of PC12 cells (overexpressing either GFP or GFP-MVI) was not detected in cells overexpressing the cargo domain. These data indicate that MVI-DOCK7 interaction could have functional implications in the protrusion outgrowth, and full length MVI seems to be important for delivery and maintenance of DOCK7 along the protrusions, and exerting its GEF activity.

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics

“…recently shown a role for MYO6 and GIPC1 in regulating Akt signalling on this compartment [34] and it will be interesting to further address the role of the LIFT complex in this pathway. The second RhoGEF, DOCK7, is a GEF for both RAC1 and CDC42 [41] and has previously been associated with MYO6 [35,36]. Here, we show DOCK7 links via LRCH3 to MYO6 to form the septin regulatory DISP (DOCK7-Induced Septin disPlacement) complex.…”

“…Our in situ proximity labelling and mutational profiling resolved another putative MYO6-associated protein complex composed of DOCK7 and LRCH family proteins. DOCK7 is a GEF for RAC1 and CDC42, which forms a complex with MYO6 to regulate neurite outgrowth [35,36]. Secondary screens in BirA*-LRCH3 expressing cells identified five high-confidence interactions, as well as several lower confidence hits, including DOCK7, a number of septin family members, protein phosphatase 6 (PP6) components as well as utrophin and syntrophins (Figs 2 and 5A).…”

Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria

“…A role for this myosin in regulating actin filament dynamics and assembly has been reported previously; for example, MYO6 has the capacity to promote the local accumulation of actin structures at cell-cell contacts, such as the zonula adherens of polarized epithelial cells ( Mangold et al., 2011 ), and in actin cones of segregating cells during Drosophila spermatid individualization ( Noguchi et al., 2006 ). Although the exact mechanism of MYO6-dependent actin regulation is currently not known, MYO6 has been identified in a complex with DOCK7, a guanine nucleotide exchange factor for Rac and cdc42, which performs important functions in the spatial regulation of actin organization ( Majewski et al., 2012 , Sobczak et al., 2016 ). In addition, we demonstrate a strong requirement for cdc42, Arp2/3, formins, and N-WASP in F-actin cage formation around damaged mitochondria ( Figure 4 ), suggesting tight control of actin filament assembly around mitochondria.…”

Myosin VI in the nucleus of neurosecretory PC12 cells: Stimulation-dependent nuclear translocation and interaction with nuclear proteins

“…We showed important roles for MVI in cell migration and proliferation, but not in catecholamine secretion [ 31 ]. Moreover, we demonstrated that interaction of MVI with the newly identified partner, DOCK7, was crucial for the NGF-stimulated outgrowth formation [ 32 , 33 ]. In the present study, we demonstrate for the first time that upon PC12 cell stimulation MVI translocates to the nucleus, where it colocalizes not only with transcriptionally active regions, but also with PML bodies and speckles.…”