The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110␣ and p110␤, and do not contain p110␦. Injection of specific inhibitory antibodies to p110␣ induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110␤ antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110␣ antibodies. In summary, the p110␣ isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.PI 3-kinases 1 are important signaling intermediates in a variety of regulated cellular processes (1). They are classified based on their regulation and substrate specificity (2). Class I enzymes produce PI[3]P, PI[3,4]P 2 , and PI[3,4,5]P 3 , whereas class II and III enzymes produce PI[3]P and PI[3,4]P 2 , or only PI[3]P, respectively. Class Ia enzymes exhibit the greatest diversity of the known PI 3-kinases, with multiple isoforms of both the regulatory (p85) and catalytic (p110) subunits (2). Differential phosphorylation of p85␣ and p85 and differential activation of p85␣-and p85␤-associated PI 3-kinase have been reported (3, 4). Knockouts of p85␣ further suggest that the p85␣ and p85␤ are not redundant (5, 6). Distinct class Ia catalytic subunit isoforms also have different functions. Both p110␣ and p110␤ play a role in mitogenesis, although p110␣ is required for responses to a broader range of growth factors (7,8). Recently, distinct signaling properties for p110 isoforms have been demonstrated in macrophages (9).We examined the specific functions of p110␣ and p110␤ in MTLn3 cells, a metastatic variant of the 13762NF rat mammary adenocarcinoma. MTLn3 cells undergo chemotaxis in an EGF gradient (10,11). This response involves the actin-dependent extension of a lamellipod in the direction of increasing EGF concentrations, with a zone of newly polymerized F-actin at the leading edge (12). Using isoform-specific inhibitory antibodies against p110␣ and p110␤, we now show that EGFstimulated lamellipod extension requires p110␣ but not p110␤. Significantly, anti-p110␣ antibodies blocked the formation of new barbed ends during an in situ actin nucleation assay. These studies provide direct evidence that p110␣ is required for the regulation of actin nucleation by EGF.

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity. Phosphoinositide 3 (PI3)1 -kinases are critical regulators of cell growth, motility, and survival. Phosphoinositide 3-kinases are classified by their preferred substrates and their mode of regulation (1, 2). Class IA PI3-kinases, which are activated by receptor tyrosine kinases, are heterodimers consisting of an SH2 domain-containing regulatory subunit (p85␣, p85␤, p55␣, p50␣, or p55␥) and a catalytic subunit (p110␣, p110␤, or p110⌬).Class IA PI3-kinases are obligate heterodimers in vivo, because the p110 catalytic subunits are labile at 37°C and are unstable as monomers (3). Dimerization of a p110 catalytic subunit with p85 (or the shorter isoforms) maintains the enzyme in a low activity state, as the regulatory subunit inhibits p110 (3). Subsequent activation of p85/p110 involves a translocation of the normally cytosolic enzyme to a membrane as well as an increase in the specific activity of the dimer. The best understood mechanism for these two events is the high affinity binding of the two SH2 domains in p85 to phosphorylated YXXM motifs in receptor tyrosine kinases or their substrates (4, 5). The activity of p85/p110 dimers is also increased by binding of activated Rac or...