Rac1
Proteins interacting with Rac: PAK The p21-activated kinase (PAK) is a downstream target of both Rac and CDC42, two members of the Ras-related Rho subfamily that mediate the signaling pathway leading to cytoskeletal reorganization. To investigate PAK's function in Caenorhabditis elegans development, the cDNA coding for the p21-activated kinase homologue (CePAK) was isolated from a C. elegans embryonic cDNA library. This 2.35-kilobase pair cDNA encodes a polypeptide of 572 amino acid residues, with the highly conserved N-terminal p21-binding and the C-terminal kinase domains. Similar to its mammalian and Drosophila counterparts, the CePAK protein expressed in E. coli exhibits binding activity toward GTP-bound CeRac1 and CDC42Ce. Polyclonal antibodies raised against the recombinant CePAK recognize a specific 70-kDa protein from embryonic extracts that displays CeRac1/CDC42Ce-binding and kinase activities. Immunofluorescence analysis indicates that CePAK is specifically expressed at the hypodermal cell boundaries during embryonic body elongation. Elongation involves dramatic cytoskeletal reorganization. Interestingly, CeRac1 and CDC42Ce are also found at the hypodermal cell boundaries, evidence suggesting their common involvement in hypodermal cell fusion, a crucial morphogenetic event for nematode development (Chen, 1996).
The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. In epithelial HeLa cells, alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42 and Rac1, proteins that colocalize to these same sites. By deletion analysis, the N terminus of PAK has been shown to contain targeting sequences for focal adhesions, indicating that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras have also been obtained. Plasmids encoding these different constitutively active alpha-PAKs cause loss of stress fibers when introduced into both HeLa cells and fibroblasts, similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions are also observed. These combined effects result in retraction of the cell periphery after plasmid microinjection. These data support previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes (Manser, 1997).
Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase) that, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak [p65(Pak)].
Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. Agents that activate or block specific components downstream of Ras were tested and a Ras > PI 3-kinase >
Rac/Cdc42 > Pak signal has been demonstrated. Although these studies suggest that the signal from Ras through PI 3-kinase is
sufficient to activate Pak, additional studies suggest that other effectors contribute to Pak activation.
RasV12S35 and RasV12G37, two effector mutant proteins that fail to activate PI 3-kinase, do not activate
Pak when tested alone but activate Pak when they are cotransfected. Similarly, RacV12H40, an effector
mutant that does not bind Pak, and Rho both cooperate with Raf to activate Pak. A dominant negative Rho
mutant also inhibits Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector
mutants that transform cells cooperatively stimulate ERK. Cooperation is Pak dependent, since all
combinations are inhibited by kinase-deficient Pak mutants in both transformation assays and ERK
activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each
other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative
transformation suggests that Pak activation is necessary, although not sufficient, for cooperative
transformation of Rat-1 fibroblasts by Ras, Rac, and Rho (Tang, 1999).
New members (X-PAKs) of the Ste20/PAK family of protein kinases have been identified in Xenopus, and an investigation has been made of their role in the process that maintains oocytes arrested in the cell cycle. Microinjection of a catalytically inactive mutant of X-PAK1 with a K/R substitution in the ATP binding site greatly facilitates oocyte release from G2/prophase arrest by progesterone and insulin. (Also deleted was its N-terminal-half that contains the conserved domains responsible for binding of both Cdc42/Rac GTPases and SH3-containing proteins). Addition of the same X-PAK1 mutant to cell cycle extracts from unfertilized eggs induces apoptosis, as shown by activation of caspases (See Drosophila Caspase 1) and cytological changes in in vitro-assembled nuclei. This is suppressed by adding Bcl-2 or the DEVD peptide inhibitor of caspases, and rescued by competing the dominant-negative mutant with its constitutively active X-PAK1 counterpart. Such results indicate that X-PAK1 (or another member of the Xenopus Ste20/PAK family of protein kinases) is involved in arrest of oocytes at G2/prophase and prevention of apoptosis; thus, there may be a link between death by apoptosis and release of healthy oocytes from cell cycle arrest. The fact that cell cycle arrest protects oocytes from apoptosis is consistent with the finding that extracts from metaphase II-arrested oocytes are less sensitive to apoptotic signals than those from activated eggs (Faure, 1997).
Proteolytic activation of hPAK65, a p21-activated kinase, induces morphological changes and elicits apoptosis. hPAK65 is cleaved both in vitro and in vivo by caspases at a single site between the N-terminal regulatory p21-binding domain and the C-terminal kinase domain. The C-terminal cleavage product becomes activated, and exhibits a kinetic profile that parallels caspase activation during apoptosis. This C-terminal hPAK65 fragment also activates the c-Jun N-terminal kinase pathway in vivo. Microinjection or transfection of this truncated hPAK65 causes striking alterations in cellular and nuclear morphology, which subsequently promotes apoptosis in both CHO and Hela cells. Conversely, apoptosis is delayed in cells expressing a dominant-negative form of hPAK65. These findings provide direct evidence that the activated form of hPAK65 generated by caspase cleavage is a proapoptotic effector that mediates morphological and biochemical changes seen in apoptosis (Lee, 1997).
The pathway involving the signaling protein p21Ras propagates a range of extracellular signals from receptors on the cell membrane to the cytoplasm and
nucleus. The Ras proteins regulate many effectors, including members of the Raf family of protein kinases. Ras-dependent activation of Raf-1 at the plasma
membrane involves phosphorylation events, protein-protein interactions and structural changes. Phosphorylation of serine residues 338 or 339 in the
catalytic domain of Raf-1 regulates Raf-1's activation in response to Ras, Src and epidermal growth factor. The p21-activated protein kinase
Pak3 phosphorylates Raf-1 on serine 338 in vitro and in vivo. The p21-activated protein kinases are regulated by the Rho-family GTPases Rac and Cdc42.
These results indicate that signal transduction through Raf-1 depends on both Ras and the activation of the Pak pathway. Since guanine-nucleotide-exchange
activity on Rac can be stimulated by a Ras-dependent phosphatidylinositol-3-OH kinase, a mechanism might exist through which one Ras effector pathway
can be influenced by another (King, 1998).
Mitogens promote cell growth through integrated signal transduction networks that alter cellular metabolism, gene expression and cytoskeletal organization. Many such signals are propagated through activation of MAP kinase cascades that are partly regulated by upstream small GTP-binding proteins. Interactions among cascades are suspected but not defined. Rho family small G proteins such as Rac1 and Cdc42hs, which activate the JNK/SAPK pathway, cooperate with Raf-1 to activate the ERK pathway. This causes activation of ternary complex factors (TCFs), which regulate c-fos gene expression through the serum response element. Examination of ERK pathway kinases shows that neither MEK1 nor Ras will synergize with Rho-type proteins, and that only MEK1 is fully activated, indicating that MEKs are a focal point for cross-cascade regulation. Rho family proteins utilize PAKs for this effect, as expression of an active PAK1 mutant can substitute for Rho family small G proteins, and expression of an interfering PAK1 mutant blocks the Rho-type protein stimulation of ERKs. PAK1 phosphorylates MEK1 on Ser298, a site important for the binding of Raf-1 to MEK1 in vivo. Expression of interfering PAK1 also reduces stimulation of TCF function by serum growth factors, while expression of active PAK1 enhances EGF-stimulated MEK1 activity. This demonstrates interaction among MAP kinase pathway elements not previously recognized and suggests an explanation for the cooperative effect of Raf-1 and Rho family proteins on cellular transformation (Frost, 1997).
Serine/threonine protein kinases of the Ste20/PAK family have been implicated in signal transduction from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades. In the yeast Saccharomyces cerevisiae, Ste20 is involved in transmitting the mating-pheromone signal from the betagamma-subunits (encoded by the STE4 and STE18 genes, respectively) of a heterotrimeric G protein to a downstream MAP kinase cascade. A binding site for the G-protein beta-subunit (Gbeta) has been identified in the non-catalytic carboxy-terminal regions of Ste20 and its mammalian homologs, the p21-activated protein kinases (PAKs). Association of Gbeta with this site in Ste20 is regulated by binding of pheromone to the receptor. Mutations in Gbeta and Ste20 that prevent this association block activation of the MAP kinase cascade. Considering the high degree of structural and functional conservation of Ste20/PAK family members and G-protein subunits, these results provide a possible model of a role for these kinases in Gbetagamma-mediated signal transduction in organisms ranging from yeast to mammals. Ste20 acts through Ste11, a MEK kinase (Leeuw, 1998).
Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 that has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. The extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induces PC12 cells to extend neurites similar to those stimulated by NGF. This effect is independent of PAK1 ser/thr kinase activity but is dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, normal neurite outgrowth stimulated by NGF can be effectively inhibited. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system that drives polarized outgrowth of the actin cytoskeleton in the developing neurite (Daniels, 1998).
AlphaPAK in a constitutively active form can exert morphological effects resembling those of Cdc42G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in alphaPAK). alphaPAK mutants with substitutions in this motif, resulting in severely reduced Cdc42 binding, can be recruited normally to Cdc42G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), while not affecting Cdc42 binding, produce a constitutively active kinase, suggesting that this is a negative regulatory region. Indeed, a 67-residue polypeptide encoding alphaPAK83-149 potently inhibits GTPgammaS-bound Cdc42-mediated kinase activation of both alphaPAK and betaPAK. Coexpression of this PAK inhibitor with Cdc42G12V prevents the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by p21. Coexpression of PAK inhibitor with Rac1G12V also prevents loss of stress fibers, but not ruffling induced by p21. Coexpression of alphaPAK83-149 completely blocks the phenotypic effects of hyperactive alphaPAKL107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization (Zhao, 1998).
The serine/threonine kinase p21-activated kinase (PAK) has been implicated as a downstream
effector of the small GTPases Rac and Cdc42. While these GTPases evidently induce a variety of
morphological changes, an understanding of PAK's role(s) remains elusive. Overexpression of betaPAK in PC12 cells induces a Rac phenotype, including cell spreading/membrane
ruffling, and increased lamellipodia formation at growth cones and shafts of nerve
growth factor-induced neurites. These effects are still observed in cells expressing kinase-negative
or Rac/Cdc42 binding-deficient PAK mutants, indicating that kinase- and p21-binding domains are
not involved. These kinase- and p21-binding domains of PAK are considered downstream effectors of Rac action. Furthermore, lamellipodia formation in all cell lines, including those expressing Rac
binding-deficient PAK, is inhibited significantly by dominant-negative RacN17. Equal inhibition is
achieved by blocking PAK interaction with the guanine nucleotide exchange factor PIX, using a
specific N-terminal PAK fragment. It is concluded that PAK, via its N-terminal non-catalytic domain,
acts upstream of Rac mediating lamellipodia formation through interaction with PIX. PAK's role in Rac signaling is 2-fold: (1) PAK is an effector of Rac; as such, acting through its kinase domain, PAK has been shown to be able to dissolve focal complexes and may also induce transcriptional up-regulation through activation of JNK and other MAP kinase cascades in appropriate cellular settings. (2) PAK can promote Rac activity and thereby has the potential to regulate many of the effects ascribed to Rac, including membrane ruffling, focal complex assembly and lamellipodia formation (Obermeier, 1998).
Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture. A
challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the
p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase
pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established. Mutants of the budding yeast Saccharomyces cerevisiae, lacking the PAK homologs Ste20 and Cla4 exhibit actin
cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression
suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of
permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the
cortical actin cytoskeleton (Eby, 1998).
The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all,
eukaryotic cells. Pak's effects on these parameters are mediated by
changes in the organization of cortical actin. Because cell motility requires polarized rearrangements of the
actin/myosin cytoskeleton, the role of Pak1 in regulating cell movement was examined. Clonal tetracycline-regulated NIH-3T3 cell lines were established that inducibly express either wild-type Pak1, a kinase-dead form of Pak1,
or a constitutively-active form. The morphology, F-actin organization, and
motility of these cells were then examined. Expression of any of these forms of Pak1 induces dramatic changes in actin
organization that are not inhibited by coexpression of a dominant-negative form of Rac1. Cells
inducibly expressing wild-type or constitutively-active Pak1 have large, polarized lamellipodia at the leading
edge, are more motile than their normal counterparts when plated on a fibronectin-coated surface, and
display enhanced directional movement in response to an immobilized collagen gradient. In contrast, cells
expressing a kinase-dead form of Pak1 project multiple lamellipodia emerging from different parts of the
cell simultaneously. These cells, though highly motile, display reduced persistence of movement when
plated on a fibronectin-coated surface and have defects in directed motility toward immobilized collagen.
Expression of constitutively activated Pak1 is accompanied by increased myosin light chain (MLC)
phosphorylation, whereas expression of kinase-dead Pak1 has no effect on MLC. These results suggest
that Pak1 affects the phosphorylation state of MLC, thus linking this kinase to a molecule that directly
affects cell movement (Sells, 1999).
The small GTPase Rac regulates cytoskeletal organization, cell cycle progression, gene expression and oncogenic transformation,
processes that depend upon both soluble growth factors and adhesion to the extracellular matrix (ECM). Growth
factors and adhesion to the ECM both contribute independently and approximately equally to Rac activation. However, activated Rac in
non-adherent cells fails to stimulate the Rac effector PAK. V12 Rac or Rac activates by serum translocated to the membrane fraction
of adherent cells but remains mainly cytoplasmic in suspended cells. An activated Rac mutant lacking a membrane-targeting sequence
does not activate PAK in adherent cells, while mutations that force membrane targeting restore PAK activation in suspended cells. In
vitro, V12 Rac shows greater binding to membranes from adherent relative to suspended cells, indicating that cell adhesion regulates membrane binding sites for
Rac. These results show that ECM regulates the ability of Rac to couple with PAK via an effect on membrane binding sites that facilitate their interaction (del Pozo, 2000).
Membrane localization is critical for function of Ras-family proteins, even when mutationally activated. Recent studies have
indicated that membrane localization of Ras is more complex than previously suspected, involving transit through the endoplasmic reticulum and Golgi. However, Rac and other Rho-family GTPases differ from Ras in that, despite the presence of a hydrophobic prenyl group at their C-termini, they rapidly
translocate between membrane and cytosolic compartments. The factors that regulate association of Rho-family GTPases
with membranes are incompletely understood, but RhoGDI clearly plays a role in this process since it promotes dissociation of GTPases from membranes and
sequesters them in the cytosol. Nucleotide exchange factors have also been proposed to be involved, since they are
often membrane bound and are believed to interact with GTPases at the plasma membrane. The data presented, however, argue
against a critical role for GEFs in the adhesion-dependent localization, since the effect is clearly separable from GTP loading (del Pozo, 2000).
Forced membrane localization of PAK also strongly enhances its activation. Thus, available evidence argues that interaction
of Rac with its effectors occurs preferentially in an adhesion-dependent membrane compartment that enhances the efficiency of their interaction. Overexpression of
Rac probably overcomes the requirement for adhesion by mass action, so that high levels of Rac can promote the activation of PAK even if the association is less
efficient. The observation that this occurs by regulating membrane binding sites also raises the interesting possibility that adhesion may control the subcellular
localization of Rac-effector interactions within cells. Thus, growth factors could globally activate Rac but the locations at which cytoskeletal structures form could be
determined by adhesive interactions (del Pozo, 2000).
The major biological functions of Rac, including cell growth, migration and gene expression, are strongly dependent on cell interactions with ECM. For example,
stimulation of JNK by cytokines or c-fos expression by growth factors is minimal in non-adherent endothelial cells or fibroblasts. In the case of migration, subcellular control of Rac interaction with effectors would allow cells to determine where to extend lamellipodia on the basis of
contacts with ECM, which would facilitate efficient cell movement. Elucidating how Rac-dependent pathways integrate information from integrins and growth factors
is therefore essential to understanding their functions in the context of intact tissues (del Pozo, 2000).
Rho family GTPases (Cdc42, Rac1, and RhoA) function downstream of Ras, and in a variety of cellular processes. Studies to examine these functions have not directly linked endogenous protein interactions with specific in vivo functions of Rho GTPases. Endogenous Rac1 and two known binding partners, Rho GDP dissociation inhibitor (RhoGDI) and p21-activated kinase (PAK), fractionate as distinct cytosolic complexes. A Rac1:PAK complex is translocated from the cytosol to ruffling membranes upon cell activation by serum. Overexpression of dominant-negative (T17N) Rac1 does not affect the assembly or distribution of this Rac1:PAK complex. This is the first direct evidence of how a specific function of Rac1 is selected by the assembly and membrane translocation of a distinct Rac1:effector complex (Hansen, 2001).
This is the first study to examine the organization of endogenous Rac1 in cells in
response to a specific extracellular signal. Proteins were separated by sucrose gradient centrifugation and native PAGE, which provide quantitative and nonselective surveys of protein complexes. RhoGDI and PAK cofractionated with Rac1. The cofractionation of these proteins under different conditions indicates the formation of specific protein complexes comprising Rac1, RhoGDI, and PAK in the cytosol, and Rac1 and PAK at the membrane. In the cytosol, it is proposed that Rac1GDP:RhoGDI is
activated, forming a Rac1GTP:RhoGDI:PAK complex, in which PAK does not appear to be active. The possibility that PAK is complexed only with Rac1 or with other proteins cannot be formally excluded; available PAK antibodies identified MDCK PAK by immunoblotting but not by immunoprecipitation. In support of the Rac1:RhoGDI:PAK complex, it is noted that PAK binds only active Rac1, while RhoGDI binds both active and inactive Rac1. Furthermore, RhoGDI and PAK both bind Rac1 via multiple domains, allowing for the formation of a trimeric complex. In the absence of signals to translocate the Rac1:PAK complex to the membrane, GTP hydrolysis would result in dissociation of the trimeric complex into Rac1GDP:RhoGDI and PAK. Serum could activate factors at the membranes that are necessary to break the interaction between Rac1 and RhoGDI, allowing the Rac1GTP:PAK complex to translocate to specific membrane sites. Of many structures that stain with PAK antibodies, only staining at ruffling membranes is lost following serum withdrawal, which coincides with the loss of the Rac1:PAK complex from membranes. It is concluded that the Rac1:PAK complex localizes to ruffling membranes in response to serum. Dissociation of the Rac1:PAK complex would leave monomeric Rac1 to bind RhoGDI, displacing Rac1 into the cytosol and leaving PAK to further act on downstream targets (Hansen, 2001).
These results show that serum activation of Rac1 induces the localized membrane recruitment of distinct effector complex with a specialized function. This suggests that, as a general mechanism, the mechanism of Rho GTPase activation selects defined interactions in order to elicit a particular biological function (Hansen, 2001).
The small GTPase Rac and the second messenger cGMP (guanosine 3',5'-cyclic monophosphate) are critical regulators of diverse cell functions. When activated by extracellular signals via membrane signaling receptors, Rac executes its functions through engaging downstream effectors such as p21-activated kinase (PAK), a serine/threonine protein kinase. However, the molecular mechanism by which membrane signaling receptors regulate cGMP levels is not known. A signaling pathway linking Rac to the increase of cellular cGMP has been uncovered. Rac uses PAK to directly activate transmembrane guanylyl cyclases (GCs), leading to increased cellular cGMP levels. This Rac/PAK/GC/cGMP pathway is involved in platelet-derived growth factor-induced fibroblast cell migration and lamellipodium formation. These findings connect two important regulators of cellular physiological functions and provide a general mechanism for diverse receptors to modulate physiological responses through elevating cellular cGMP levels (Guo, 2007).
Protrusion of the leading edge of migrating epithelial cells requires precise regulation of two actin filament (F-actin) networks, the lamellipodium and the lamella. Cofilin is a downstream target of Rho GTPase signaling that promotes F-actin cycling through its F-actin-nucleating, -severing, and -depolymerizing activity. However, its function in modulating lamellipodium and lamella dynamics, and the implications of these dynamics for protrusion efficiency, has been unclear. Using quantitative fluorescent speckle microscopy, immunofluorescence, and electron microscopy, this study established that the Rac1/Pak1/LIMK1 signaling pathway controls cofilin activity within the lamellipodium. Enhancement of cofilin activity accelerates F-actin turnover and retrograde flow, resulting in widening of the lamellipodium. This is accompanied by increased spatial overlap of the lamellipodium and lamella networks and reduced cell-edge protrusion efficiency. It is proposed that cofilin functions as a regulator of cell protrusion by modulating the spatial interaction of the lamellipodium and lamella in response to upstream signals (Delorme, 2007).
The Akt/PKB isoforms have different roles in animals, with Akt2 primarily regulating metabolic signaling and Akt1 regulating growth and survival. This study shows distinct roles for Akt1 and Akt2 in mouse embryo fibroblast cell migration and regulation of the cytoskeleton. Akt1-deficient cells responded poorly to platelet-derived growth factor while Akt2-deficient cells had a dramatically enhanced response, resulting in a substantial increase in dorsal ruffling. Swapping domains between Akt1 and Akt2 demonstrated that the N-terminal region containing the pleckstrin homology domain and a linker region distinguishes the two isoforms, while the catalytic domains are interchangeable. Akt2 knock-out cells also migrated faster than wild-type cells, especially through extracellular matrix (ECM), while Akt1 knock-out cells migrated more slowly than wild-type cells. Consistently, Akt2 knock-out cells had elevated Pak1 and Rac activities, suggesting that Akt2 inhibits Rac and Pak1. Both Akt2 and Akt1 associated in complexes with Pak1, but only Akt2 inhibited Pak1 in kinase assays, suggesting an underlying molecular basis for the different cellular phenotypes. Together these data provide evidence for an unexpected functional link between Akt2 and Pak1 that opposes the actions of Akt1 on cell migration.
This study demonstrates that planar cell polarity signaling regulates morphogenesis in Xenopus embryos in part through the assembly of the fibronectin (FN) matrix. A regulatory pathway is outlined that includes cadherin adhesion and signaling through Rac and Pak, culminating in actin reorganization, myosin contractility, and tissue tension, which, in turn, directs the correct spatiotemporal localization of FN into a fibrillar matrix. Increased mechanical tension promotes FN fibril assembly in the blastocoel roof (BCR), while reduced BCR tension inhibits matrix assembly. These data support a model for matrix assembly in tissues where cell-cell adhesions play an analogous role to the focal adhesions of cultured cells by transferring to integrins the tension required to direct FN fibril formation at cell surfaces (Dzamba, 2009).
Both PCP signaling and FN are required for gastrulation movements in Xenopus. The current study shows that normal assembly of FN matrix is inhibited following expression of dnWnt11. It is proposed that PCP signaling acts upstream to regulate FN fibrillogenesis by increasing cadherin adhesive activity and tension in BCR cells. Thus, one function of the PCP pathway in these embryos is to regulate FN matrix assembly in the marginal zone of the BCR. In both cultured mammalian cells and in the embryo, FN is first observed as diffuse punctae across cell surfaces. With time, both in cultured cells and on the BCR, fine fibrils are found initially at cell-cell junctions. These newly assembled FN fibrils are soluble in 2% DOC, but with time become detergent insoluble. The fibrils identified morphologically at gastrulation are DOC soluble, but, by neurula stages, they display DOC insolubility, suggesting that this progression is, in fact, similar to the progression of FN assembly and DOC solubility reported for cultured cells. Moreover, FN fibrils are required for radial intercalation and epiboly in the BCR, and nonfibrillar FN promotes high-speed migration of mesendodermal cells. Therefore, while early embryonic fibrillar and non-fibrillar FNs are indistinguishable in terms of DOC solubility, differences in biological functions supported by these two physical states of FN are evident in vivo (Dzamba, 2009).
The small GTPase, Rac, is a critical component of the pathway through which cadherins contribute to tissue tension. Both cadherin ligation and Wnt/PCP signaling can promote the activation of the small GTPases, Rac and Rho. While Rho has been shown to promote FN fibril assembly in cultured cells by promoting contractility through the phosphorylation of MLC, in the current system, Rac is the critical GTPase for FN assembly. Tension is generated via regulation of the actin cytoskeleton and MLC phosphorylation by Rac and its downstream effector, Pak. Inhibiton of either Rac or Pak abrogated cortical actin assembly in BCR cells. Activated Pak colocalized with FN fibrils. When Pak was inhibited, the phosphorylation of MLC at cell-cell junctions was reduced. Taken together, these data indicate that Pak is the key downstream effector of Rac in this system regulating cell tension and FN assembly (Dzamba, 2009).
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