PerspectiveSignal Transduction

Crosstalk Between Rac and Rho

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Science  26 Mar 1999:
Vol. 283, Issue 5410, pp. 2028-2029
DOI: 10.1126/science.283.5410.2028

The cytoskeletal changes that alter the adhesion, spreading, and motility of cells depend on a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. Members of the Rho family of guanine nucleotide triphosphate (GTP)-binding proteins—including RhoA, Rac, and Cdc42—are important regulators of cytoskeletal organization (1). RhoA controls the assembly of stress fibers (bundles of actin and myosin filaments that attach to the plasma membrane at points called focal adhesions), Rac regulates the formation of membrane ruffles, and Cdc42 governs the extension of slender membrane protrusions (filopodia or microspikes) (1). Although a hierarchy has been established in which activated Cdc42 stimulates Rac activity and Rac stimulates RhoA activation (1), during cell movement these proteins work antagonistically. Rac and Cdc42 promote protrusive events at the leading edge of cells, whereas RhoA induces retraction of the leading edge. Little is known about how Rac or Cdc42 oppose the action of RhoA. On page 2083 of this issue, Sanders et al. identify a possible pathway through which Rac may counteract RhoA (2). The authors demonstrate that p21-activated kinase (PAK), which is activated by either Rac or Cdc42, blocks the phosphorylation of myosin light chains induced by RhoA. This results in decreased myosin activity, a reduction in contractility, and the disassembly of stress fibers (3).

In nonmuscle cells, the activity of myosin II (the myosin found in all cell types that is composed of two heavy chains and four light chains) is regulated by phosphorylation of myosin light chains. This stimulates myosin adenosine triphosphatase activity and induces a conformational change in myosin that promotes its assembly into bipolar filaments. The activated myosin generates tension on the actin filaments and bundles them into stress fibers. Myosin light chain phosphorylation is regulated both by kinases (which add a phosphate group) and phosphatases (which remove a phosphate group).

Historically, most attention has been paid to the myosin light chain kinase (MLCK), an enzyme regulated by calcium and calmodulin. But a new player arrived on the scene with the discovery that Rho kinase (a downstream effector of RhoA) inhibits a myosin phosphatase that removes phosphate groups from myosin light chains and blocks myosin activity (see the figure) (4). Thus, a pathway emerged in which RhoA elevates myosin light chain phosphorylation by inhibiting its dephosphorylation. This scheme became even more complex with the finding that Rho kinase could also directly phosphorylate myosin light chains, potentially usurping the role of MLCK (5). It is clear that myosin light chain phosphorylation is elevated in vivo in response to RhoA activation, but whether this is due primarily to inhibition of the myosin phosphatase or to direct phosphorylation of light chain, or to a combination of both, has not yet been established.

Cells on the move.

PAK, a kinase activated by either Rac or Cdc42, inhibits myosin light chain (MLC) phosphorylation and cell contractility. It does this by phosphorylating the myosin light chain kinase (MLCK) and inhibiting its activity. Rho kinase, a downstream effector of RhoA, promotes MLC phosphorylation by blocking the activity of myosin phosphatase. Rho kinase can also directly phosphorylate MLC, bypassing the MLCK pathway.

In previous work, PAK was shown to promote the disassembly of stress fibers and focal adhesions (57). Sanders et al. now demonstrate that MLCK is a substrate for PAK. Phosphorylation of MLCK by PAK decreases its activity, which in turn results in decreased myosin light chain phosphorylation and a decrease in actin-myosin filament assembly (see the figure). Just as elevated myosin activity promotes the assembly of stress fibers, it has been shown that inhibiting actin-myosin interactions with pharmacological reagents causes the disassembly of these structures (3). Consequently, PAK's ability to inhibit myosin light chain phosphorylation accounts for the disassembly of stress fibers and focal adhesions observed in cells overexpressing activated PAK.

Most of the kinases stimulated by Rac, Cdc42, or RhoA have multiple targets, and so it is likely that there are additional ways in which PAK opposes or modifies the actions of RhoA. Indeed, the cytoskeletal rearrangements induced by activated PAK are dramatic (68)—reminiscent of those seen in cells treated with the actin filament-disrupting drug cytochalasin D—suggesting that cytoskeletal proteins as well as MLCK are targets for PAK.

The observations of Sanders et al. are important for understanding cell motility. During this complex process, protrusive and contractile forces must be coordinated. Prominent focal adhesions and stress fibers are associated with cells that do not move. Rac and Cdc42 stimulate cell movement, and to be effective these proteins must not only stimulate protrusion, but must also promote disassembly and turnover of focal adhesions and stress fibers. The current work is important because it suggests how contractile forces in the cell can be restrained by Rac and Cdc42, and how stress fibers and focal adhesions may be disassembled through the action of PAK on MLCK.

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