Actin Polymerization Is Regulated by Proteins That Bind GActin

In the artificial world of a test tube, experimenters can start the polymerization process by adding salts to G-actin or can depolymerize F-actin by simply diluting the filaments. Cells, however, must maintain a nearly constant cytosolic ionic concentration and thus employ a different mechanism for controlling actin polymerization. The cellular regulatory mechanism includes several actin-binding proteins that either promote or inhibit actin polymerization. Here, we consider two such proteins that have been isolated and characterized.

Inhibition of Actin Assembly by Thymosin (34 Calculations based on the Cc of G-actin (0.1 ^M), a typical cytosolic total actin concentration (0.5 mM), and the ionic conditions of the cell indicate that nearly all cellular actin should exist as filaments; there should be very little G-actin. Actual measurements, however, show that as much as 40 percent of actin in an animal cell is unpolymerized. What keeps the cellular concentration of G-actin above its Cc? The most likely explanation is that cytosolic proteins sequester actin, holding it in a form that is unable to polymerize.

Because of its abundance in the cytosol and ability to bind ATP-G-actin (but not F-actin), thymosin is considered to be the main actin-sequestering protein in cells. A small protein (5000 MW), thymosin binds ATP-G-actin in a 1:1 complex. The binding of thymosin p4 blocks the ATP-binding site in G-actin, thereby preventing its polymerization. In platelets, the concentration of thymosin p4 is 0.55 mM, approximately twice the concentration of unpoly-merized actin (0.22 mM). At these concentrations, approximately 70 percent of the monomeric actin in a platelet should be sequestered by thymosin p4.

Thymosin ß4 (Tß4) functions like a buffer for monomeric actin, as represented in the following reaction:

In a simple equilibrium, an increase in the cytosolic concentration of thymosin ß4 would increase the concentration of sequestered actin subunits and correspondingly decrease F-actin, because actin filaments are in equilibrium with actin monomers. This effect of thymosin ß4 on the cellular F-actin level has been experimentally demonstrated in live cells.

Promotion of Actin Assembly by Profilin Another cytosolic protein, profilin (15,000 MW), also binds ATP-actin monomers in a stable 1:1 complex. At most, profilin can buffer 20 percent of the unpolymerized actin in cells, a level too low for it to act as an effective sequestering protein. Rather than sequestering actin monomers, the main function of profilin probably is to promote the assembly of actin filaments in cells. It appears to do so by several mechanisms.

First, profilin promotes the assembly of actin filaments by acting as a nucleotide-exchange factor. Profilin is the only actin-binding protein that allows the exchange of ATP for ADP. When G-actin is complexed with other proteins, ATP or ADP is trapped in the ATP-binding cleft of actin. However, because profilin binds to G-actin at a site opposite the

ATP-binding cleft, it can recharge ADP-actin monomers released from a filament, thereby replenishing the pool of ATP-actin (Figure 19-10).

Second, as a complex with G-actin, profilin is postulated to assist in the addition of monomers to the (+) end of an actin filament. This hypothesis is consistent with the three-dimensional structure of the profilin-actin complex in which profilin is bound to the part of an actin monomer opposite the ATP-binding end, thereby leaving it free to associate with the (+) end of a filament (see Figure 19-3). After the complex binds transiently to the filament, the profilin dissociates from actin.

Finally, profilin also interacts with membrane components taking part in cell-cell signaling, suggesting that it may be particularly important in controlling actin assembly at the plasma membrane. For example, profilin binds to the membrane phospholipid phosphoinositol 4,5-bisphosphate (PIP2); this interaction prevents the binding of profilin to G-actin. (As discussed in Chapter 13, PIP2 is hydrolyzed in response to certain extracellular signals.) In addition, profilin binds to proline-rich sequences that are commonly found in membrane-associated signaling proteins such as Vasp and Mena. This interaction, which does not inhibit the binding of profilin to G-actin, localizes profilin-actin complexes to the membrane.

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