title: 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.
authors.affiliation: Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Sobczak
authors.affiliation: Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland; Laboratory of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences of Ukraine, 14-16 Drahomanov St., 79005 Lviv, Ukraine.
authors.forename: Vira
authors.initials: V
authors.lastname: Chumak
authors.affiliation: Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland.
authors.forename: Paweł
authors.initials: P
authors.lastname: Pomorski
authors.affiliation: Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland.
authors.forename: Emilia
authors.initials: E
authors.lastname: Wojtera
authors.affiliation: Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland.
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Majewski
authors.affiliation: Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Nowak
authors.affiliation: Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo, Japan; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo, Tokyo, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Yamauchi
authors.affiliation: Laboratory of Molecular Basis of Cell Motility, Department of Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02-093 Warsaw, Poland. Electronic address: j.redowicz@nencki.gov.pl.
authors.forename: Maria Jolanta
authors.initials: MJ
authors.lastname: Rędowicz
mesh_terms: D000818:Animals; D022081:Cell Surface Extensions / Q000187:drug effects* / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D057809:HEK293 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D020932:Nerve Growth Factor / Q000494:pharmacology*; D055495:Neurogenesis / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: C514577:DOCK7 protein, human; C000610446:DOCK7 protein, rat; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; C089785:myosin VI; D020932:Nerve Growth Factor; D048031:JNK Mitogen-Activated Protein Kinases; D018995:Myosin Heavy Chains; D020744:rac GTP-Binding Proteins
keywords: DOCK7; GEF for Rac; Myosin VI; NGF; PC12 cells; Protrusions