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Vol. 19, Issue 12, 5032-5046, December 2008

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Rap1 Activation in Collagen Phagocytosis Is Dependent on Nonmuscle Myosin II-A

Pamela D. Arora^*, Mary Anne Conti, Shoshana Ravid, David B. Sacks, Andras Kapus^||, Robert S. Adelstein, Anne R. Bresnick^¶, and Christopher A. McCulloch^*

*CIHR Group in Matrix Dynamics, University of Toronto, Toronto, ON M5S 3E2, Canada; Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel; Harvard Medical School, Brigham and Women's Hospital, Boston, MA 02115; ^||St. Michael's Hospital Research Institute, Toronto, ON, M5B 1W8, Canada; and ^¶Albert Einstein College of Medicine, Bronx, NY 10461

Submitted April 25, 2008; Revised August 26, 2008; Accepted September 9, 2008
Monitoring Editor: Erika Holzbaur

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 β1-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.

Address correspondence to: Christopher A. McCulloch (mccculloch{at}utoronto.ca).

Abbreviations used: GST, glutathione-S-transferase; RBD, Ras-binding domain; MLC, myosin light chain; NM II-A, non-muscle myosin-II-A; NMHC II-A, non-muscle myosin II-A heavy chain; NMHC II-B, non-muscle myosin II-B heavy chain; MLCK, myosin light-chain kinase.

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