title: Rap1 activation in collagen phagocytosis is dependent on nonmuscle myosin II-A.
abstract: Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or beta1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.
authors.affiliation: CIHR Group in Matrix Dynamics, University of Toronto, Toronto, ON M5S 3E2, Canada.
authors.forename: Pamela D
authors.initials: PD
authors.lastname: Arora
authors.forename: Mary Anne
authors.initials: MA
authors.lastname: Conti
authors.forename: Shoshana
authors.initials: S
authors.lastname: Ravid
authors.forename: David B
authors.initials: DB
authors.lastname: Sacks
authors.forename: Andras
authors.initials: A
authors.lastname: Kapus
authors.forename: Robert S
authors.initials: RS
authors.lastname: Adelstein
authors.forename: Anne R
authors.initials: AR
authors.lastname: Bresnick
authors.forename: Christopher A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D003094:Collagen / Q000378:metabolism*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D024507:Nonmuscle Myosin Type IIA / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D016608:Talin / Q000235:genetics / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D006576:Heterocyclic Compounds, 4 or More Rings; D019012:Integrin beta1; D010455:Peptides; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D016608:Talin; C472645:blebbistatin; D003094:Collagen; D009219:Myosin-Light-Chain Kinase; D024507:Nonmuscle Myosin Type IIA; D020827:rap1 GTP-Binding Proteins
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