Phosphorylated form of MacMARCKS is essential to LFA-1-dependent cell-cell adhesion of U937 monocytic cells

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Macrophage spreading requires the microtubule cytoskeleton and protein kinase C (PKC). The mechanism of involvement of the microtubules and PKC in this event is not fully understood. Dynamitin is a subunit of dynactin, which is important for linking the microtubuledependent motor protein dynein to vesicle membranes. We report that dynamitin is a Ca 2؉ /calmodulin-binding protein and that dynamitin binds directly to macrophage-enriched myristoylated alanine-rice C kinase substrate (MacMARCKS), a membrane-associated PKC substrate involved in macrophage spreading and integrin activation. Dynamitin was found to copurify with Mac-MARCKS both during MacMARCKS purification with conventional chromatography and during the immunoabsorption of MacMARCKS using anti-MacMARCKS antibody. Vice versa, MacMARCKS was also found to cosediment with the 20 S dynactin complex. We determined that the effector domain of MacMARCKS is required to interact with the N-terminal domain of dynamitin. MacMARCKS and dynamitin also partially colocalized at peripheral regions of macrophages and in the cell-cell border of 293 epithelial cells. Treatment with phorbol esters abolished this colocalization. Disrupting the interaction with a short peptide derived from the MacMARCKS-binding domain of dynamitin caused macrophages to spread and flatten. These data suggest that the dynamitin-MacMARCKS interaction is involved in cell spreading. Furthermore, the regulation of this interaction by PKC and Ca 2؉ /calmodulin provides a possible regulatory mechanism for cell adhesion and spreading.

Section: Discussionmentioning

confidence: 71%

Section: Discussionmentioning

confidence: 99%

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Protein Kinase C-regulated Dynamitin-Macrophage-enriched Myristoylated Alanine-Rice C Kinase Substrate Interaction Is Involved in Macrophage Cell Spreading

Yue¹,

Lu²,

Garcés³

et al. 2000

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“…4 Furthermore, PKC-mediated phosphorylation of the cytoskeletal remodeling effector MacMarcks and its related Marcks (48) has been shown to increase LFA-1 mobility and avidity to ICAM-1 (44). However, phorbol ester stimulation of LFA-1 avidity in U937 cells required the presence of functional MacMarcks (49). We found that both CD81-expressing and -null U937 clones expressed negligible amounts of MacMarcks or the related Marcks.…”

Section: Discussionmentioning

confidence: 78%

The CD81 Tetraspanin Facilitates Instantaneous Leukocyte VLA-4 Adhesion Strengthening to Vascular Cell Adhesion Molecule 1 (VCAM-1) under Shear Flow

Feigelson

Grabovsky

Shamri

et al. 2003

Leukocyte integrins must rapidly strengthen their binding to target endothelial sites to arrest rolling adhesions under physiological shear flow. We demonstrate that the integrin-associated tetraspanin, CD81, regulates VLA-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells. CD81 strengthens multivalent VLA-4 contacts within subsecond integrin occupancy without altering intrinsic adhesive properties to low density ligand. CD81 facilitates both VLA-4-mediated leukocyte rolling and arrest on VCAM-1 under shear flow as well as VLA-5-dependent adhesion to fibronectin during short stationary contacts. CD81 also augments VLA-4 avidity enhancement induced by either chemokine-stimulated G i proteins or by protein kinase C activation, although it is not required for G i protein or protein kinase C signaling activities. In contrast to other proadhesive integrin-associated proteins, CD81-promoted integrin adhesiveness does not require its own ligand occupancy or ligation. These results provide the first demonstration of an integrin-associated transmembranal protein that facilitates instantaneous multivalent integrin occupancy events that promote leukocyte adhesion to an endothelial ligand under shear flow.

“…Both actin and microtubule filaments are involved in constraining integrin molecules. The PKC signal is most likely transduced to cytoskeleton by MacMARCKS protein, a member of the MARCKS (myristoylated alanine-rich protein kinase C substrate) family of PKC substrates (4 -6), because mutation or lack of this protein prevents PKC-stimulated molecular mobility of integrin (2), as well as ligand binding by integrin (7)(8)(9). In addition, MARCKS protein, the homologue of MacMARCKS, has been shown to be involved in cell spreading (10,11).…”

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confidence: 99%

In Vivo Interaction between Dynamitin and MacMARCKS Detected by the Fluorescent Resonance Energy Transfer Method

Jin

Yue

2001

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Dynamitin is a subunit of the dynactin complex regulating microtubule-dependent motor functions, and MacMARCKS (Macrophage-enriched myristoylated alanine-rich protein kinase C substrate) is a major protein kinase C substrate regulating integrin activation. The interaction between dynamitin and MacMARCKS has been implicated in integrin-dependent cell spreading. However, the in vivo interaction of these two proteins in living cells has not been demonstrated. Spatial and temporal information about the interaction is also lacking. In this study, we used the fluorescent resonance energy transfer method to demonstrate in vivo interaction between MacMARCKS and dynamitin with cyan fluorescent protein (CFP)-conjugated dynamitin as the donor fluorophore and yellow fluorescent protein (YFP)-conjugated MacMARCKS as the acceptor fluorophore. The interaction of these two fusion proteins was studied both in vitro and in vivo, and typical fluorescent resonance energy transfer was observed; the CFP emission peak increased while the YFP emission peak decreased when protein interaction was abolished. Spatial and temporal information was obtained in RAW macrophage cells. In resting macrophage cells, dynamitin-Mac-MARCKS interaction is concentrated at the cell periphery, although the majority of dynamitin is distributed at the perinuclear region of the cells. When cells were treated with phorbol 12-myristate 13-acetate, both proteins concentrated to perinuclear regions of the cells, and yet the interaction disappeared as the cell spread. Similar events were also observed in 293 cells. Thus, we conclude that dynamitin and MacMARCKS indeed interact in living cells.The cytoskeleton plays a pivotal role in activating ␤ 2 integrin molecules by restraining the integrin molecules on the cell membrane in an inactivated state (1-3). Upon stimulation by PKC 1 activation, the cytoskeleton constraint is released and integrin is activated. Both actin and microtubule filaments are involved in constraining integrin molecules. The PKC signal is most likely transduced to cytoskeleton by MacMARCKS protein, a member of the MARCKS (myristoylated alanine-rich protein kinase C substrate) family of PKC substrates (4 -6), because mutation or lack of this protein prevents PKC-stimulated molecular mobility of integrin (2), as well as ligand binding by integrin (7-9). In addition, MARCKS protein, the homologue of MacMARCKS, has been shown to be involved in cell spreading (10, 11). MacMARCKS protein contains an N-terminal membrane-targeting domain that is myristoylated at its N-terminal glycine residue. The effector domain, in the center part of the protein, contains the PKC phosphorylation sites, two serine residues at positions 93 and 104.Interaction between MacMARCKS and dynamitin, a subunit of dynactin, has been observed (12). Dynactin (reviewed in Refs. 13-16), a complex of proteins, is an important regulator of dynein, a microtubule-dependent minus end-directed motor protein (17). The dynactin complex has been suggested as a "molecular bridge" connecting...