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

Abstract

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...

Cross-talk unfolded: MARCKS proteins

“…In agreement with the model proposed for MARCKS, a myristoyl moiety and the MRP ED combined are sufficient to target a protein to the plasma membrane in i o, as was demonstrated in MDCK cells using a myristoylated GFP-MRP-ED construct [50]. Also, a chimaera of yellow fluorescent protein with MRP translocates rapidly from the plasma membrane to the cytosol and the perinuclear region of macrophages when phosphorylated, similarly to the typical behaviour of MARCKS [51]. However, differences in subcellular localization and membrane binding have been reported for MARCKS and MRP.…”

A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells

“…Marcksl1 overexpression also imposed a substantial delay in MTOC reorientation, consistent with the idea that PKC activity plays a central role in the process. This delay was quite intriguing to us because Marcksl1 has been shown to associate with dynein through the regulatory dynactin complex 39, 40 , and therefore could conceivably play a role in dynein regulation during the polarization response. However, siRNA-mediated suppression of Marcksl1 had no effect on MTOC reorientation (data not shown), indicating Marcksl1 is probably not involved in this process at physiological concentrations.…”

Two‐Hybrid Fluorescence Cross‐Correlation Spectroscopy Detects Protein–Protein Interactions In Vivo

“…[9] For FCCS, using two AFPs in vivo has a great advantage over organic dyes: No external manipulation is necessary to introduce the proteins into the cell-furthermore, they are interacting in their natural environment. In contrast to fluorescence resonance energy transfer (FRET) measurements, which have often been used to show interactions in vivo, [22][23][24][25] FCCS does not depend on the close proximity of the interacting partners in the nanometer range, but only on their correlated motion. Even interactions inside large protein complexes can be measured easily by these correlated events.…”