PAK4 was originally identified as a molecular target for the Rho GTPase Cdc42, and it plays a key role in Cdc42's ability to induce filopodia (1
). Here we have generated a constitutively active mutant of PAK4 [PAK4(S445N)] in order to better characterize the function of this serine/threonine kinase. Fibroblasts expressing PAK4(S445N) transiently induced filopodia when they were plated onto fibronectin, which is consistent with PAK4's function as a target for Cdc42. Activated PAK4 also had a number of other functions which were not revealed previously by studying wild-type PAK4. Most importantly, expression of PAK4(S445N) caused a transformed morphology in fibroblasts. Fibroblasts stably expressing PAK4(S445N) lost their anchorage-dependent growth requirement and formed colonies in soft agar. Similar results were shown previously for cells expressing activated Cdc42 (19
). Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for the Rho GTPases. We propose that PAK4(S445N)'s ability to transform cells is due at least in part to its ability to induce morphological changes, including cell rounding, loss of stress fibers and focal adhesions, and decreased spreading.
PAK4(S445N) is the first full-length activated PAK4 to be generated. PAK4 as well as the PAK4 Drosophila melanogaster
homologue mushroom body tiny (mbt) (25
) and an uncharacterized Caenorhabditis elegans
homologue, C45B11.1, all encode for proteins with a serine at residue 445. Most serine/threonine protein kinases, including other members of the PAK family, however, have an asparagine residue at the corresponding site. The asparagine at this position serves to stabilize the catalytic loop by hydrogen bonding to a conserved aspartate (corresponding to D440 in PAK4) within the loop (47
). The serine-to-asparagine conversion in PAK4 is therefore thought to function by strengthening the catalytic loop. Although PAK4(S445N) has strong kinase activity, its substrate specificity appears to be unaltered. Like wild-type PAK4, for example, PAK4(S445N) does not phosphorylate MLCK. Furthermore, PAK4(S445N) does not activate the ERK or p38 pathways and does not activate the JNK pathway any further than wild-type PAK4 does. This is consistent with our previous prediction that PAK4 is primarily a mediator of the cytoskeletal changes induced by Cdc42 rather than a key player in the Cdc42-to-JNK pathway (1
The morphological changes resulting from PAK4(S445N) expression do not appear to be nonspecific effects due to the high activity of this kinase. Rather, they seem to be an amplification of wild-type PAK4's activity. Overexpression of even wild-type PAK4 in some cells, such as HeLa cells, results in a slightly rounded shape and lowered adhesion (N. Gnesutta and A. Minden, unpublished results). Likewise, another activated PAK4 mutant, PAK4Δ, in which the PAK4 regulatory domain is deleted (1
), induced some of the morphological changes induced by PAK4(S445N), such as a decrease in stress fibers and focal adhesions, but not filopodium formation (data not shown). Furthermore, some of the functions of PAK4(S445N) are consistent with PAK4's role as an effector for Cdc42. For example, the transient induction of filopodia and the ability to confer anchorage-independent growth are functions which are shared by PAK4 and activated Cdc42 (17
). Importantly, the activities of PAK4(S445N) appear to be specific to PAK4. For example, unlike activated PAK4, constitutively active PAK1(T423E) does not induce anchorage-independent growth (data not shown) (45
). PAK4(S445N) also differs from activated PAK1 in substrate specificity and the mechanism by which it induces cytoskeletal changes. In particular, we have found that unlike PAK1, the dissolution of stress fibers by PAK4 does not appear to be mediated by MLCK phosphorylation and the subsequent regulation of MLC phosphorylation.
While many of the functions of PAK4(S445N) are characteristic of Cdc42 functions, some of its functions, such as cell rounding and the dissolution of stress fibers, may be distinct from Cdc42 functions. The identification of new PAK4 activators other than Cdc42 will be important in order to better understand whether it also serves as a target for other types of signaling and cytoskeletal regulatory proteins. The mechanisms by which PAK4 induces its various morphological effects are not yet clearly understood. For example, as discussed above, unlike for PAK1, the dissolution of stress fibers by PAK4 is not due to a decrease in MLC phosphorylation. It is interesting to note, however, that PAK4 does lead to a consistent decrease in MLC expression which could potentially contribute to the dissolution of stress fibers. Another possibility is that PAK4 could inhibit Rho activity, which normally functions to stimulate stress fiber formation. We have in fact found that Rho activity is decreased (but not abolished) in PAK4(S445N)-expressing cells (data not shown) and it will be interesting to determine whether this is related to PAK4's role in stress fiber dissolution. It should be noted, however, that activated Rho triggers stress fiber formation by a mechanism that involves multiple steps which include increased phosphorylation of MLC (15
) and activation of LIMK1 (21
), yet we do not see a decrease in MLC phosphorylation (Fig. ) or an inhibition of LIMK1 activity (data not shown) in PAK4(S445N)-expressing cells. Ultimately it will be important to identify new substrates for PAK4 in order to fully understand the mechanism by which it induces specific morphological changes.
The ability of PAK4(S445N) to induce a transformed phenotype in fibroblasts is especially intriguing because the regulation of cell proliferation, progression through the cell cycle, and oncogenic transformation are important functions of the Rho proteins (14
). Most of the Dbl family exchange factors are potent oncogenes, and all three GTPases have important contributions to oncogenic transformation by Dbl (20
). A number of target proteins for the Rho GTPases have been identified which may be necessary for oncogenic transformation, such as the Rho effector protein ROCK (39
) and the Cdc42 target protein γ-COP (49
). Neither of these, however, is sufficient to transform cells on its own. Likewise, activated PAK1 does not induce transformation on its own (44
), even though it has been shown to be necessary for transformation by oncogenic Ras in some cells (44
). Furthermore, effector loop mutants for Cdc42 and Rac which do not bind to PAKs 1, 2, or 3 can still induce many of the hallmarks of oncogenic transformation (13
), suggesting that these PAK family members are not required for transformation by the Rho family GTPases.
In contrast to other PAKs, we have found that activated PAK4 confers anchorage-independent growth on fibroblasts and leads to focus formation in soft agar assays. This work is the first study to show a direct role for a PAK protein in transformation. Importantly, constitutively active and cycling mutants of Cdc42 also induce anchorage-independent growth in fibroblasts (19
). The induction of anchorage-independent growth has in fact been shown to be a major contribution of Cdc42 to transformation by oncogenic Dbl (20
). Not only does activated PAK4 trigger anchorage-independent growth, but dominant-negative PAK4 also inhibits transformation by oncogenic Dbl, a potent activator of all three Rho family GTPases. This inhibition is not due merely to sequestering Cdc42, because a mutant that lacks the GBD also inhibits transformation. Dominant-negative PAK4 was less efficient at inhibiting Dbl-induced foci than was dominant-negative Cdc42 (Cdc42N17). However, it should be noted that the PAK4 mutants and Cdc42N17 cannot be directly compared with each other because we do not know whether they have the same capacities to act as dominant-negative mutants, i.e., to inhibit their endogenous counterparts. Furthermore, it should be noted that Cdc42N17 can bind directly to Dbl and therefore may be a particularly effective inhibitor of Dbl activity.
The mechanism by which PAK4 can regulate oncogenic transformation is not yet known. Our results suggest that activated PAK4 can induce anchorage-independent growth in the absence of any strong activation of several known signal transduction pathways, such as the JNK, p38, and ERK pathways, that lead to changes in gene expression patterns. Rather, we propose that changes in cell morphology and adhesion play a major role in PAK4's ability to transform cells. We do not know, however, whether all of the different cytoskeletal changes induced by PAK4(S445N) contribute to the oncogenic process. Further investigation will be necessary in order to determine which of the morphological changes induced by PAK4 are directly related to its role in transformation. Furthermore, as discussed above, although PAK4 was originally identified as a target for Cdc42, we cannot rule out the possibility that it could regulate transformation by a mechanism that is either partly or entirely independent of Cdc42. For example, recently we have found that PAK4 can protect cells against apoptosis and inhibit caspase activation in response to a variety of different stimuli, including serum withdrawal, UV irradiation, and tumor necrosis factor alpha stimulation (12
). Since inhibition of apoptosis is an important part of oncogenic transformation, such a protective role is also likely to contribute to PAK4's role in oncogenic transformation.
We do not rule out the possibility that PAK4 may also mediate oncogenic transformation or morphological changes in response to other signaling enzymes besides Cdc42. We have found, however, that dominant-negative PAK4 is an inefficient inhibitor of Ras transformation. This is initially surprising because previous work using dominant-negative Cdc42N17 has indicated that Ras requires Cdc42 to produce foci (34
). It should be noted, however, that the requirement for Cdc42 by Ras and Dbl cannot be directly compared, because Cdc42N17 is a much more effective inhibitor of Dbl foci than of Ras foci (Fig. ). In future work it will be interesting to determine whether other oncogenes require signals through PAK4 to induce transformation and whether PAK4 can mediate responses that are independent of Cdc42.